single GJ channel conductance
BSA modified with 1,2-cyclohexanedione
cytosolic phospholipase A2
fetal bovine serum
- g j
Hank's balanced salt solution
- I j
long carbon chain n-alkanols
methyl arachidonyl fluorophosphonate
modified Krebs–Ringer solution
- N F
number of functional channels
- N o
number of open channels
- N T
total number of channels
polyunsaturated fatty acid
region of interest
stochastic four-state model
short carbon chain n-alkanols
- V j
- • Under control conditions only one of ∼128 connexin (Cx)36 gap junction channels assembled in junctional plaques are open at a given time. This ratio was increased several times by short carbon chain n-alkanols and BSA, and reduced by long carbon chain n-alkanols.
- • Bovine serum albumin (BSA) increased junctional conductance (gj) by removal of polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA) from the plasma membrane of HeLaCx36-EGFP cells. BSA modified with 1,2-cyclohexanedione, which does not bind PUFAs, did not affect gj.
- • A primary culture of pancreatic β-cells, expressing solely Cx36, shows similar properties as HeLa transfectants, i.e. gj increases under an exposure to BSA and hexanol, while decanol and nonanol caused full uncoupling.
- • Methyl arachidonyl fluorophosphonate (MAFP) and thapsigargin, inhibitor and activator of AA synthesis, increased and reduced gj, respectively.
- • The gj-enhancing effect of hexanol did not change during combined application with MAFP and BSA, whereas AA and thapsigargin reduced the potentiating effect of hexanol.
Abstract We examined junctional conductance (gj) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic β-cells. Only a very small fraction (∼0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased gj, while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and gj increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and β-cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase gj by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.
Connexins (Cxs) are members of a large family of membrane proteins that oligomerize into hexamers called connexons or hemichannels (HCs). Docking of two HCs from cells in contact results in the formation of a gap junction (GJ) channel with a pore diameter of ∼1.3 nm connecting cytosols of neighbouring cells for direct electrical and metabolic cell–cell communication (Harris, 2007). GJs formed of different Cx isoforms exhibit differences in single GJ channel conductance (γ), perm-selectivity and dependence of junctional conductance (gj) on transjunctional voltage (Vj; Gonzalez et al. 2007). Although sensitivity and kinetics of Vj-gating depend on the Cx type, homotypic GJs show a symmetric gj–Vj dependence, which has been ascribed to the presence of fast and slow Vj-sensitive gates in each apposed/docked HC (aHC; Bukauskas & Verselis, 2004; Paulauskas et al. 2012). Typically, the fast gate exhibits ∼1 ms or faster gating transitions between the open state with conductance, γopen, to the residual state, γres. The slow gate is characterized by slower kinetics (∼10 ms) between open and fully closed states. In addition to Vj sensitivity, GJ channels are sensitive to intracellular H+, cationic divalents, posttranslational modifications and a variety of chemical reagents (Harris, 2001). The mechanisms by which these factors exert their effect on GJs remain unclear, although H+ and Ca2+ ions may both act through the slow Vj gate (Peracchia, 2004). It was proposed that chemical uncoupling is performed by the gating element of the slow gate, which is triggered by different sensorial elements/Cx-domains specific for voltage as well as chemical reagents (Bukauskas & Verselis, 2004).
