SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    This study describes properties of monovalent cation currents through ECaC, a recently cloned epithelial Ca2+-permeable channel from rabbit.
  • 2
    The kinetics of currents through ECaC was strongly modulated by divalent cations. Currents were inhibited in the presence of extracellular Ca2+. They showed an initial voltage-dependent decay in the presence of 1 mm Mg2+ at hyperpolarizing steps in Ca2+-free solutions, which represents a voltage-dependent Mg2+ block through binding of Mg2+ to a site localized in the electrical field of the membrane (δ= 0.31) and a voltage-dependent binding constant (at 0 mV 3.1 mm Ca2+, obtained from a Woodhull type analysis).
  • 3
    Currents were only stable in the absence of divalent cations and showed under these conditions a small time- and voltage-dependent component of activation.
  • 4
    Single channel currents in cell-attached and inside-out patches had a conductance of 77.5 ± 4.9 pS (n= 11) and reversed at +14.8 ± 1.6 mV (n= 9) in the absence of divalent cations.
  • 5
    The permeation sequence for monovalent cations through ECaC was Na+ > Li+ > K+ > Cs+ > NMDG+ which is identical to the Eisenmann sequence X for a strong field-strength binding site.
  • 6
    It is concluded that the permeation profile of ECaC for monovalent cations suggests a strong field-strength binding site that may be involved in Ca2+ permeation and Mg2+ block.

Transcellular Ca2+ transport in polarized epithelia present in kidney, intestine and placenta, is of vital importance for Ca2+ homeostasis. Depending on the tissue, Ca2+ transport is regulated by long- and short-term acting hormones such as 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH) and calcitonin (Friedman & Gesek, 1995; Hoenderop et al. 1999). At the cellular level transcellular Ca2+ transport is generally described as a three-step process consisting of passive apical entry of Ca2+, cytosolic diffusion of Ca2+ bound to vitamin D3-sensitive calcium-binding proteins and active extrusion of Ca2+ mediated by a Na+-Ca2+ exchanger and/or a high affinity Ca2+-ATPase present in the opposite basolateral membrane (Friedman & Gesek, 1995; Hoenderop et al. 2000).

The apical influx of Ca2+ is the rate-limiting step in this process and, therefore, a prime regulatory target for stimulatory and inhibitory hormones (Raber et al. 1997; Hoenderop et al. 2000). Recently, an epithelial Ca2+ channel (ECaC) was cloned from rabbit kidney which is an excellent candidate for this elusive apical Ca2+ influx mechanism (Hoenderop et al. 1999). ECaC shares a low homology with structurally related cation channels including transient receptor potential channels (TRPs), capsaicin receptors and the growth-factor-regulated channel (Caterina et al. 1997, 1999; Zhu & Birnbaumer, 1998; Kanzaki et al. 1999). These families comprise both Ca2+ selective and non-selective cation channels, and are structurally characterized by six putative transmembrane domains including a pore-forming region. They share a homology of only 30 % that is mainly confined to the pore-forming region and flanking transmembrane segments. The presence of several potential phosphorylation sites for protein kinase C, cAMP- and cGMP-dependent protein kinase, and calcium-calmodulin-dependent protein kinase suggests that ECaC could be subject to hormonal regulation (Hoenderop et al. 1999).

Initial electrophysiological studies, performed in Xenopus oocytes and human embryonic kidney (HEK)293 cells heterologously expressing ECaC demonstrated functional characteristics of ECaC that are consistent with its putative role as an apical Ca2+ entry channel mediating transcellular Ca2+ transport in 1,25(OH)2D3-responsive epithelia (Hoenderop et al. 1999; Vennekens et al. 2000). These distinctive properties include a constitutively activated Ca2+ permeability at physiological membrane potentials, a high calcium selectivity, and hyperpolarization-stimulated and Ca2+-dependent feedback regulation of channel activity.

