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Keywords:

  • binding;
  • cocaine analog;
  • depolarization;
  • dopamine;
  • dopamine transporter;
  • sodium ion

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The present study addresses the effect of intracellular Na+ and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT-expressing cells with gramicidin diminished DA uptake and decreased the potency (increases Ki) of DA in inhibiting the binding of cocaine analog [3H]2β-carbomethoxy-3β-(4-fluorophenyl)tropane (CFT). It also compromised the ability of external Na+ to reduce DA Ki. No substantial effect on DA Ki was observed upon gramicidin treatment in Na+-free buffer, membrane depolarization with high [K+]o, or elevation of [Na+]i with monensin under non-depolarizing conditions. Elevation of DA Ki was greater at more positive potentials when [Na+]i was raised to a similar level, or at higher [Na+]i when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA Ki was significantly higher but less sensitive to gramicidin than that in wild-type (WT) cells. In contrast, DA Ki in cell-free membranes was insensitive to Na+, gramicidin, and D313N mutation. The data suggest that (i) intracellular Na+ plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell-free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na+ and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA.

Abbreviations used
BSA

bovine serum albumin

CFT

2β-carbomethoxy-3β-(4-fluorophenyl)tropane

DA

dopamine

DAT

dopamine transporter

DiBAC2(3)

bis-(1,3-diethylthiobarbituric acid)trimethine oxonol

gramicidin

Gram

HEK

human embryonic kidney

KRH

Krebs/Ringer/HEPES buffer

monensin

Mon

[Na+]i, and [K+]i

intracellular concentration of Na+ and K+, respectively

[Na+]o, and [K+]o

extracellular concentration of Na+ and K+, respectively

NMDG-Cl

N-methyl-D-glucamine chloride

ouabain

Oua PBFI, potassium-binding benzofuran isophthalate

PBS

Phosphate-buffered saline

SBFI

sodium-binding benzofuran isophthalate

WT

wild-type

The dopamine (DA) transporter (DAT) belongs to the superfamily of the Na+/CL-dependent neurotransmitter transporters that couple substrate transport to co-transport of Na+ and Cl (Amara and Kuhar 1993). DAT-mediated re-uptake of released DA is the main mechanism for termination of dopaminergic neurotransmission (Amara and Kuhar 1993; Giros et al. 1996). In addition to its classic action in clearing extracellular DA, DAT also plays a role in mediating somatodentritic DA release, with the reversal of DA transport as one of the potential mechanisms (Falkenburger et al. 2001). Factors determining the direction and magnitude of DA transport appear to be the transmembrane Na+ gradient and membrane potential. DA uptake requires the presence of the inward-directed Na+ gradient and the inside-negative membrane potential (Krueger 1990; McElvain and Schenk 1992; Gu et al. 1994; Sonders et al. 1997; Chen et al. 1999). By contrast, DA efflux is elicited by diminishing or reversing the Na+ gradient (Pifl et al. 1997; Pifl and Singer 1999) and enhanced by membrane depolarization (Khoshbouei et al. 2003).

Reversible binding interaction between DA and DAT is thought to be the key event initiating DA transport cycle. In the conventional model proposed for monoamine transporters (Rudnick 2002), the transporter protein is assumed to alternate between at least two different conformational states, which differ in the accessibility of the binding site for substrates. For inward transport, external substrates are proposed to initially bind at the outward-facing state where the binding site for substrates is exposed only to the external medium. For outward transport, it is thought to be a prerequisite that internal substrates bind to the inward-facing state where the site is only exposed to the cytoplasmic fluid (Levi and Raiteri 1993; Chen and Justice 1998). Given their tight coupling to inward and outward transport of DA, Na+ and membrane potential may play a role in regulating DA binding in addition to serving as the thermodynamic driving forces to influence the velocity of DA transport.

Analyzing competition for binding of a radiolabeled high-affinity blocker is the major approach for quantitatively assessing substrate binding at monoamine transporters. Recently, we have characterized the potency for DA to inhibit the binding of a radiolabeled DAT blocker and cocaine analog, [3H]2β-carbomethoxy-3β-(4-fluorophenyl) tropane ([3H]CFT), to intact cells (Chen et al. 2003). This potency is insensitive to temperature and displays a similar Na+-dependence as Km for DA uptake. These properties support the interpretation that DA inhibits CFT binding mainly via interactions at binding-associated steps for the Na+-regulated transport process. Mutational and chimeric studies have provided substantial evidence that domains on the DAT for DA transport and for cocaine analog binding are closely associated although they are not identical (for reviews see Chen and Reith 2002; Uhl and Lin 2003). Thus, the apparently competitive interactions between DA and cocaine analogs could result from the two compounds binding to distinct but overlapping sites, to distinct sites involving steric hindrance, or to distinct sites linked conformationally.

A surprising finding from studies on DA binding is that, although DA binding to the plasma membrane of cells expressing the DAT is stimulated by Na+ (Chen et al. 2003), DA binding to membranes isolated from the same DAT-expressing cell line is Na+-insensitive (Chen et al. 2002, 2003; Li et al. 2002). Another intriguing finding of our preliminary experiments is that several mutations affected the potency of DA in inhibiting CFT binding to cells but not cell-free membranes. These differences between intact cells and broken membranes prompted our speculation that the transmembrane ion gradient or membrane potential plays a role in DA binding to DAT. Thus, their loss in cell-free DAT preparations could influence the DA–DAT interaction and the impact of Na+. The present study was undertaken to address the effect of transmembrane ion gradients and membrane potential on the binding of DA to the plasma membrane DAT. Information advanced by this study might also be helpful to understanding the impact of ionic gradients and membrane potential on substrate binding to other members of the Na+/CL-dependent neurotransmitter transport family.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Generation of cell lines stably expressing wild-type and mutant DATs

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The pCIN4 vector and the pCIN4- synthetic human DAT construct were generous gifts from Dr Jonathan A. Javitch of Columbia University. The DNA sequences encoding c-myc (EQKLISEEDL) epitope tag was inserted by inverse PCR mutagenesis just after the translational start sequence at the N-terminal of the synthetic human DAT cDNA. The MluI–EspI fragment containing the c-myc epitope was cut and subcloned into the wild-type (WT) pCIN4-DAT construct. The D313N mutant was generated previously using site-directed mutagenesis (Chen et al. 2001). The BstEII–Bsu361 fragment containing the mutation was cut and subcloned into the c-myc tagged pCIN4-DAT construct. All constructs were screened by restriction mapping, and confirmed in both directions by dye terminator cycle sequencing the region flanking the subcloning region (Research Resource Center, University of Illinois). Stable pools of HEK-293 cells expressing DATs were established as described previously (Chen et al. 2001). In preliminary experiments, the uptake and binding activity as well as the effects of ionophores were virtually the same in the absence or presence of the N-terminal c-myc tag.

[3H]CFT binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Cell suspensions or membrane preparations were prepared as described previously (Chen et al. 2001, 2002). Modified Krebs–Ringer–HEPES buffer (KRH) was used in all experiments, which contained 10 mm HEPES (adjusted to pH 7.4 with Tris), 130 mm NaCl unless otherwise stated, 1.2 mm KH2PO4, 1.2 mm MgSO4, 1 mm CaCl2, 3 mm KCl, 10 mm glucose, 0.1 mm tropolone, and 1 mm ascorbic acid. For experiments involving different Na+ concentrations, NaCl in the buffer was isotonically replaced with NMDG-Cl, LiCl, or KCl. Binding assays were conducted in 96-well plates. Cells or their membrane preparations were incubated with 3–4 nm[3H]CFT (84.5 Ci/mmol, Perkin-Elmer Life Sciences, Boston, MA, USA) for 15 or 20 min at 25°C in a total volume of 200 µL. For saturation or competition analysis, varying concentrations of non-radioactive CFT (0.3–1000 nm) or DA (0.3–1000 µm) were included in the assay mixture. The binding reactions were terminated by filtration followed by five washes with ice-cold saline on 0.1% polythyleneimine pre-soaked Wallac B filtermats (Wallac, Gaithersburg, MD, USA) with a 96-well Brandel cell harvester (Gaithersburg, MD, USA). The radioactivity was counted in a 1405 Microbeta liquid scintillation counter (Wallac) after adding 10 mL of Betaplate Scint (Wallac) to each filtermat. Cells transfected with vector alone were tested in parallel and used to estimate non-specific binding. Every experiment was performed in triplicate. Nonlinear regression fitting of data was performed using LIGAND (Biosoft, Cambridge, UK) and ORIGIN (OriginLab Co., Northampton, MA, USA) to estimate Kd, Bmax, and IC50 values. Apparent Ki values were calculated by the Cheng–Prusoff equation.