Here, we examined modulation of functional efficiency, defined as the fraction of channels that are functional, gj and Vj-gating of Cx36 GJ channels by n-alkanols with different carbon chain (C-chain) lengths from pentanol to decanol, arachidonic acid (AA) and compounds that increase or reduce its endogenous production. We demonstrated that only a very small fraction of Cx36 channels (∼0.8%) are open under control conditions. This fraction increased several fold under application of pentanol, hexanol or heptanol, representatives of short C-chain n-alkanols (SCCAs), while it decreased under application of octanol, nonanol or decanol, representatives of long C-chain n-alkanols (LCCAs). Cell–cell coupling substantially increased during exposure to fatty acid-free BSA, which efficiently extracts polyunsaturated fatty acids (PUFAs), including AA, from the plasma membrane, but not by BSA modified with 1,2-cyclohexanedione (CHD), which has greatly reduced binding of PUFAs. Moreover, an increase of endogenous AA production by thapsigargin reduced gj, while reduction of its production by inhibition of cytosolic phospholipase A2 (cPLA2) with methyl arachidonyl fluorophosphonate (MAFP) increased gj. We propose that under control conditions, levels of endogenous PUFAs, especially AA, hold Cx36 GJ channels preferentially in a closed state. Thus, reducing AA concentration in the plasma membrane with BSA or MAFP increases cell–cell coupling, while increasing AA concentration with thapsigargin decreases coupling.
Cell lines and culture conditions
Experiments were performed on HeLa (human cervix carcinoma) cells stably transfected with wild-type Cx36 (Cx36) or its fusion form with green fluorescent protein tagged to the C-terminus (Cx36-EGFP). Stable cell lines of HeLaCx36 were obtained as described earlier (Teubner et al. 2000). HeLaCx36-EGFP transfectants were obtained by using pCx36EGFP-P plasmid, and stable cell lines were selected based on EGFP fluorescence visible as junctional plaques (JPs). Cells were maintained in DMEM medium containing 10% fetal bovine serum (FBS), penicillin/streptomycin mix (100 U ml−1 penicillin and 100 μg ml−1 streptomycin; Gibco Laboratories) and puromycin 1 μg ml−1. HeLa cell lines carrying wild-type mCx30.2, Cx36, Cx45 and Cx47, and a Novikoff cell line endogenously expressing Cx43 (Meyer et al. 1992) were kindly provided by Dr. K. Willecke and Dr. R. Johnson.
Pancreatic β-cell isolation and culture
Ethical approval in all animal experiments was provided by the Institutional Animal Care and Use Committee at Albert Einstein College of Medicine, and conforms to the Guide for the Care and Use of Laboratory Animals (National Institute of Health). Dispersed β-cell cultures were obtained by isolating CD1 mouse islets of Langerhans by collagenase, as previously described (Jarchum et al. 2008), and subsequent dissociation with trypsin. Briefly, mice were killed by asphyxiation with CO2. Then the pancreas was perfused through the common bile duct with ∼2 ml of cold Hank's balanced salt solution (HBSS) containing 0.6 mg ml−1 collagenase P (Roche, Indianapolis, NJ, USA), and after removal was placed in HBSS containing collagenase, and incubated for 15 min at 37°C. Then, the digested pancreas was mechanically dispersed by gentle pipetting with HBSS enriched with 2% FBS. After centrifugation, the supernatant was removed and 10 ml of new HBSS with 2% FBS was added and gently mixed with the pellet. Washed islets were resuspended in HBSS with 10 μg ml−1 DNAse I (Worthington Biochemical, Lakewood, NJ, USA) to avoid aggregation. Islets free of exocrine tissue were handpicked using a glass pipette and placed in a dish with RPMI-1640 medium. Each pancreas yielded approximately, 100–200 islets, which were then trypsinized for 10 min (0.25%; Invitrogen). Dissociated β-cells were seeded onto glass coverslips placed in culture dishes containing RPMI-1640 medium supplemented with 10% FBS, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (GIBCO Laboratories). Cells remained in a 5% CO2 incubator for at least 8 h before electrophysiological and imaging recordings.