One of the most striking features of ECaC currents in the presence of extracellular Ca2+ is the fast and reversible decay of the current with time (Vennekens et al. 2000), which seriously hampered further detailed analysis of the biophysical properties of the ECaC pore in the presence of Ca2+. The aim of the present study was, therefore, to measure distinct features of ECaC channels in the absence of extracellular divalent cations. Under these conditions ECaC exhibits large currents that do not show run down and allow measurements of single channel characteristics.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Vector construction for ECaC-GFP co-expression

The open reading frame from rbECaC was cloned as a Pvu II-Bam HI fragment in the pCINeo/IRES-GFP vector (Trouet et al. 1997; Hoenderop et al. 1999). This bicistronic expression vector pCINeo/IRES-GFP/rbECaC was used to co-express rbECaC and enhanced green fluorescent protein (GFP).

Cell culture and transfection

Human embryonic kidney cells, HEK293, were grown in Dulbecco's modified Eagle's medium containing 10 % (v/v) human serum, 2 mm L-glutamine, 2 U ml−1 penicillin and 2 mg ml−1 streptomycin at 37°C in a humidity controlled incubator with 10 % CO2. HEK293 cells were transiently transfected with the pCINeo/IRES-GFP/rbECaC vector using methods as described previously (Vennekens et al. 2000). Transfected cells were visually identified in the patch clamp set up. GFP was excited at a wavelength between 450 and 490 nm and the emitted light was passed through a 520 nm long-pass filter. The ECaC-expressing cells were identified by their green fluorescence and GFP negative cells from the same batch were used as controls.

Electrophysiology

Electrophysiological methods and Ca2+ measurements have been described in detail previously (Nilius et al. 1994). Electrode resistance was between 2 and 5 MΩ. Whole-cell currents were measured with an EPC-9 (HEKA Elektronik, Lambrecht, Germany) or an L/M-EPC-7 (List Elektronics, Darmstadt, Germany) using ruptured patches. Cell capacitance and access resistance were monitored continuously. The internal (pipette) solution contained (mm): 20 CsCl, 100 caesium aspartate, 1 MgCl2, 10 BAPTA, 4 Na2ATP and 10 Hepes, pH 7.2 with CsOH.

The standard extracellular solution (Krebs) contained (mm): 150 NaCl, 6 CsCl, 1 MgCl2, 1.5 CaCl2, 10 Hepes and 10 glucose, pH 7.4 with CsOH. The hyperosmolarity of the extracellular solution is routinely used to prevent activation of volume-regulated anion channels. The free Ca2+ concentration in these nominally Ca2+-free solutions was spectrophometrically measured and amounted to about 50 nm. For measuring currents carried by various monovalent cations, NaCl was equimolarly replaced by LiCl, KCl, CsCl or NMDG-Cl. Extracellular pH was changed to 6 or 8.5 by adjusting the standard solution with HCl or NaOH, respectively.

Cells were kept in a nominally Ca2+-free medium to prevent Ca2+ overload and exposed for a maximum of 5 min to a Krebs solution containing 1.5 mm Ca2+ before sealing the patch pipette to the cell. All experiments were performed at room temperature (20–22°C).

We applied either ramp protocols, consisting of linear voltage changes from −100 or −150 mV to +100 mV within 400 ms, applied every 5 s, or step protocols, consisting of a series of 60 ms voltage steps applied every 5 s from a holding potential (Vh) of +20 mV to voltages between +60 and −140 mV with a decrement of 40 mV. The sampling interval was 1 ms for the ramp protocols, 0.2 ms for the step protocol. Data were filtered at the appropriate frequency before digitization. For comparing data obtained from different cells, current amplitudes were expressed per unit cell capacitance.

For single channel measurements in cell-attached mode, cells were perfused with the following solution to zero the membrane potential (mm): 150 KCl, 1 MgCl2 and 10 Hepes. For inside-out patches, excision was done in the same solution. In both configurations, the pipette contained (mm): 150 NaCl, 0.1 EDTA and 10 Hepes, pH 7.4 with NaOH. Thus, the outside solution was nominally free of divalent cations. The sampling rate was 5 kHz; currents were filtered at 1 kHz.

The software package ASCD (G. Droogmans, Leuven) was used for analysis of whole-cell and single channel data.