[3H] DA uptake

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Uptake assays were also conducted in 96-well plates. Cells were incubated with 10 nm[3H]DA (60 Ci/mmol, Perkin–Elmer Life Sciences, Foster City, CA, USA) in KRH buffer for 5 min at 25°C in a total volume of 200 µL. Except that the uptake reactions were terminated and washed on the harvester under lower pressure, the other procedures and data analysis were the same as described for binding assays. In a separate experiment determining the intracellular DA concentration, cells (1.9 × 105/well) were incubated with a high concentration of DA (50 nm[3H]DA plus 100 µm unlabeled DA, 0.03 Ci/mmol) for various times. Intracellular concentrations were calculated from the accumulated radioactivity in the harvested cells and the intracellular volume (1.25 pL per HEK cell) reported by Sitte et al. (2001).

Treatment with ionophores

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Monensin or gramicidin (mixture of gramicidin A, B, C, and D) were prepared freshly and dissolved in ethanol as a 10-mm stock. The stocks were quickly diluted to proper concentrations in 8% ethanol and added to the incubation mixtures resulting in 1% final ethanol for all concentrations of the ionophore tested. Approximately 10 min after being suspended in Na+-free KRH/NMDG+ buffer, aliquots of cells were transferred into appropriate assay mixture and incubated with the ionophore tested or 1% ethanol at approximately 0.5 mg protein/mL for 6 or 30 min at 25°C while gently shaking. For cells treated with gramicidin, the incubation mixture also contained 100 µm ouabain to inhibit Na+, K+-ATPase. Afterwards, a mixture of [3H]CFT, various concentrations of unlabeled CFT or DA was added to the incubation media and the incubation was continued for 15 or 20 min. No cell rupture was found after gramicidin treatment as judged under the microscope by the Trypan blue dye exclusion test.

Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The cell-permeant acetoxymethyl ester (AM) forms of Na+-sensitive dye SBFI and K+-sensitive dye PBFI (Molecular Probes, Eugene, OR, USA) were dissolved in dimethyl sulphoxide (DMSO) to a 1000-fold concentrated stock solution and mixed with an equal volume of 20% w/v Pluronic F-127 solution (Molecular Probes) immediately prior to its addition to the loading buffer [KRH containing 1% bovine serum albumin (BSA)]. Serum-starved cells on the glass bottom of a dish were loaded with 10 µm SBFI/AM or 5 µm PBFI/AM for 2 h at 25°C. The cells were then washed with 1% BSA-containing KRH twice and incubated for 30 min in regular KRH to allow de-esterification of the fluorophores. Measurements of [Na+]i and [K+]i were performed using a dual-excitation fluorescence imaging system (Intracellular Imaging, Cincinnati, OH, USA) in conjunction with a Nikon TMS-F invert microscope. SBFI- or PBFI-loaded cells were alternately excited via a Nikon Fluor 20 Ph3DL objective at 340 and 380 nm by a computer-controlled filter changer and the fluorescence emissions from individual cells were monitored at 510 nm by a COHU high-performance CCD camera. The excitation light level was attenuated electronically until the emitted fluorescence intensity remained constant for 80 min. The camera sensitivity was optimized by adjusting exposure times (usually 3 s for SBFI and 1 s for PBFI). Sequential image pairs were collected every 10 s. Calibration of the excitation ratio in terms of the tested ion concentration was accomplished in situ by equilibrating the dye-loaded cells with KRH containing various Na+ or K+ concentrations, 10 µm gramicidin, and 100 µm ouabain. The Na+ calibration solutions were made by mixing NaCl and KCl to a total concentration of 140 mm, and the K+ calibration solution, by mixing KCl and LiCl. The full in situ calibration was performed in sister cultures using six Na+ or K+ concentrations in a range from 0 to 140 mm. The recordings collected from the 2-min plateau phase at each intracellular ion concentration ([ion]i) were averaged and normalized to [ion]i = 140 mm. The normalized data were plotted according to the equation: RR0 = (RmaxR0) –Km(RR0)/[ion]i. In this equation, R is the ratio of 340 nm fluorescence to 380 nm fluorescence at a particular [ion]i, R0 is the fluorescence ratio in the absence of the ion, Rmax is the fluorescence ratio of the ion-saturated dye, and Km is the Michaelis–Menten constant that reflects the affinity of the dye for that ion. At the end of every experiment, cells were exposed sequentially to calibration solutions containing 140 and 0 mm Na+ or K+, and the R-values from the entire experiment for a given cell were divided by the R-value at [Na+]i or [K+]i = 140 mm for that cell. The normalized R-values were then used to calculate the intracellular concentration, utilizing the above-mentioned equation and the Km and Rmax value fitted from the full calibration. To improve signal–noise ratio, individual points represent the average of six ratio determinations taken every 10 s (six pairs/min). Experiments were repeated at least on three different dishes, each allowing collection of data from 10 to 30 individual cells.

Membrane potential (Em) was calculated from outside and inside monovalent ion concentrations. In the absence of gramicidin, Em = 59 log [K+]o/[K+]i (Nernst equation). In the presence of gramicidin, Em = 59log([Na+]o + 0.29[Li+]o + 3.5[K+]o)/([Na+]i +0.29[Li+]i + 3.5[K+]i) (Goldman–Hodgkin––Katz equation), in which the relative permeability of the gramicidin pore to Na+, Li+, and K+ is 1, 0.29, and 3.5, respectively (Hille 2001). The H+ concentration and H+ permeability were not included in the Goldman–Hodgkin––Katz equation, because due to its vary low concentration, the impact of H+ on the membrane potential was negligible under our conditions.

Fluorescent imaging of membrane potential changes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The lipophilic anionic fluorescent dye DiSBAC2(3) (Molecular Probes) was used to detect changes in membrane potential. This dye is reported to accumulate in the cytoplasm of depolarized cells via a Nernst equilibrium-dependent uptake (Dall'Asta et al. 1997). In addition, it is excluded from the mitochondria due to the negative charge, making it superior to other fluorescence dyes for measuring plasma membrane potential. Serum-starved cells on the glass coverslip were pre-incubated with 1 µm DiSBAC2(3) in KRH at 25°C for 1 h, and then subjected to experimental manipulations for 30 or 50 min in the presence of the same concentration of DiSBAC2(3). Cellular DiSBAC2(3) fluorescence was viewed with an Olympus BX61 fluorescence microscope equipped with a neutral density filter U-25ND25, excitation filter BP 470–490, and barrier filter BA510-550. Images were captured with a CCD camera operated with Spot software (Olympus, Melville, NY, USA). In some experiments, cellular fluorescence intensity was quantified with AlphaEasy software (Alpha Innotech, San Leandro, CA, USA). The average pixel intensity for each of 50 cells in the middle area of the image was measured and the background fluorescence was subtracted. The cellular pixel intensity was expressed as the average value of 50 individual cells.

Immunocytochemistry and confocal microscopy

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Stably transfected HEK-293 cells were grown to 50–70% confluence in eight-chamber slides. Cells were incubated with 1% ethanol, 10 µm gramicidin, or 134.2 mm KCl (isotonically replacing NaCl) for 50 min at 25°C or with 100 nm PMA for 30 min at 37°C in KRH buffer. After incubation, cells were rinsed twice with ice-cold phosphate-buffered saline (PBS), and fixed with methanol for 10 min followed by acetone for 1 min/at20°C. The cells were rinsed with PBS and blocked with 10% normal goat serum and 1% BSA in PBS containing 0.05% Triton X-100 (TPBS) for 1 h at room temperature. Cells were then incubated with the mouse monoclonal anti c-myc antibody (9E10, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1: 200 dilution for 1 h. After three washes in TPBS, cells were incubated with Alexa Fluor 488-conjugated antimouse secondary antibody (Molecular Probes) at 1 : 500 dilution for 1 h. The slide was then washed three times in TPBS and once in PBS, and mounted with SlowFade Light antifade reagent (Molecular Probes). Confocal microscopy was performed by using an Olympus confocal imaging system operated via FluoView software (Melville, NY). The sample was excited via argon laser at 488 nm and viewed using a 510 nm long pass filter and a 530 nm short pass filter.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The role of transmembrane ion gradients and membrane potential in the interaction of external ligands with the DAT was explored with gramicidin, a polypeptide antibiotic that forms pores in the cell membrane but allows only monovalent cations to pass (Wallace 1990). Treatment of cells with gramicidin in the presence 130 mm Na+ strongly affected both DA uptake (Fig. 1a) and the ability of DA to inhibit CFT binding (Fig. 1b, see the increase in DA Ki values). Both effects became evident at 0.1 µm and approached a plateau at 1 µm. In contrast, even at 10 µm, gramicidin showed only a minor effect on the Bmax and Kd values for CFT binding (Figs 1c and d). In both absence and presence of gramicidin, CFT binding reached equilibrium within 15 min, and the binding data were best fit by a one-site model (data not shown).

image

Figure 1. Effect of gramicidin on interactions of DA and CFT with DAT. (a) [3H]DA uptake; (b) DA Ki in inhibiting [3H]CFT binding; (c) CFT Bmax; (d) CFT Kd. Treatment of cells with various concentrations of gramicidin or vehicle (1% ethanol) started 30 min before the addition of radiolabeled and unlabeled ligands. Uptake and binding assays were conducted at 25°C in KRH buffer containing 100 µm ouabain. The results are expressed as the percentage of the value in the presence of vehicle and ouabain. Data are means ± SE for three to six experiments.