For simultaneous electrophysiological and fluorescence recordings, cells grown on coverslips were transferred to an experimental chamber mounted on the stage of an inverted Olympus IX-70 microscope equipped with an imaging system. Experiments were performed in a modified Krebs–Ringer (MKR) solution containing (in mm): NaCl, 140; KCl, 4; CaCl2, 2; MgCl2, 1; glucose, 5; pyruvate, 2; Hepes, 5 (pH 7.4). Patch pipettes were filled with a solution containing (in mm): KCl, 140; NaAsp, 10; MgATP, 2; MgCl2, 1; CaCl2, 0.2; EGTA, 2; Hepes, 5 (pH 7.2, free [Ca2+]i= 5 × 10−8 m). Cells were perfused with MKR solution at room temperature. gj was measured in selected cell pairs using dual whole-cell patch-clamp. Vj was induced by stepping the voltage in cell-1 (ΔV1) and keeping the voltage in cell-2 constant, Vj=ΔV1. Junctional current (Ij) was measured as the change in current in the unstepped cell-2, Ij=ΔI2. Thus, gj was obtained from the ratio, −Ij/Vj, where the negative sign indicates that Ij measured in cell-2 is oppositely oriented to the one measured in cell-1. To minimize effects of series resistance on gj measurements (Wilders & Jongsma, 1992), we maintained the pipette resistances below 3 MΩ. Signals were acquired and analysed using custom-made software (Trexler et al. 1999) and an A/D converter (National Instruments, Austin, TX, USA).
Fluorescence signals were acquired using an ORCA digital camera (Hamamatsu Corp., Bridgewater, NJ, USA) with UltraVIEW software for image acquisition and analysis (Perkin Elmer Life Sciences, Boston, MA, USA). Fluorescence imaging was used to select HeLaCx36-EGFP cell pairs exhibiting at least one JP. Typically, cell pairs without JPs showed no coupling, while gj was higher in cell pairs with larger JPs. Furthermore, fluorescence imaging was used to evaluate fluorescence of a single GJ channel and consequently the total number of channels assembled in JPs. Selected filters for EGFP excitation and emission were housed in Sutter filter wheels (LAMBDA 10-2).
Preparation and application of different compounds used in this study
AA, BSA, n-alkanols (1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol and 1-decanol), MAFP and CHD were purchased from Sigma-Aldrich (Seelze, Germany). Thapsigargin was obtained from EMD Millipore (Billerica, USA). Stock solutions of both AA and MAFP were prepared in DMSO, while thapsigargin was prepared in 100% ethanol. Stock solutions were stored at −80°C until use. Solubilities of pentanol, hexanol, heptanol and octanol in water (g/100 g H2O) at 20°C are as follows: 2.7, 0.6, 0.093, 0.054, respectively (Ebbing & Gammon, 2009). Therefore, n-alkanols from pentanol to octanol were diluted directly in MKR solution (final concentrations were below the solubility limit). Nonanol and decanol exhibit very low solubility in H2O, hence stock solutions (2 m) were prepared in DMSO and further diluted to working concentrations in MKR. The final concentration of DMSO was not higher than 0.1%, which shows no effect on gj or toxicity on cells (Picoli et al. 2012). Furthermore, all solutions containing n-alkanols were vortexed for at least 10 min. All drugs were applied after establishing the patch recording and gj had reached a constant value.
It was reported that to eliminate or significantly reduce binding of AA and other PUFAs to BSA, positively charged guanidine groups of arginine residues should be modified using CHD (Toi et al. 1967; Beck et al. 1998). To follow this protocol, we dissolved 825 mg of BSA in 25 ml of 0.2 m NaOH solution. Then, 330 mg of CHD was added and the mixture was incubated for 3 h at room temperature. Afterwards, the solution was titrated to pH 7.0 and dialysed with MKR solution at 4°C for 24 h. The resulting solution was titrated to pH 7.4 and SDS–PAGE was used to confirm that BSA was modified as previously shown (Michel et al. 1985; Katz & La Marche, 1995; Beck et al. 1998); the level of modification was >90%. SDS–PAGE showed a very small quantity of dimers and trimers of BSA and no oligomers of its modified form (BSA–CHD), which is consistent with previous results (Katz & La Marche, 1995). SDS–PAGE data show that BSA–CHD migrates with a broader spread than BSA, indicating that capping of the positively charged guanidine groups of the arginine residues was successful. The final concentration (mg ml−1) of the BSA–CHD stock solution was evaluated using a standard Bradford protein assay (Sigma Aldrich, USA). Molar concentrations of BSA and BSA–CHD solutions used in this study were calculated assuming that the molecular weight of BSA is equal to ∼66 kDa (Hirayama et al. 1990; Beck et al. 1998; Ninomiya & Kayama, 1998).