Statistical analysis

In all experiments the data are expressed as the mean ±s.e.m. Overall statistical significance was determined by analysis of variance (ANOVA). In case of significance (P < 0.05), individual groups were compared by Student's t test.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Effects of extracellular divalent cations on the whole-cell currents through ECaC

One of the most striking features of the current through ECaC is its fast and reversible decline in the presence of physiological extracellular Ca2+ concentration (Vennekens et al. 2000). This ‘Ca2+-induced current decay’ is illustrated in Fig. 1A (period b–c), which shows that addition of 1 mm Ca2+ to the nominally Ca2+-free solution induces an immediate increase of the current followed by a rather fast and almost complete decline of the current, which completely recovers upon washout of Ca2+ and Mg2+. The amplitude of the current measured at −80 mV during the first ramp protocol after application of Ca2+ was increased by 134 ± 31 % compared to that before Ca2+ administration (n= 12 cells). The reversal potential (Fig. 1B, trace b) is clearly shifted to more positive potentials in the presence of 1 mm Ca2+ (+30 ± 2 mV, n= 11, compared with +8 ± 1 mV, n= 21, in 0 mm Ca2+, 1 mm Mg2+, Fig. 1Aa), consistent with a permeation of Ca2+ ions through ECaC. The Ca2+-induced current decay seriously hampers a detailed analysis of the biophysical properties of ECaC in the presence of Ca2+.

image

Figure 1. Whole-cell currents in ECaC-expressing HEK293 cells under different ionic conditions

A, time course of the changes in ECaC current at −80 mV. The current was initially recorded in a Ca2+-free, 1 mm Mg2+-containing solution. The free Ca2+ concentration in nominally Ca2+-free solutions was estimated at 50 nm (see Methods). Note the initial fast increase of the current in the presence of 1 mm Ca2+ which is then followed by an almost complete run down. Wash-out of both divalent cations increased the current, an effect that was potentiated in the presence of 0.1 mm EDTA. Data points were obtained from the voltage ramp protocol which was applied every 5 s. B, current-voltage curves derived from voltage ramps taken at the times marked ▪ in panel A. C, current traces from another cell during voltage steps from +60 to −140 mV in a nominally Ca2+-free, 1 mm Mg2+-containing solution (decrement is 40 mV, holding potential is +20 mV). D, current traces from the same cell as in C in a solution containing 1 mm Ca2+ and 1 mm Mg2+. Note the larger current at the end of the 60 ms pulse compared with panel C. E, currents through ECaC recorded in a nominally Mg2+- and Ca2+-free extracellular solution. Experimental protocol same as in C and D. F, in the presence of 0.1 mm EDTA (same cells as in E) the current at negative potentials not only was increased in amplitude, but also showed a slowly activating component.

Download figure to PowerPoint

The currents were, however, stable and much larger in nominally Ca2+-free solutions (Fig. 1A), and their amplitude was even enhanced in the absence of extracellular Mg2+ or if EDTA (or EGTA) was added, suggesting that Mg2+ and/or micromolar concentrations of Ca2+ (contaminating Ca2+ in the nominally Ca2+-free solution) may induce a partial block of the current carried by monovalent cations. A peculiar but consistent finding was the shift of the reversal potential in the presence of EDTA to more positive values (Fig. 1Ad). Such a shift was not observed in non-transfected cells, suggesting that EDTA may exert some hitherto unknown effect on the channel.

The currents through ECaC showed a fast and pronounced decay to a much lower steady-state level at negative potentials in cells exposed to a nominally Ca2+-free, 1 mm Mg2+-containing solution (Fig. 1C). This initial current transient is much slower and less pronounced in the presence of 1 mm Ca2+ (Fig. 1D, obtained immediately after addition of extracellular Ca2+), but the current amplitudes at the end of the voltage steps were larger than those in the absence of Ca2+ (Fig. 1C) indicating again that Ca2+ permeates through the channel. The initial decay also disappeared completely after removing extracellular Mg2+ (Fig. 1E), whereas addition of 0.1 mm EDTA to the nominally Ca2+- and Mg2+- free solution further increased the current (Fig. 1F). These data may indicate that Ca2+ and Mg2+ inhibit the current of monovalent cations through ECaC, although a direct effect of EDTA cannot be excluded. In line with a more direct action of EDTA is the observation that it also shifts the reversal potential to more positive values. We have at present no explanation for these peculiar findings.