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In order to exclude any chemical effect on the transporter, we also investigated the effect of gramicidin in membranes isolated from cells expressing the DAT. Treatment of isolated membranes with 10 µm gramicidin had no adverse effect on CFT binding (control: Kd = 63 ± 4 nm and Bmax = 5.72 ± 0.32 pmol/mg; gramicidin: Kd = 46 ± 4 nm and Bmax = 5.84 ± 0.17 pmol/mg n = 4). It also had negligible effect on the DA Ki in inhibiting this binding (control: Ki = 7.4 ± 0.4 µm; gramicidin: Ki = 8.5 ± 0.3 µm n = 4). In further support for the lack of a direct effect on DA–DAT interactions, in the absence of external Na+, gramicidin failed to increase the DA Ki in inhibiting CFT binding to whole cells (Table 1, treatment 2). The later data also imply that Na+ influx via gramicidin pores plays a crucial role in affecting DA–DAT interactions.

Table 1.  Effect of transmembrane ion concentrations and membrane potential on DA Ki in inhibiting [3H]CFT binding
TreatmentExtracellular ions, mmIntracellular ions, mmEm, mVDA Ki for inhibiting CFT binding, µm
[Na+]o ([Li+]o)[K+]o[Na+]i ([Li+]i)[K+]iControlTreatmentFold change
  1. Control groups were generally run in parallel with treatment groups. Except for treatments 1 and 6, all controls included a 30-min pre-incubation of cells with vehicle (1% ethanol). The control was water without the pre-incubation for treatments 1 and 6, and was 50 or 190 mm Na+ plus 3 mm K+ for treatments 4 or 5. When the total concentration of Na+, K+, and Li+ was lower than 134.2 mm, NMDG+ was added to compensate the buffer osmolarity. The concentration for gramicidin and ouabain was 10 and 100 µm, respectively. The intracellular ion concentration reflects the steady state concentration after a 30-min treatment unless otherwise stated. Membrane potential was calculated as Em = 59 log [K+]o/[K+]i in the absence of gramicidin, and Em = 59 log([Na+]o + 0.29[Li+]o + 3.5[K+]o)/([Na+]i + 0.29[Li+]i + 3.5[K+]i) in the presence of gramicidin. Data are means ± SE for four to ten binding experiments or for 38–112 individual cells used in [ion]i determinations. *p < 0.05 versus control (paired t-test with Bonferroni correction as appropriate). a Corresponding to binding measurements during 6–21 min after addition of gramicidin as opposed to 30–50 min for all other treatments (also see Fig. 2b). b Fluorescence emission results support a membrane potential close to control value (– 88 mV). c[K+]i was measured by replacement of external Na+ with the same concentration of Li+ to avoid interference of Na+ influx with [K+]i determination.

 1Ethanol1304.23.4 ± 0.19131 ± 2.3− 885.41 ± 0.517.38 ± 0.521.36
 2Gram + Oua04.23.3 ± 0.13125 ± 13− 8755.4 ± 9.3254.8 ± 15.50.99
 3Gram + Oua0 (5)4.2approximately 0 (approximately 54)22.9 ± 1.1− 4659.1 ± 11.273.5 ± 10.21.24
 4High K+5094.2approximately 3.4approximately 130− 88.60 ± 0.299.61 ± 0.911.12
 5High K+50144.2approximately 3.4approximately 1302.65.10 ± 0.5911.2 ± 1.65*2.20
 6Ouabain1304.28.05 ± 0.81128 ± 7.2− 885.41 ± 0.515.78 ± 0.461.07
 7Mon 5 µmb1304.230.7 ± 2.8unknownapproximately − 8810.9 ± 0.788.86 ± 1.090.81
 8Mon 20 µmb1304.252.2 ± 4.8unknownapproximately − 8810.9 ± 0.788.39 ± 0.970.77
 9Gram + Oua5094.2approximately 50approximately 94.209.61 ± 0.91132 ± 22.6*13.7
10Gram + Oua50144.2approximately 50approximately 144.2011.2 ± 1.65231 ± 45*21.0
11Gram + Oua50 (90)4.2approximately 50 (approximately 90)approximately 4.2014.9 ± 2.5697.1 ± 15.3*6.52
12Gram + Oua5c4.254.1 ± 2.122.9 ± 1.1− 4921.2 ± 3.397.0 ± 8.6*4.58
13Gram + Ouaa5c4.290 [RIGHTWARDS ARROW] 5428 [RIGHTWARDS ARROW] 23− 58 [RIGHTWARDS ARROW]− 4924.4 ± 1.7316 ± 65*12.9
14Gram + Oua20c4.268.0 ± 4.212.4 ± 0.47− 309.05 ± 0.3790.3 ± 9.5*9.98
15Gram + Oua60c4.292.1 ± 3.95.13 ± 0.92− 108.25 ± 0.2069.0 ± 7.8*8.36
16Gram + Oua130c4.2124 ± 3.72.58 ± 0.072.28.54 ± 0.7861.2 ± 5.9*7.17

Intracellular Na+ concentrations upon gramicidin treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Na+-sensitive dye SBFI was used to examine [Na+]i. The intracellular Na+ signal under conditions described for Fig. 1 was examined in preliminary experiments. Lower concentrations (0.01–0.1 µm) of gramicidin appeared to decrease the success rate of membrane perforation, because the increase in intracellular Na+ signal was generally slower and, in some cells, did not reach the maximal level or even dropped towards basal level during a 50-min incubation. At 1 and 10 µm gramicidin, the intracellular Na+ signal reached the maximal level in all cells after 30 min of exposure. Thus, 10 µm gramicidin was used in further experiments to ensure a rapid change in intracellular ions in all sampled cells. Basal [Na+]i ranged from 0.5 to 5 mm with an average of 3.4 mm (Table 1, treatment 1). The effect of gramicidin on [Na+]i was further investigated at various extracellular Na+ concentrations with non-permeable NMDG+ as the Na+ substitute. In the absence of gramicidin, reducing [Na+]o to 5 mm or completely removing extracellular Na+ had little effect on [Na+]i (data not shown). Exposure of cells to gramicidin was unable to alter [Na+]i when all extracellular Na+ was replaced with NMDG+ (Table 1, treatment 2). However, gramicidin elicited a rapid Na+ influx when Na+ was present in assay buffers (Fig. 2a). Noticeably, a great increase (> 80 mm) in [Na+]i was observed even when [Na+]o was only 5 mm, and this increase became less pronounced (approximately 54 mm) after 30 min incubation with gramicidin (Fig. 2a). When [Na+]o was increased during the gramicidin exposure, [Na+]i promptly adjusted to a higher level (Fig. 2a). Again, at 20 and 60 mm extracellular Na+, [Na+]i was higher than [Na+]o (Table 1, treatments 14 and 15). In a separate experiment with gramicidin, the change in [Na+]i at 5 mm extracellular Na+ was monitored for 50 min. [Na+]i rose quickly and then dropped gradually from its peak value to approximately 50 mm during the first 25 min exposure; afterwards, it stayed at approximately 50 mm for the remaining time (Fig. 2b).

image

Figure 2. Gramicidin-induced alterations in intracellular Na+ and K+ levels. (a) Intracellular Na+ or K+ levels at various concentrations of extracellular Na+ or Li+ with NMDG+ as the substitution. Cells were pre-loaded with fluorescent Na+ indicator SBFI or K+ indicator PBFI as descried in Materials and methods. When a stable signal was obtained (Time 0), the incubation medium was substituted with KRH buffer containing 5 mm Na+ (for determination of [Na+]i) or Li+ (for determination of [K+]i). When a new stable signal was attained, cells were exposed to 10 µm gramicidin and 100 µm ouabain for 30 min. Afterwards, the buffer Na+ or Li+ was increased stepwise to 20, 60, and 130 mm in the presence of gramicidin as indicated. Each curve shows a representative experiment. The straight line and the number above the line denote the time for the presence of a concentration of buffer Na+ or Li+. Data are means ± SE from 21 (Na+ curve) or 12 (K+ curve) individual cells. Each experiment was repeated more than three times. (b) Time course for intracellular Na+ change upon exposure to 10 µm gramicidin and 100 µm ouabain in the buffer containing 5 mm Na+ and 125 mm NMDG+. The double arrow lines denote the time frames for binding assays performed in treatments 12 and 13 in Table 1. Data are means ± SE from 38 cells.