Data analysis and statistics
Dose–response curves obtained with different concentrations of tested compounds were fitted to a three-parameter logistic sigmoid equation, and a concentration of the compound required to produce 50% of maximal effect (EC50) was derived. To evaluate changes of voltage-gating parameters, the experimental gj–Vj dependences were fitted using a stochastic four-state model (S4SM; Paulauskas et al. 2009). Data are reported as means ± SEM. Unpaired Student's t test was used for statistical evaluation. P < 0.05 was considered significant.
Functional efficiency of Cx36 GJ channels
HeLa cells stably expressing Cx36-EGFP allowed us to select cell pairs exhibiting JPs in apposition between neighbouring cells. Experiments were performed on the second and third days after plating cells on glass coverslips. The fraction of GJ channels that is open at any given time over the total number of physical channels in the JP (functional and non-functional) was initially measured for Cx43-EGFP and denominated as the functional efficiency (K) of GJ channels (Bukauskas et al. 2000). K was defined as the ratio of the number of open channels at Vj= 0 (No) to the total number of channels in the JP (NT), K = No/NT. These measurements were achieved by combining a dual whole-cell patch-clamp for estimating the number of open channels, where No=gj/γ, and fluorescence imaging of EGFP tagged to Cxs to estimate NT. It has been shown that only ∼1/10 of Cx43, ∼1/50 of Cx45 and ∼1/100 of Cx57 GJ channels are open at any time at Vj= 0 (Bukauskas et al. 2000; Palacios-Prado et al. 2009, 2010). In this study, we used the same approach. Briefly, we selected cell pairs with JPs that were oriented parallel to the focal plane and could be viewed en face (Fig. 1A). To estimate NT it was necessary to assess the fluorescence produced by a single GJ channel (Fγ). We selected large JPs with uniform fluorescence in order to measure fluorescence per unit area in a central region of the JP and avoid edge effects (Fig. 1A, inset). We assessed fluorescence per unit area (FJP) in this region in arbitrary fluorescent units (a.u.) and subtracted background fluorescence outside the JP. We assumed that each Cx36-EGFP channel occupied 100 nm2 (corresponding to 10 nm centre-to-centre spacing in a square array and 104 channels per μm2) as an approximation of values seen in atomic force microscopy (Lal et al. 1995; Muller et al. 2002). Thus, we assessed Fγ from the ratio, FJP/10,000; on average Fγ= 0.140 ± 0.004 a.u. (n= 29). Light intensity and recording conditions were kept constant in all experiments to maintain a.u. consistent.
In combined imaging and electrophysiological studies, we measured gj and the total fluorescence intensity (FT) of JPs, independently of their spatial orientation. FT was estimated by measuring the total fluorescence in the region of interest (ROI) enclosing a JP (dashed ellipse in Fig. 1B). To collect all light including that from out-of-focus regions, the ROI was made several times larger than the size of the JPs (Bukauskas et al. 2000). The total number of GJ channels present in each JP was determined from the ratio, NT = FT/Fγ. JPs from ∼2 to 9 μm in diameter contained from ∼5.6 × 103 to 19 × 104 GJ channels (n= 27), respectively. In estimating No, we used the latest report on the single channel conductance of Cx36, which was estimated in pancreatic β-cells (Moreno et al. 2005) expressing solely Cx36; γ= 6 pS. In summary, we found that under control conditions K=No/NT= 0.0078 ± 0.0005 (n= 21), i.e. only ∼1/128 of Cx36-EGFP channels were open at any given time (Fig. 1C, filled circles).