Block of currents through ECaC by extracellular Mg2+

The time constant of current decay in the presence of 1 mm Mg2+, as shown in Fig. 1C, is clearly voltage dependent and therefore consistent with an open channel block of ECaC by Mg2+. Figure 2A shows similar current traces obtained at various extracellular Mg2+ concentrations. It is obvious that both the rate of current decay and the extent of current inhibition are strongly enhanced by increasing extracellular Mg2+ and by hyperpolarization. To quantify these effects, we have fitted the current traces at negative potentials to a single exponential, and applied the classical Woodhull technique (Hille, 1992) to analyse the time constants of current inhibition, from which the rate constants of block and unblock as well as the voltage dependence of block can be derived. Binding of Mg2+ to a site, RECaC, in the channel pore can be described by:

  • image

where k1 and k-1 are the rate constants of block (m−1 s−1) and unblock (s−1), respectively. The apparent binding constant is Kmg=k-1/k1. This model predicts a linear relation between the reciprocal time constant of current inhibition and the concentration of Mg2+ given by:

  • image
image

Figure 2. Voltage-dependent block of currents through ECaC by Mg2+

Current traces in response to voltage steps from +60 to −180 mV (decrement 40 mV, Vh+20 mV) in nominally Ca2+-free solution containing different Mg2+ concentrations (mm) as indicated above each panel. Mono-exponential fits of the current traces at negative potentials are superimposed as thin continuous lines. B, reciprocal time constants as a function of Mg2+ concentrations at four different potentials. Continuous lines represent linear fits, from which rate constants for block and unblock were obtained (data from 6 cells, means ±s.e.m.). C, voltage dependence of the apparent binding constant for Mg2+ and Kmg. The continuous line represents the best fit to the Woodhull equation with a location of the binding site at 0.31 within the electrical field from the extracellular site and an apparent binding constant of 3.1 mm at 0 mV.

Download figure to PowerPoint

This correlation is shown in Fig. 2B for time constants observed at four different potentials. From the rate constants of block and unblock derived from linear fits to the data points at each potential, we calculated the corresponding apparent binding constant, Kmg. The voltage dependence of Kmg, as shown in Fig. 2C, was fitted to:

  • image

where Kmg(0) represents the apparent binding constant at 0 mV and δ the electrical distance of the binding site inside the channel. The parameters of this fit were δ= 0.31 and Kmg(0) = 3.1 mm.

Monovalent cation currents through ECaC channels

Since Mg2+ and Ca2+ strongly influence currents through ECaC, we have omitted both divalent cations to study monovalent cation currents through this channel. Figure 3 shows the salient features of these macroscopic currents through ECaC in the absence of divalent cations. Except for a small time-dependent component, activation is instantaneous which suggests that a considerable fraction of the channels is in a constitutively open configuration. The small activating component could be fitted to an exponential. Its time constant was decreased and its amplitude expressed as a fraction of the total current decreased at more negative potentials. The nature of this slow activation phase is so far unknown.

image

Figure 3. Kinetic analysis of currents through ECaC in the absence of Mg2+ and Ca2+ (monovalent cation currents)

A, voltage steps from −60 to −140 mV (Vh=+20 mV) in nominally Ca2+- and Mg2+-free extracellular solutions. Currents show an immediate increase and a delayed activation phase. This time-dependent component was fitted with an exponential (curves are superimposed). B, the time constant is significantly decreased at more negative potentials (*P< 0.05). C, the relative amplitude of the time-dependent component (amplitude of the slow component divided by the size of the total current 60 ms after the hyperpolarizing step, Aslow/Atot) is decreased at negative potentials (data from 14 cells, means ± s.e.m).