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Intracellular K+ concentration upon gramicidin treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

We considered that the uphill Na+ influx in the presence of gramicidin (Fig. 2b) is driven by the inside-negative electrical gradient. At the same time, the depolarizing Na+ influx would allow intracellular K+ to leave cells down its chemical gradient. This process may continue until [Na+]i is higher than [Na+]o such that the tendency of intracellular Na+ to leave cells counteracts the electrical force (inside negative) attracting Na+ into cells. Although [Na+]o was lowered to 5 mm, the supply for intracellular Na+ is sufficient because there is a vast excess of extracellular versus intracellular volume. If this interpretation is true, in the presence of 5 mm[Na+]o with NMDG+ as the substitute, gramicidin must increase [Na+]i at the expense of [K+]i. Thus, K+-sensitive dye PBFI was used to examine [K+]i. Basal [K+]i was usually higher than 130 mm (Table 1, treatment 1). In the absence of gramicidin, reducing or completely replacing extracellular Na+ with NMDG+ had little detectable effect on [K+]i (data not shown). Exposure of cells to gramicidin did not cause a substantial loss of intracellular K+ when all extracellular Na+ was replaced with NMDG+ (Table 1, treatment 2). This result confirmed that NMDG+ could not pass gramicidin pores. When cells were exposed to gramicidin in buffers containing various concentrations of Na+ and NMDG+, a decrease in intracellular K+ signal was observed at 5 mm[Na+]o but not at 20–130 mm[Na+]o (data not shown). Evidently, because of the high affinity of PBFI for Na+ (Minta and Tsien 1989), the entered Na+ interfered with the determination of [K+]i. Therefore, Li+, which passes gramicidin pores (Hille 2001) but has low affinity for PBFI (Minta and Tsien 1989; Kiedrowski 1999), was used to substitute Na+ in the experiments for Fig. 2(a) to facilitate determination of the gramicidin-induced change in [K+]i. Indeed, the presence of 5 mm extracellular Li+ was sufficient to reduce [K+]i to approximately 23 mm (Table 1, treatment 3), and further stepwise increases in [Li+]o (to 20, 60, and 130 mm) caused rapid drops of [K+]i to lower and lower plateau levels (Fig. 2a). Because the gramicidin pore has a threefold higher permeability for Na+ over Li+ (Hille 2001), a faster loss of K+ from the cytoplasm would be expected to occur when 5–130 mm Na+ was present in the medium. However, the steady-state [K+]i in the presence of external Na+ during gramicidin treatment is expected to be similar to that in the presence of external Li+ (Fig. 2a).

Membrane potentials upon gramicidin treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Based on the known concentrations of extracellular and intracellular ions, we calculated the membrane potential. In the absence of gramicidin, the membrane potential was approximated as K+ diffusion potential using the Nernst equation, and was approximately − 88 mV. In the presence of gramicidin, the membrane potential was estimated using the Goldman-Hodgkin-Katz voltage equation, which considers the relative permeability of gramicidin pores to each ion. With NMDG+ as the Na+ substitute, the addition of gramicidin did not alter the membrane potential in Na+-free medium, but reduced the membrane potential modestly in low-Na+ medium and drastically in high-Na+ medium (Table 1, compare treatment 2 with treatments 12–16). The gramicidin-induced change in the membrane potential was verified with the potential-sensitive fluorescent dye DiSBAC2(3). The dye enters depolarized cells where it binds to intracellular proteins or membranes and exhibits enhanced fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. Figure 3 illustrates the changes in cellular DiSBAC2(3) fluorescence. Membrane depolarization induced by isotonically replacing extracellular Na+ with K+ (4.2–134.2 mm) enhanced the cellular fluorescence, confirming the potential-sensitivity of the dye (Figs 3a–d). Based on K+ permeability, the Nernst equation predicted that elevation of extracellular K+ to 34.2, 74.2, and 134.2 mm depolarized the membrane to −34, − 15, and 0.6 mV, respectively. These K+ diffusion potentials were close to the membrane potentials calculated using the Goldman-Hodgkin-Katz voltage equation for gramicidin-treated cells at 20, 60, and 130 mm extracellular Na+ (− 30, − 10, and 2.2 mV; Table 1, treatments 14–16). If the membrane potential predicted by Goldman–Hodgkin–Katz voltage equation for gramicidin-treated cells is reasonable, changes in the cellular DiSBAC2(3) fluorescence should be similar between the two sets of experiments. Indeed, as for high K+-induced depolarization, the fluorescence signal in the presence of gramicidin was progressively enhanced upon increasing extracellular Na+ or Li+ concentration (Figs 3e–l). Further, when the predicted depolarization was similar, the enhancement in the DiSBAC2(3) fluorescence was also comparable between high K+ and gramicidin (Fig. 3b versus f, c versus g, and d versus h). At 5 mm external Na+ or Li+, although the calculated membrane potential was less negative in the presence of gramicidin (Table 1, treatments 12 and 13), the difference in DiSBAC2(3) fluorescence was not evident between control and gramicidin after 30 min treatment (Figs 3a, e and i). It is possible that a small change in the membrane potential may not allow the slow-response dye DiSBAC2(3) to develop fluorescence strong enough to be detected under a short time of camera exposure (2 s in Fig. 3). The DiSBAC2(3) signal was therefore determined after 50 min incubation (equal to the entire time length for exposure of cells to gramicidin in binding experiments), using a longer camera exposure time (Fig. 4). Under these conditions, gramicidin modestly enhanced the cellular DiSBAC2(3) fluorescence at 5 mm external Na+ or Li+ (Figs 4b and d). This increase was observed more clearly upon quantitation of the fluorescence intensity (Fig. 4i). A sole reduction in external Na+ level showed no effect on the cellular DiSBAC2(3) fluorescence (Fig. 4a and c versus e).

image

Figure 3. Cellular DiSBAC2(3) fluorescence emission after 30 min treatment with high K+ or gramicidin. (a) Regular KRH with 4.2 mm K+ (control). (b–d) High K+. The indicated K+ concentrations were achieved by isotonically replacing NaCl with KCl. (e–h) A total of 10 µm gramicidin and 100 µm ouabain at various [Na+]o. (I–l) A total of 10 µm gramicidin and 100 µm ouabain at various [Li+]o. The indicated Na+ or Li+ concentration was achieved by isotonically replacing NaCl or LiCl with NMDG-Cl. Images were taken with a 2-s exposure. Enhanced fluorescence indicates plasma-membrane depolarization. Experiments were repeated three times with comparable results. No quantitation of cellular fluorescence intensity was conducted because the images in panels (d), (h), and (l) were saturated.

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image

Figure 4. Cellular DiSBAC2(3) fluorescence emission after 50 min treatment with gramicidin or monensin. (a) Ethanol, 5 mm Na+ (b) 10 µm gramicidin and 100 µm ouabain (Gram + Oua), 5 mm Na+ (c) Ethanol, 5 mm Li+ (d) 10 µm gramicidin and 100 µm ouabain, 5 mm Li+. The indicated Na+ or Li+ concentration was achieved by isotonically replacing NaCl or LiCl with NMDG-Cl. (e) Control (130 mm Na+ plus 1% ethanol). (f) Ethanol, 34.2 mm K+. The K+ concentrations were achieved by isotonically replacing NaCl with KCl. (g and h) Monensin (Mon), 130 mm Na+. (i) Quantitation of cellular fluorescence intensity. Data are the average intensity of 50 cells in the middle area of panels (a–h). The letter on the x-axis represents the corresponding panel in this figure. *p < 0.05 between (a) and (b), (c) and (d), and (e) and (f) (Dunn's test). Images were taken with a 4-s exposure. Enhanced fluorescence indicates plasma-membrane depolarization while reduced fluorescence indicates plasma-membrane hyperpolarization. The experiments were repeated three times with comparable results.