Regulation of gj and Vj-gating by n-alkanols
Macroscopic gating properties of Cx36 homotypic GJs were reported earlier (Srinivas et al. 1999; Teubner et al. 2000; Moreno et al. 2005), and it was shown that Cx36 is among the least Vj-sensitive Cxs. To measure gj and Vj-gating of Cx36 GJ channels, we evoked Ij by repeated Vj ramps from 0 to –105 mV (assuming that gj–Vj is symmetric around Vj= 0 mV) of 35 s in duration and consecutive 20 mV steps (0.5 s) in between them (Fig. 2A, upper trace).
Lipophilic agents, such as n-alkanols, are broadly used for cell–cell uncoupling (Bruzzone et al. 1987; Burt & Spray, 1988; Weingart & Bukauskas, 1998; Rozental et al. 2001). In addition, it was reported that some n-alkanols had no or little effect on cell–cell coupling depending on the cell type and Cx isoform (Chanson et al. 1989; Rup et al. 1993; Ehrlich et al. 2000); however, there are no reports showing an increase in gj under an exposure to n-alkanols. Thus, we were surprised when application of pentanol (5 mm) strongly and reversibly increased Ij in Cx36-expressing cells (Fig. 2A, lower trace). To study the effect of n-alkanols on gj and the Vj-gating in more detail, we used a homologous series of n-alkanols from pentanol to decanol. Hexanol, as well as pentanol, induced a concentration-dependent increase in gj of Cx36-EGFP GJ channels (Fig. 2B). The value of EC50 for gj increase under an exposure to pentanol or hexanol was 4.6 ± 0.5 mm (n= 5) or 2.8 ± 0.4 mm (n= 6), respectively. These data are in good agreement with measurements described in Fig. 1 where K was increased ∼four times under an exposure to hexanol (up to 0.030 ± 0.003; n= 11; Fig. 1C). The size of JPs ranged from ∼5.4 × 103 to 8.6 × 104 GJ channels, and neither the size nor the total fluorescence intensity of JPs changed during application of n-alkanols (not shown), indicating no changes in NT.
Figure 2C shows typical gj–Vj plots measured in response to Vj ramps from 0 to –100 mV under control and ∼7 min after application of 3 and 5 mm hexanol, which caused an increase in Ij. Similar gj–Vj plots were obtained in three other experiments. All experimental gj–Vj plots (n= 4) were fitted using a S4SM of GJ channels combined with a global optimization algorithm (Paulauskas et al. 2009; see at http://connexons.aecom.yu.edu performance of the S4SM and Movies 1–3). During the fitting process, we assumed that only slow gates function in Cx36. This assumption is based on low sensitivity to Vj-gating and slow kinetics of gj decay during application of Vj steps that resembles properties of Cx43-EGFP in which tagged EGFP eliminates function of the fast gate (Bukauskas et al. 2001). Furthermore, there are no reliable data demonstrating gating of Cx36 to the residual state, which is the main property of the fast gate. The S4SM allowed us to estimate gating parameters characterizing sensitivity to Vj for each aHC/gate (AH and Vo,H), open probability of aHC (Po,H) and the number of functional channels (NF). AH characterizes the steepness of changes in Po,H as a function of voltage across the aHC (VH), while Vo,H is VH at which Po,H= 0.5. Values of AH and Vo,H estimated during the fitting process allowed calculation of NF and Po,H as a function of Vj. Data fitting (Table 1) shows that hexanol (5 mm) reduced the sensitivity of Vj-gating by enhancing Vo,H from ∼42 to 75 mV. Figure 2D shows the dependence of Po,H on Vj obtained using gating parameters from fitting experimental gj–Vj plots shown in Fig. 2C. The averaged data from four Po,H–Vj plots show that under control conditions at Vj= 0, approximately 61% (0.78 × 0.78) of functional GJ channels are open at any given time. An increase in Vo,H resulted in an increase in Po,H at Vj= 0 mV by ∼1.1-fold at 3 and 5 mm (Table 1), which in part explains the observed gj increase. In addition, gj increase was due to an increase in NF, on average from 377 channels at control to 891 and 1292 channels at 3 and 5 mm hexanol, respectively. This is also reflected in an increase of a steepness of the No–NT relationship under an effect of 5 mm hexanol (Fig. 1C) obtained in a separate series of experiments.