Download figure to PowerPoint

We have also studied single channel events that underlie these monovalent macroscopic currents because it has been postulated that CaT1, a recently cloned homologue of ECaC from rat intestine, functions as a transporter rather than as an ion channel. The main argument in favour of this hypothesis was that no single channel events could be recorded in cell-attached patches (Peng et al. 1999). Measurement of single channel events seems, therefore, mandatory to support the channel-like nature of ECaC. The large macroscopic currents in ECaC-expressing HEKcells in the absence of divalent cations (see also Vennekens et al. 2000) are reminiscent of the permeation pattern for highly Ca2+ selective L-type Ca2+ channels (Almers & McCleskey, 1984; McCleskey & Almers, 1985; Hess et al. 1986; Lansman et al. 1986), which also exhibit a tiny conductance in the presence of extracellular Ca2+ (Fenwick et al. 1982; Hess et al. 1986; Droogmans & Nilius, 1989) but a much higher conductance in the absence of divalent cations (Hess et al. 1986). We have, therefore, first tried to identify single channel events in divalent cation-free solutions containing 0.1 mm EDTA. It turned out to be extremely difficult to record the activity of a small number of channels because of the high channel density, which is in line with the observed high macroscopic current densities. Figure 4A shows an example of a cell-attached patch containing only a few channels at different holding potentials in the absence of any additional stimulation to evoke channel activity, and on the right the corresponding amplitude histograms from which we calculated the single channel current amplitudes. Such activity was never observed in non-transfected HEK293 cells investigated in parallel with the transfected ones. Figure 4B shows an i–V relation measured from a voltage ramp from −100 to +100 mV. It has a single channel slope conductance of 74 pS. A consistent finding in all these traces was the lack of channel activity at positive potentials, suggesting that open probability is low at these potentials. Figure 4C shows the i–V relation derived from measurements of single channel currents at fixed potentials, as shown in the traces of Fig. 4A. The single channel conductance from six patches ranged between 55 and 107 pS with a mean value of 79.2 ± 4.9 pS; the reversal potential was +12.5 ± 3.4 mV. Figure 5 shows an example of an inside-out patch and the corresponding amplitude histogram. The single channel conductance in the inside-out configuration was 75.4 ± 7.2 pS (n= 5, ranging between 55 and 95 pS); the reversal potential was +19.3 ± 2.2 mV (n= 3). These values were not significantly different from those in cell-attached patches, indicating that it is probably the same channel. From the pooled data of both configurations we have calculated a conductance of 77.5 ± 4.9 pS (n= 11) and a reversal potential of +14.8 ± 1.6 mV (n= 9). This reversal potential nicely fits that of the macroscopic current under the same conditions (14.3 ± 1.8 mV, n= 15 this study, see also Vennekens et al. 2000). Also the pattern of the ensemble averaged current (Fig. 5B) matches that of the whole-cell current measured under similar conditions (see Figs 1F and 3). Unfortunately, all attempts to record single channel currents in the presence of extracellular divalent cations failed.

image

Figure 4. Single channel currents through ECaC in divalent cation-free solutions

A, current traces from a cell-attached patch at different holding potentials, and the corresponding amplitude histograms from which the current amplitudes were measured. B, current-voltage relationship obtained from a ramp under the same conditions as shown in panel A. C, current-voltage relationship from data points obtained at fixed potentials, such as shown in panel A. Pipette and extracellular solutions (mm) are shown in the inset of panel C.

Download figure to PowerPoint

image

Figure 5. Single channel currents through ECaC from an inside-out patch

A, current traces from an inside-out patch in response to hyperpolarizing steps to −80 mV from a holding potential of +20 mV. Pipette and intracellular solutions (mm) are shown in the inset of panel C. B, ensemble averaged currents from 38 sweeps. C, amplitude histogram from the same inside-out patch.

Download figure to PowerPoint

To characterize the pore properties of ECaC further, we have measured macroscopic currents in the presence of various monovalent cations. Figure 6 shows a series of current measurements after replacement of Na+ by Li+, K+, Cs+, or NMDG+. The largest current was observed with Na+ as the charge carrier (Fig. 6A), and its amplitude decreased in the order Na+ > Li+ > K+ > Cs+ > NMDG+ (Fig. 6AC). This sequence, as shown in Fig. 6C is identical to the Eisenmann conductance sequence X for a strong field-strength binding site (Eisenmann, 1962). This is in striking contrast with the background non-selective cation currents in non-transfected HEK cells which follow an Eisenmann I sequence for a weak field-strength binding site (B. Nilius, unpublished observations).