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Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

As the gramicidin-induced inhibition of DA interactions with DAT was associated with changes in intracellular ion concentrations and membrane potential, we sought to address which change is important. To explore the role of the membrane potential, we manipulated the membrane potential without increasing [Na+]i by two strategies. One was to remove Na+ but add 5 mm Li+ to the buffer and use NMDG+ to compensate the osmolarity of the buffer (Table 1, treatment 3). In Na+-free medium, the existence of 5 mm Li+ itself had no adverse effect on DA Ki in inhibiting CFT binding (Table 1, compare treatment 3 with treatment 2), though it reduced CFT binding affinity (Table 2, treatments 2 and 3). In spite of the significant drop in [K+]i (Fig. 2a) and membrane potential (Fig. 4d), the DA Ki value was not significantly altered by gramicidin (Table 1, treatment 3). The other was to raise extracellular K+. For non-excitable HEK-293 cells, this approach reduced the K+-diffusion potential (Figs 3a–d) without changing [Na+]i and[K+]i. In these experiments, the extracellular Na+ was fixed at 50 mm, and the extracellular K+ was increased to either 94.2 or 144.2 mm, which depolarized the membrane approximately to −8 mV or 3 mV. However, the DA Ki value was not significantly increased by 94.2 mm K+ (Table 1, treatment 4), and was increased only twofold at 144.2 mm K+ (Table 1, treatment 5).

Table 2.  Effect of transmembrane ion concentrations and membrane potential on [3H]CFT binding
Treatment (see details in Table 1)CFT Kd, nmCFT Bmax, pmol/mg
ControlTreatmentFold changeControlTreatmentFold change
  1. For transmembrane ion concentrations and membrane potential under each treatment, see corresponding numbered treatments in Table 1. For other informations, see notes in Table 1. Data are means ± SE for four to ten binding experiments. p < 0.05 versus control (paired t-test with Bonferroni correction as appropriate).

 1Ethanol16.6 ± 1.318.0 ± 1.41.082.73 ± 0.182.90 ± 0.241.06
 2Gram + Oua149 ± 19167 ± 281.122.62 ± 0.291.68 ± 0.31*0.64
 3Gram + Oua228 ± 16289 ± 22*1.264.79 ± 0.602.80 ± 0.25*0.56
 4High K+33.5 ± 2.359.3 ± 7.01.774.88 ± 0.103.04 ± 0.16*0.62
 5High K+35.5 ± 1.270.8 ± 2.8*1.995.01 ± 0.053.17 ± 0.31*0.63
 6Ouabain16.6 ± 1.315.6 ± 0.100.943.97 ± 0.284.54 ± 0.141.15
 7Mon 5 µm24.1 ± 3.621.1 ± 2.30.883.97 ± 0.283.43 ± 0.250.86
 8Mon 20 µm24.1 ± 3.620.5 ± 4.00.853.97 ± 0.283.54 ± 0.090.89
 9Gram + Oua59.3 ± 7.096.3 ± 9.71.623.04 ± 0.161.86 ± 0.16*0.61
10Gram + Oua70.8 ± 2.876.6 ± 6.31.083.17 ± 0.311.15 ± 0.22*0.36
11Gram + Oua44.9 ± 5.5120 ± 8.3*2.673.78 ± 0.143.75 ± 0.340.99
12Gram + Oua72.9 ± 2.890.3 ± 7.61.244.90 ± 0.222.84 ± 0.10*0.58
13Gram + Oua91.5 ± 8.389.6 ± 5.90.984.46 ± 0.352.92 ± 0.21*0.65
14Gram + Oua41.9 ± 2.358.4 ± 111.393.69 ± 0.292.08 ± 0.16*0.56
15Gram + Oua35.6 ± 2.953.8 ± 9.91.513.38 ± 0.262.50 ± 0.20*0.74
16Gram + Oua28.9 ± 3.542.3 ± 4.2*1.463.68 ± 0.292.82 ± 0.28*0.77

Increasing [Na+]i without membrane depolarization has no effect on DA binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

To explore the role of intracellular Na+, we first examined the effect of ouabain, which was included in all experiments involving gramicidin. Ouabain, at 100 µm, slowly increased [Na+]i to 8 mm (Table 1, treatment 6) after 30 min incubation and to 12.0 ± 0.81 mm after 50 min incubation. Such a small change in [Na+]i indicates an ouabain-induced reduction in intracellular K+, if any, must be small, too. Indeed, no appreciable change in [K+]i was observed, and the membrane potential was near normal as calculated from the measured intracellular ion concentrations (Table 1, treatment 6) or visualized by DiSBAC2(3) fluorescence imaging (data not shown). The relative lack of effect of ouabain on intracellular ions and membrane potential suggests that the background Na+ permeability and thus the basal activity of Na+, K+-ATPase were low under our experimental conditions. In addition, the small increase in [Na+]i with ouabain did not significantly affect the uptake rate of 10 nm[3H]DA (data not shown). It also had no effect on the DA Ki in inhibiting CFT binding (Table 1, treatment 6). Intracellular Na+ can also be raised by the Na+-specific ionophore monensin. Monensin produces initial Na+in/H+out or Na+in/K+out exchange (Rochdi et al. 1996). However, the effect on intracellular pH and K+ is moderate and transient (Lichtshtein et al. 1979; Ereciñska et al. 1991), whereas the elevation of intracellular Na+ is stable and depends on the concentration of monensin. Because of this feature, monensin has been widely used to chemically ‘clamp’ the intracellular Na+ at various levels (Haber et al. 1987). Figure 5(a) shows the intracellular Na+ concentration during a 50-min exposure of cells to different concentrations of monensin in KRH buffer containing 130 mm Na+. Monensin, at 5 or 20 µm, produced a gradual increase in [Na+]i up to 30 min, and then the [Na+]i remained stable for at least another 30 min (Fig. 5a). The average plateau level of intracellular Na+ from three independent experiments was approximately 30 mm at 5 µm monensin and 52 mm at 20 µm monensin (Table 1, treatments 7 and 8). The lack of membrane depolarization was demonstrated by the near normal DiSBAC2(3) or on occasion even slightly reduced fluorescence in cells treated with monensin (Fig. 4g and h versus e). There was no difference in DiSBAC2(3) fluorescence between 5 and 20 µm monensin. The DiSBAC2(3) results are consistent with the electrophysiological studies in which monensin produces similarly modest membrane hyperpolarization at this range of concentrations (Doebler 2000). Thus, increasing the concentration of monensin from 5 to 20 µm increased [Na+]i without changing membrane potential. Despite its obvious effect on intracellular Na+ level, monensin, up to 60 µm, showed no effect on the uptake rate of 10 nm[3H]DA (Fig. 5b). In saturation analysis, monensin was found to modestly affect Vmax (Fig. 5d) without a significant effect on Km for DA uptake (Fig. 5c). In agreement, monensin, at both 5 and 20 µm, did not significantly alter the DA Ki in inhibiting CFT binding (Table 1, treatments 7 and 8). Unlike the situation with gramicidin, the ability of both ouabain and monensin to increase intracellular Na+ level was strictly dependent of the extracellular Na+ level. At lower [Na+]o, ouabain and monensin failed to increase [Na+]i significantly (data not shown).

image

Figure 5. Effect of monensin on intracellular Na+ concentration and on DA uptake. (a) Monensin-induced increase in intracellular concentration of Na+. Cells were pre-loaded with fluorescent Na+ indicator SBFI as described in Materials and methods. When a stable signal was obtained, the incubation medium was substituted with KRH buffer containing 5 or 20 µm monensin. The arrow indicates the time point corresponding to the addition of monensin. Each curve shows a representative experiment. Data are means ± SE from 20 individual cells. Each experiment was repeated three times. (b) DA uptake rate of 10 nm[3H]DA in the presence of various concentrations of monensin. (c) Effect of monensin (20 µm) on Km for DA uptake. (d) Effect of monensin (20 µm) on Vmax for DA uptake. In uptake assays, treatment of cells with monensin or vehicle (1% ethanol) started 30 min before addition of the radiolabeled and unlabeled DA. Uptake assays were conducted for 5 min at 25°C in KRH buffer. Data are means ± SE for three to six experiments. *p < 0.05 vs. control group (t-test).