image

Figure 6. Permeation of monovalent cations through ECaC

A, currents in responses to voltage steps from +60 to −140 mV (decrement 40 mV, Vh+20 mV) in a nominally Ca2+-and Mg2+-free solution containing 150 mm of the cations as indicated by the labels. B, I–V curves derived from linear voltage ramps for the same conditions as in panel A. C, pooled data from 12 cells of the current amplitudes at −80 mV normalized to the corresponding value from the same cell in the 150 mm Na+-containing solution.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

We have recently shown that ECaC is approximately 100 times more permeable for Ca2+ than for Na+ (Vennekens et al. 2000). The observed shift of +20 mV in reversal potential in the presence of 1 mm extracellular Ca2+ is consistent with this contention. This permeation ratio is, however, much smaller than that of voltage-dependent Ca2+ channels (Hess et al. 1986; Lansman et al. 1986; Tsien et al. 1987) but higher than that of non-selective cation channels. A study of the biophysical properties of ECaC in the presence of divalent cations is extremely difficult because of the fast and pronounced Ca2+-induced current decay under these conditions (Vennekens et al. 2000). We have, therefore, investigated its properties using monovalent cations as the main charge carriers. Not only was the current more stable under these conditions, but it also had a much higher amplitude than in the presence of divalent cations (Vennekens et al. 2000). Currents through ECaC are relatively small at physiological Mg2+ and Ca2+ concentrations, but still sufficient to elevate [Ca2+]i substantially at hyperpolarized potentials (Vennekens et al. 2000). Therefore, block by Mg2+ and decrease of the current by extracellular Ca2+ might be physiologically important to prevent overloading of ECaC-expressing cells with Ca2+.

We have recorded single channel events in both cell-attached and inside-out patches in Ca2+- and Mg2+-free solutions containing 0.1 mm EDTA with a conductance of about 77 pS and a reversal potential similar to that of the whole-cell currents. This result is important because calcium transporter 1 (CaT1), a recently cloned rat intestine ECaC homologue (Peng et al. 1999), has been proposed to be a transporter rather than a channel. This conclusion was based on its macroscopic kinetic properties and the absence of single channel events.

The macroscopic current carried by monovalent cations is characterized by an initial rapid decay at negative potentials if the extracellular solution contains 1 mm Mg2+. This decay is absent in Mg2+- and Ca2+-free solutions and less pronounced in the presence of 1 mm Ca2+ (Vennekens et al. 2000). The dramatic changes in the time course of currents through ECaC in the absence of extracellular Ca2+ can be explained by an open channel block whereby Mg2+ binds to a site within the electrical field of the membrane (δ= 0.31) which results in an enhanced apparent affinity at hyperpolarized potentials. The sensitivity of ECaC to Ca2+ and Mg2+ is further supported by the observation that addition of 0.1 mm EDTA, which reduces the divalent concentrations to zero, substantially increases the current through ECaC (Fig. 1E and F).

The permeation of monovalent cations through ECaC shows some similarities to L-type Ca2+ channels inasmuch as they also conduct Na+ better than Li+, K+ and Cs+ (Hess et al. 1986; Lansman et al. 1986; Tsien et al. 1987). The sequence Na+ > Li+ > K+ > Cs+ > NMDG+ is identical to the Eisenmann sequence X, which is characteristic for a strong field-strength binding site.

In conclusion, we show here for the first time single channel events through ECaC, giving unequivocal proof for the channel nature of this protein. Its permeability sequence for monovalent cations suggests a strong field-strength binding site, which might be involved in Ca2+ permeation and block by Mg2+. The molecular mechanism responsible for this permeation fingerprint of ECaC needs further investigation.

  • Almers, W. & McCleskey, E. W. (1984). Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. The Journal of Physiology 353, 585608.
  • Caterina, M. J., Rosen, T. A., Tominaga, M., Brake, A. J. & Julius, D. (1999). A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398, 436441.
  • Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D. & Julius, D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816824.
  • Droogmans, G. & Nilius, B. (1989). Kinetic properties of the cardiac T-type calcium channel in the guinea-pig. The Journal of Physiology 419, 627650.
  • Eisenmann, G. (1962). Cation selective glass electrodes and their mode of action. Biophysical Journal 2, 259323.
  • Fenwick, E. M., Marty, A. & Neher, E. (1982). Sodium and calcium channels in bovine chromaffin cells. The Journal of Physiology 331, 599635.
  • Friedman, P. A. & Gesek, F. A. (1995). Cellular calcium transport in renal epithelia: measurement, mechanisms, and regulation. Physiological Reviews 75, 429471.
  • Hess, P., Lansman, J. B. & Tsien, R. W. (1986). Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells. Journal of General Physiology 88, 293319.
  • Hille, B. (1992). Ionic Channels of Excitable Membranes. Sinauerm, Sunderland , MA , USA .
  • Hoenderop, J. G., van der Kemp, A. W., Hartog, A., Van de Graaf, S. F., Van Os, C. H., Willems, P. H. & Bindels, R. J. (1999). Molecular identification of the apical Ca2+ channel in 1,25- dihydroxyvitamin D3-responsive epithelia. Journal of Biological Chemistry 274, 83758378.
  • Hoenderop, J. G. J., Willems, P. H. G. M. & Bindels, R. J. M. (2000). Towards a comprehensive molecular model of active calcium reabsorption. American Journal of Physiology 278, F352360.
  • Kanzaki, M., Nagasawa, M., Kojima, I., Sato, C., Naruse, K., Sokabe, M. & Iida, H. (1999). Molecular identification of a eukaryotic, stretch-activated nonselective cation channel. Science 285, 882886.
  • Lansman, J. B., Hess, P. & Tsien, R. W. (1986). Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore. Journal of General Physiology 88, 321347.
  • McCleskey, E. W. & Almers, W. (1985). The Ca channel in skeletal muscle is a large pore. Proceedings of the National Academy of Sciences of the USA 82, 71497153.
  • Nilius, B., Oike, M., Zahradnik, I. & Droogmans, G. (1994). Activation of a Cl current by hypotonic volume increase in human endothelial cells. Journal of General Physiology 103, 787805.
  • Peng, J. B., Chen, X. Z., Berger, U. V., Vassilev, P. M., Tsukaguchi, H., Brown, E. M. & Hediger, M. A. (1999). Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption. Journal of Biological Chemistry 274, 2273922746.
  • Raber, G., Willems, P. H. G. M., Lang, F., Nitschke, R., Van Os, C. H. & Bindels, R. J. M. (1997). Coordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system. Cell Calcium 22, 157177.
  • Trouet, D., Nilius, B., Voets, T., Droogmans, G. & Eggermont, J. (1997). Use of a biscistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells. Pflügers Archiv 434, 632638.
  • Tsien, R. W., Hess, P., McCleskey, E. W. & Rosenberg, R. L. (1987). Calcium channels: mechanisms of selectivity, permeation, and block. Annual Review of Biophysics and Biophysical Chemistry 16, 265290.
  • Vennekens, R., Hoenderop, J. G.-J., Prenen, J., Stuiver, M., Willems, P. H. G. M., Droogmans, G., Nilius, B. & Bindels, R. J. M. (2000). Permeation and gating properties of the novel epithelial channel, ECaC. Journal of Biological Chemistry 275, 39633969.
  • Zhu, X. & Birnbaumer, L. (1998). Calcium channels formed by mammalian Trp homologues. News in Physiological Sciences 13, 211217.

Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

We thank Dr J. Eggermont for providing the IRES-GFP vector, and D. Hermans, M. Crabbé, H. Van Weijenbergh and M. Schuermans for their skilful technical assistance and help with the cell cultures. This work was supported by the Belgian Federal Government, the Flemish Government and the Onderzoeksraad KU Leuven (GOA 99/07, F.W.O. G.0237.95, F.W.O. G.0214.99, F.W.O. G.0136.00; Interuniversity Poles of Attraction Programme, Prime Ministers Office IUAP Nr.3P4/23, and C.O.F./96/22-A069), by ‘Levenslijn’ (7.0021.99), a grant from the ‘Alphonse and Jean Forton – Koning Boudewijn Stichting’ R7115 B0 and the Dutch Organization of Scientific Research (NWO-ALW 805-09.042).