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Increasing intracellular Na+ under depolarizing conditions affects DA binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

In a series of experiments with gramicidin, DA Ki was determined in the presence of various concentrations of extracellular Na+ (Table 1, treatments 9–16). In these experiments, the gramicidin-induced increase in DA Ki was consistently observed. The impact of changes in [K+]i on gramicidin-induced increase in DA Ki could be inferred from treatments 9–11 (Table 1) where the Na+ concentrations across the membrane were the same (50 mm) and the membrane potential was totally abolished, but the intracellular K+ concentrations ranged from 4 to 140 mm upon gramicidin treatment. Increasing internal K+ actually potentiate the adverse effect of gramicidin on DA Ki (Table 1, treatments 9–11). Thus, at least, a reduction in [K+]i did not contribute to the gramicidin-induced increase in DA Ki. To evaluate the impact of membrane potential under conditions with increased intracellular Na+, DA Ki was measured in treatments 8–12 (see ‘fold change’ column in Table 1). In these experiments, [Na+]i was similar (50–54 mm), but the inside-negative membrane potential varied, being near normal (Table 1, treatment 8), partially lost (treatment 12), and completely abolished (treatments 9–11). It was found that the less negative the membrane potential was, the greater the fold increase was in DA Ki. We also compared results from treatments 4 and 15 or from treatments 5 and 10 (Table 1). In either pair, [Na+]o (approximately 50–60 mm) and membrane depolarization were similar but [Na+]i was very different (3.4 vs. 92 mm or 3.4 vs. 50 mm). A great increase in DA Ki was observed only in treatment 15 or 10 where [Na+]i was high. These comparisons indicate that an increase in [Na+]i under depolarizing conditions is critical for an increase in DA Ki. To further substantiate this conclusion, we tested the effect of gramicidin on DA Ki after a 6-min pretreatment in a buffer containing 5 mm Na+ and 125 mm NDMG+ (treatment 13). Under this condition, the intracellular Na+ level experienced triple phases: rise, drop, and steady-state (Fig. 2b). Such multiple phases could be interpreted as to indicate the following events. Phase 1, the inside-negative electrical field drives uphill Na+ influx. Phase 2, partial membrane depolarization reduces the electrical force to hold Na+ inside and some Na+ fluxes out of cells. Phase 3, the transmembrane ion gradient (inside > outside) exactly balances the electrical driving force, and no net change occurs in intracellular Na+. We reasoned that if an increase in [Na+]i is more important, DA Ki could be more affected during the second phase of the gramicidin treatment when the intracellular [Na+] was higher. Thus, the binding assay was initiated at 6 min corresponding to the intracellular Na+ peak after gramicidin treatment and continued for 15 min to cover most of the declining phase (Fig. 2b). Indeed, this approach considerably increased DA Ki (Table 1, treatment 13).

An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The impact of an increase in [Na+]i under depolarizing conditions on the external Na+ stimulation of DA binding was investigated by measuring DA Ki for inhibiting CFT binding at various concentrations of external Na+ in the presence of gramicidin. The Na+-dependence of the CFT Kd was similar between control and gramicidin groups (Fig. 6a), suggesting that the external Na+ binding was not affected. However, gramicidin severely compromised the stimulatory effect of Na+ on DA Ki. In the absence of gramicidin, DA Ki decreased dramatically in response to addition of 5 mm Na+, approaching its minimal value at 20–130 mm Na+. In the presence of gramicidin, the addition of 5 mm Na+ significantly increased DA Ki; a further elevation of external Na+ level to 20 mm failed to reduce DA Ki; and the highest Na+ concentration tested (130 mm) caused a statistically significant reduction in DA Ki compared with its peak value but not the value in the absence of Na+ (Fig. 6b).

image

Figure 6. Effect of gramicidin on affinity of [3H]CFT binding and potency of DA in inhibiting [3H]CFT binding at various external Na+ concentrations. (a) CFT Kd; (b) DA Ki. Data are expressed as the percentage of the value at 0 mm external Na+ (for absolute values at 0 mm Na+, see Table 1 and Table 2). Treatment of cells with 10 µm gramicidin or vehicle started 30 min before addition of [3H]CFT and unlabeled compounds. All assays were conducted at 25°C in KRH buffer with NaCl isotonically replaced by NMDG-Cl. Data are means ± SE for more than four experiments. *p < 0.05 versus the percentage at 0 mm Na+ (Dunnett's test).

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Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

In most experiments, treatment of cells with 10 µm gramicidin resulted in an approximately 30–40% reduction in CFT Bmax (Table 2). Membrane depolarization with high K+ also modestly reduced CFT Bmax (Table 2, treatments 4 and 5). To examine whether the reduced Bmax is a consequence of alterations in DAT subcellular localization due to disturbance of ion gradients and/or membrane potential, DAT localization was observed under confocal microscope using the antibody against the c-myc epitope tagged to the N-terminal of the transporter protein. Cells transfected with vector had no detectable immunofluorescence (Fig. 7a). In vehicle-treated DAT-expressing cells, the immunofluorescence was almost exclusively on the cell surface (Fig. 7b). Exposure of cells to 10 µm gramicidin or 140 mm KCl for 50 min under conditions for binding assays did not markedly alter the surface staining pattern of the DAT (Figs 7c and d). Additionally, the co-presence of CFT (100 nm) or DA (100 µm) with gramicidin during the last 20 min incubation also had no effect (data not shown). As a positive control, the DAT internalization upon activation of protein kinase C with PMA caused intracellular appearance of the immunofluorescence (Fig. 7e).

image

Figure 7. Confocal microscopy images of DAT immunofluorescence emission from cells expressing DAT. (a) Control cells (transfected with vector only); (b–e) cells stably expressing DAT. DAT cells were pre-incubated with 1% ethanol (b), 10 µm gramicidin (c), 134.2 mm KCl (d) in KRH buffer at 25°C for 50 min, or 100 nm PMA (e) at 37°C for 30 min. Shown are the Z sections at 5 µm from the top of the cell. The results are representative of two independent experiments.

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Intracellular DA concentrations

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Although there was little measurable DA uptake in cells treated with 10 µm gramicidin (Fig. 1a), it is possible that external DA could leak into cells, leading to an intracellular concentration equal to the extracellular one. In this case, a gramicidin-induced alteration in DA binding from both sides of the membrane needs be considered. Thus, we measured intracellular DA concentration in DAT-expressing cells after applying 100 µm external DA, a concentration at the higher end of Ki values observed for DA for the inhibition of CFT binding to gramicidin-treated cells, within a time range comparable with that for CFT binding assays (maximal 20 min). For vehicle-treated cells, there was a time-dependent increase in the intracellular DA concentration, reflecting uptake of the external DA by the DAT (Fig. 8). After a 20-min incubation, the DA concentration inside vehicle-treated cells reached 71 ± 3 µm. However, this concentration was still lower than the extracellular concentration (100 µm), most likely due to the impact of temperature (25°C rather than 37°C) and intracellular clearance of DA. Incidently, it can be calculated that at lower extracellular (DA) substantial accumulation occurs with inside concentrations far exceeding those outside. In contrast to the situation for vehicle-treated cells, the intracellular DA concentration for gramicidin-treated cells was significantly lower and did not change over time (Fig. 8), suggesting the absence of active uptake into these cells. After 20 min incubation, the DA concentration inside the gramicidin-treated cells was 6.97 ± 2.6 µm, 14-fold lower than the extracellular concentration. The intracellular DA concentration was also examined in HEK cells transfected with the vector only. It was 5.81 ± 1.85 µm after 20 min incubation of the cells with 100 µm DA, which was not statistically different from that observed in gramicidin-treated DAT-expressing cells. The low concentration of intracellular DA for gramicidin-treated or mock-transfected cells suggests limited leakage and/or rapid clearance of the leaked DA under our conditions.

image

Figure 8. DA concentration inside cells treated with gramicidin or vehicle. DAT-expressing cells were treated with 10 µm gramicidin or vehicle for 30 min. Afterwards, a mixture of [3H]DA (50 nm) and unlabeled DA was added to the incubation media to achieve a final DA concentration of 100.05 µm and the incubation was continued for 5–20 min. Calculation of intracellular DA concentrations was based on the radioactivity remaining in cells at each time point, the cell number (1.9 × 105/well), and the intracellular volume for HEK cells (1.25 pL/cell). Data are means ± SE for four experiments, each performed in triplicate.

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D313N mutant

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The D313N mutation severely damaged [3H]DA uptake causing a significantly higher Km(Table 3). It also affected the ability of DA to inhibit CFT binding in intact cells but not in cell-free membrane preparations (Table 3). Furthermore, Na+ stimulation of DA uptake (data not shown) and binding (Table 3) became significantly smaller at this mutant. These features partially resembled those caused by gramicidin, raising the question whether D313N mutation and gramicidin disturb a common mechanism regulating DA binding. Thus, DA Ki was determined in D313N-expressing cells treated with gramicidin. In dose–response curves for gramicidin, the effect of gramicidin on D313N approached the plateau at 1–10 µm just as found in WT DAT (Table 3). Again, similar to what was observed in WT DAT (Table 2, treatment 16), treatment of D313N-expressing cells with 10 µm gramicidin resulted in approximately 30% reduction in CFT Bmax (data not shown). However, 10 µm gramicidin did not affect CFT Kd (data not shown) and only caused a modest increase (two- to threefold) in DA Ki for inhibiting CFT binding (Fig. 9). Thus, although it displayed a sixfold higher DA Ki value than the WT DAT in the absence of gramicidin, the mutant had a DA Ki value similar to that for WT DAT in the presence of gramicidin (Table 3), suggesting that the inhibitory effects of D313N mutation and gramicidin treatment on DA Ki are not additive.

Table 3.  Apparent affinity of D313N DAT for DA
  WTD313N
  1. For binding assays, cells or membrane preparations were incubated with 4 nm[3H]CFT, various concentration of unlabeled CFT or DA in KRH buffer containing 130 mm NaCl or 130 mm NMDG-Cl for 20 min at 25°C. Treatment of cells with 10 µm gramicidin started 30 min before addition of [3H]CFT and unlabeled compounds. For uptake assays, cells were incubated with 10 nm[3H]DA, various concentrations of unlabeled DA for 5 min at 25°C. Data are means ± SE for four to eight experiments. p < 0.05 versus wild-type (WT, t-test).

MembranesKi for inhibiting CFT binding, µm6.18 ± 0.425.10 ± 0.40
CellsKm for uptake, µm0.56 ± 0.052.38 ± 0.21*
Ki for inhibiting CFT binding, µm5.41 ± 0.5134.0 ± 7.68*
Ki decrease from 0 mm to 130 mm Na+, fold11.2 ± 1.55.7 ± 1.6*
Gramicidin treated cells (1 µm)Ki for inhibiting CFT binding, µm54 ± 1266 ± 8
Gramicidin treated cells (10 µm)Ki for inhibiting CFT binding, µm63 ± 780 ± 6
image

Figure 9. Effect of gramicidin on potency of DA in inhibiting [3H]CFT binding to cells expressing D313N. Treatment of cells with 10 µm gramicidin or vehicle started 30 min before addition of [3H]CFT and unlabeled compounds. All assays were done at 25°C in KRH buffer with NaCl isotonically replaced by NMDG-Cl. The results are expressed as a percentage of the value in the presence of vehicle. Data are means ± SE for four to six experiments. *p < 0.05 vs. wild-type (t-test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The adverse effect of gramicidin on the DA Ki in inhibiting CFT binding cannot simply be explained by membrane depolarization, because substantial membrane depolarization with high K+ only produced a minor increase in DA Ki when intracellular Na+ remained at its normal low level. In fact, gramicidin did not affect DA Ki unless extracellular Na+ (not Li+) was present. Moreover, when the membrane was depolarized to a similar level, DA Ki was elevated at increasing [Na+]i (Table 1, treatment 4 vs. 15 and 5 vs. 10). Another piece of evidence supporting the critical role of intracellular Na+ comes from the study on the time course of gramicidin treatment at 5 mm external Na+, in which an increase in DA Ki was more pronounced during the phase with higher [Na+]i. However, for a given elevated [Na+]i to be effective in inhibiting DA binding a drop in membrane potential (but not in [K+]i) is required (Table 1, treatment 8 vs. 9–12). Clearly, elevation of intracellular Na+ and depolarization together are vital. It is plausible that membrane depolarization exerts a permissive role in the binding of intracellular Na+ at the cytoplasmic surface of the DAT as reported for the Na+/K+ pump (Or et al. 1996; Barmashenko et al. 1999). Such phenomena have been attributed to the existence of an ion-well connecting the cytosol with the protein's Na+ binding sites buried in the membrane (Läuger 1991). Thus, depolarization would increase local [Na+] at the binding site.

In addition to the fact that monensin did not cause membrane depolarization, the disparity between monensin and gramicidin may also be one of degree in changing the transmembrane Na+ gradient: it being completely collapsed or reversed with gramicidin, but remaining outside > inside with monensin. It is important to note that external Na+ exerts a stimulatory effect on DA binding. Thus, even though monensin significantly increased [Na+]i, [Na+]o was still twofold higher and may have been sufficient to overcome the inhibitory effect of internal Na+. Indeed, when an increase in [Na+]i was concomitant with a decrease in [Na+]o, a strong inhibitory effect on DA Ki was observed even under conditions of modest membrane depolarization (Table 1, treatments 12–14).

Are other cellular processes involved in the effect of gramicidin?

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Acute regulation of the DAT has been identified as a receptor- or protein kinase-mediated subcellular redistribution of the transporter, which controls the availability of the DAT at the cell surface (Pristupa et al. 1998; Daniels and Amara 1999; Melikian and Buckley 1999; Grånäs et al. 2003). In experiments involving gramicidin and high K+, a reduction in CFT Bmax was observed. It could be contemplated that membrane depolarization might activate such an acute regulation process, resulting in DAT internalization. If some of the internalized transporters still allow lipophilic CFT to bind at higher [Na+]i while being unavailable to extracellular DA, the ability of DA to inhibit CFT binding would be expected to become weaker with gramicidin but not with high K+ because the latter does not increases [Na+]i. However, the transporter immunofluorescence remained on the cell surface upon treatment with gramicidin or high-K+. Additionally, the effect of gramicidin was independent of the membrane permeability of substrates (data not shown). It is also unlikely that Ca2+/calmodulin-dependent kinases play a role in inhibiting DA binding, even though the intracellular free Ca2+ level might increase due to reversed Na+/Ca2+ exchange. First, monensin, which is known to increase intracellular free Ca2+ (Ereciñska et al. 1991) through activating reversed Na+/Ca2+ exchange, did not affect DA Ki. Second, inhibition of Na+/Ca2+ exchange by removing Ca2+ from the assay did not attenuate the inhibitory effect of gramicidin on DA Ki (data not shown). Third, activation of Ca2+/calmodulin-dependent kinases appears to upregulate monoamine transporters (Zahniser and Doolen 2001), which is opposite to the effect of gramicidin. However, we cannot exclude other unknown intracellular DAT-regulating mechanisms linked to intracellular Na+ and membrane potential and will explore them in the future.

Access of DA to its binding site in cells and cell-free membranes: role of Na+

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Remarkable differences in DA binding exist between cells and cell-free membranes. Thus, removal of extracellular Na+, treatment with gramicidin, and mutation of D313 to N increased the Ki for DA to inhibit CFT binding to the former but not to the latter (Chen et al. 2002, 2003; Li et al. 2002; current study). These disparities are consistent with a model in which access of DA to its binding site is different between cells and membranes (Fig. 10).

image

Figure 10. Cartoon of access model for DA and CFT at the DAT. All panels illustrate only the initial conformational state for ligand binding. (a) WT cells in Na+-free media. Most transporters reside in inward-facing states where DA binding sites are inaccessible from the extracellular side. On these states, CFT binding sites have lower affinity. (b) WT cells at 130 mm Na+. Binding of Na+ to a more extracellularly located site increases the fraction of the outward-facing state, stimulating the binding of externally applied DA by enhancing external access. External Na+-bound transporters are in a conformation favoring CFT binding, again from the extracellular side. (c) Gramicidin-treated WT cells at 130 mm Na+. Intracellular Na+ binds to a separate site, increasing the fraction of the inward-facing state. Extracellular DA cannot gain easy access to its binding site. For the transporter bound with internal Na+ only, CFT binding sites have lower affinity because of lack of external Na+ binding. However, binding of internal Na+ to external Na+-bound transporters does not affect CFT binding. Thus, increasing [Na+]o still enhances CFT binding. (d) D313N cell at 130 mm Na+. The gray region making the external entry path narrower symbolizes the problem in external access to DA binding site. Thus, fewer of outward-facing like states can accept extracellular DA, though all of them accept CFT. (e) WT membranes in Na+-free media. Although most transporters reside in a state resembling the inward-facing state adopted in these membranes when actually part of the intact cell, this does not affect DA access because DA can approach its binding site from both sides of membrane. However, CFT has to bind to the small portion of outward-facing states from the side that normally is the extracellular side. (f) WT membranes at 130 mm Na+. Na+ binding causes conformational redistribution similar to that occurring in gramicidin-treated cells; this does not affect DA binding (access from both sides) but still enhances CFT binding (favorable Na+-bound conformation). Features of DA and CFT binding at D313N in gramicidin-treated cells or in membrane preparations resemble those described for WT.

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Cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

It is possible that extracellular and intracellular Na+ interacts with different parts of the DAT and exert opposite effects on DAT conformational orientation. Thus, binding of Na+ to its more extracellularly located site facilitates the conversion of the DAT to the outward-facing state (Figs 10a and b), while binding of Na+ to its more intracellularly located site promotes the DAT (including that bound with external Na+) to convert to the inward-facing state. In this view, the DA Ki is a time-averaged value of the binding to different states of the transporter that continuously interconverts in spontaneous conformational changes as is commonly accepted for receptor proteins (Gether 2000). It should be noted that binding of DA, regardless to outward-facing or inward-facing state, could change DAT into a conformation that is unfavorable to CFT binding. Thus, competition with CFT for the same or an overlapped site is not a prerequisite for DA to inhibit CFT binding. It is known that K+ in the cytoplasm directly inhibits DA binding and this inhibition is especially potent at low concentrations of Na+ (IC50 of K+ < 20 mm at 10 mm Na+Li and Reith 1999). Thus, in normal (not gramicidin-treated) DAT-expressing cells, despite being concentrated inside, DA is expected to bind preferentially from the extracellular side. Accordingly, removal of external Na+ would affect this process. However, under depolarizing conditions with a diminished or even reversed transmembrane Na+ gradient, the effect of intracellular Na+ may prevail. Gramicidin causes the latter scenario, likely leading to accumulation of the transporter in the inward-facing state (Fig. 10c). Under this condition, the binding site is unavailable to extracellular DA. Additionally, although gramicidin generates an intracellular ionic condition (high Na+ and low K+) favorable to binding of intracellular DA, DA cannot accumulate inside to high enough concentration. Consequently, DA becomes a weak inhibitor of CFT binding. Interestingly, the DA Ki value in gramicidin-treated cells was generally equivalent to an extracellular DA concentration of 60–100 µm. In these cells, an extracellular concentration of 100 µm was found to produce an intracellular DA concentration of 6–7 µm, which is close to the DA Ki value in membrane preparations. It could be inferred that for gramicidin-treated cells, high concentrations of DA inhibit CFT binding by leaking into cells and binding from the intracellular face. In support of this possibility, in gramicidin-treated cells, DA Ki tended to increase with increasing [K+]i, especially evident when [Na+]i and the membrane potential was kept constant (Table 1, treatments 9–11, see ‘fold change’). As for the effect of D313N mutation, it seems that the mutation specifically affects the external access. Thus, the fraction of the state accepting external DA would be lower to begin with at D313N than at WT (Fig. 10d compared with Fig. 10b), which accounts for the higher DA Ki in vehicle-treated D313N cells and the smaller relative increase in the DA Ki in gramicidin-treated D313N cells.

Membranes

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

There is no permeability barrier to DA in cell-free membrane preparations. Conceivably, manipulations that mainly affect DA's external access rather than recognition, would have little effect on DA binding to membranes if DA can gain readily access to its site from the side normally facing the intracellular milieu. Our data are consistent with this view. If access from the intracellular side were restricted in membranes, the DA Ki value should have been similarly high in cells and membranes treated with gramicidin, which was not the case: in fact, the DA Ki value was much lower in gramicidin-treated membranes. If in cells the majority of DAT resides in the inward-facing state upon elimination of the inwardly directed Na+ gradient (Figs 10a and c), this likely also occurs in membrane preparations where the Na+ gradient does not exist. Thus, a shift towards states in membrane preparations resembling inward-facing DAT states in cells would provide an opportunity for ambient DA to bind predominantly from the intracellular side (Figs 10e and f), which obliterates the effect of reduced external access by gramicidin and D313N mutation. The Na+-insensitive feature of the DA Ki in inhibiting CFT binding to membrane preparations (Chen et al. 2003) supports the notion that Na+ regulates access not recognition of DA. In membrane preparations where DA can approach its binding site from both sides regardless of the conformational orientation of DAT, full access to DA binding site could be already achieved in the absence of Na+ (Fig. 10e). Thus, Na+ does not further enhance DA binding, even though it could still cause conformational changes, as judged from the enhanced CFT affinity.

CFT binding

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

In contrast to the case for the DA Ki, there was no difference between intact cells and membranes in the effect on CFT Kd of extracellular Na+ removal, gramicidin treatment, and D313N mutation. The CFT Kd value was similar between gramicidin-treated cells and membranes (42 vs. 46 nm at 130 mm Na+), and as sensitive to Na+ stimulation and D313N mutation (higher affinity) in membranes as in cells (Chen et al. 2002; Li et al. 2002). Moreover, increasing intracellular Na+ with gramicidin did not compromise the external Na+ stimulation of CFT binding to cells. These results offer some clues to CFT binding as illustrated in Fig. 10. Thus, CFT may prefer to bind at the outward-facing state but does not share the same site with DA. The access path for CFT may be similar between cells and membranes, primarily from the extracellular side. Further, it is possible that binding of internal Na+ to the external Na+-bound transporter closes the external access path for DA but not CFT (Fig. 10c). With these properties, obstruction of DA external access with gramicidin or with D313N mutation does not necessarily affect CFT binding to cells (Figs 10c and d), and CFT binding would retain its sensitivity to Na+ in both cells (Figs 10a and b) and membrane preparations (Figs 10e and f). However, the CFT binding site may be close to (or overlap with) the external access path for DA (Fig. 10). Thus, structural rearrangements resulting from changes in external access of DA could alter the binding energy for CFT. It is also possible that a loss of the Na+ gradient and membrane potential increases certain DAT forms unfavorable to CFT (Figs 10c and f). As a result, the measured Kd and Bmax values, statistical averages of multiple ever-interconverting populations, would be changed to some degree.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

Although many of the ionic conditions created in the present study may not occur physiologically, they provide novel information on the mechanistic integration of Na+ into DA–DAT interaction. Importantly, inhibition of substrate binding to the DAT has also been observed in striatal synaptosomes upon increasing intracellular Na+ with gramicidin or opening tetrodotoxin-sensitive Na+ channels (manuscript in preparation). It follows that intracellular Na+ and membrane potential may work together to contribute to physiological modulation of DA binding/transport. For instance, on polarized membranes, an elevation of intracellular [Na+] resulting from co-transport of Na+ with DA may not severely suppress the uptake process. In contrast, a depolarizing Na+ influx resulting from pre- or post-synaptic excitation would prevent external DA from binding and transporting, which might be crucial for the released DA to activate post-synaptic receptors or for the DAT to mediate DA release. The results from D313N urge for caution in interpreting mutation-induced change in substrate binding to cells. Probing of residues, mutation of which causes Na+ gradient-dependent and DAT preparation-dependent changes in substrate binding, may provide insights into the structural determinants governing the accessibility of the binding site. It will also be interesting to assess whether the impact of intracellular Na+ on the interaction of other blockers with DAT resembles more the situation for DA than for CFT. The former would open up the possibility that drug action at DAT depends on access in addition to mere recognition.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References

The study was supported by National Institute on Drug Abuse Grants DA08379 and 13261.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation of cell lines stably expressing wild-type and mutant DATs
  5. [3H]CFT binding
  6. [3H] DA uptake
  7. Treatment with ionophores
  8. Ratiometric fluorescence measurements of intracellular Na+ and K+ concentrations and calculation of membrane potential
  9. Fluorescent imaging of membrane potential changes
  10. Immunocytochemistry and confocal microscopy
  11. Results
  12. Perforation of plasma membranes of cells with gramicidin affects DA interactions with DAT
  13. Intracellular Na+ concentrations upon gramicidin treatment
  14. Intracellular K+ concentration upon gramicidin treatment
  15. Membrane potentials upon gramicidin treatment
  16. Membrane depolarization without an increase in [Na+]i has trivial effect on DA binding
  17. Increasing [Na+]i without membrane depolarization has no effect on DA binding
  18. Increasing intracellular Na+ under depolarizing conditions affects DA binding
  19. An increase in [Na+]i under depolarizing conditions counteracts the external Na+ stimulation of the external DA binding
  20. Abolishment of ion gradient does not have detectable effect on surface distribution of DAT immunofluorescence
  21. Intracellular DA concentrations
  22. D313N mutant
  23. Discussion
  24. An increase in intracellular Na+ concentration under depolarizing conditions affects the apparent affinity of the DAT towards extracellular DA
  25. Are other cellular processes involved in the effect of gramicidin?
  26. Access of DA to its binding site in cells and cell-free membranes: role of Na+
  27. Cells
  28. Membranes
  29. CFT binding
  30. Conclusion
  31. Acknowledgements
  32. References
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