|Concentration||A H (mV−1)||V o,H (mV)||γres,H (pS)||γo,H (pS)||N F||P o,H|
|Control||0||0.029 ± 0.001||42 ± 1.3||0||12||377 ± 66||78 ± 1.0|
|Hexanol (mm)||3||0.029 ± 0.006||69 ± 4.9**||0||12||891 ± 114**||86 ± 3.8*|
|5||0.025 ± 0.004||75 ± 4.3**||0||12||1292 ± 141**||85 ± 3.7*|
|BSA (μm)||15||0.032 ± 0.004||57 ± 4.5*||0||12||582 ± 187||86 ± 3.1**|
Further studies revealed that heptanol (2 mm) increased gj by 3.0 ± 0.4-fold (n= 7), while octanol (2.5 mm; n= 4), nonanol (0.5 mm; n= 11) and decanol (0.5 mm; n= 5) caused full uncoupling; an example of the uncoupling effect of decanol is shown in Fig. 2E. We observed similar gj changes depending on the C-chain length of n-alkanols in HeLa cells expressing wild-type Cx36; 2 mm heptanol increased gj 3.6 ± 0.3-fold, n= 18. Furthermore, hexanol and pentanol increased gj, while octanol and decanol uncoupled HeLaCx36 cells (not shown). In summary, Cx36 and Cx36-EGFP GJs exhibit gj increase under application of SCCAs and gj decrease under LCCAs with the transition between heptanol and octanol.
Regulation of gj and Vj-gating by BSA
Typically, application of AA leads to full uncoupling of GJs formed from a variety of Cxs and innexins (Spray & Burt, 1990; Schmilinsky-Fluri et al. 1997; Weingart & Bukauskas, 1998; Teubner et al. 2000; Rozental et al. 2001; Contreras et al. 2002), with very slow gj recovery during washout. It is well established that fatty acid-free BSA accelerates coupling recovery (Fluri et al. 1990; Schmilinsky-Fluri et al. 1997) by directly binding AA and other PUFAs on three potential sites (Reed, 1986; Hamilton et al. 1991; Bojesen & Bojesen, 1994). In addition, BSA decreases plasma membrane fluidity by reducing the concentration of PUFAs (Beck et al. 1998) and by binding divalent metal ions (Sommer-Knudsen & Bacic, 1997; Bal et al. 1998).
We found that in HeLa cell pairs expressing Cx36-EGFP, BSA (15 μm) increased gj on average ∼1.6 ± 0.1 (n= 8) times. The maximal effect of BSA was reached ∼10 min after its application and gj returned to the initial level ∼11 min after washout; Fig. 3A shows an example of gj changes. Figure 3B shows the averaged percentage change in gj of Cx36 GJs over control depending upon BSA concentration; EC50= 2.3 μm. In subsequent experiments, we used BSA at a concentration of 15 μm, which produced a maximal effect on gj. To eliminate the possibility that BSA-dependent changes in gj are due to alterations in free [Ca2+]i (Firek & Weingart, 1995), we substituted 2 mm EGTA in the pipette solution for 3 mm BAPTA ([Ca2+]i= 5 × 10−8 m). This change in buffer had no effect on the BSA-dependent increase in gj (1.6 ± 0.3; n= 4), suggesting that the BSA-dependent effect on gj was not due to changes in [Ca2+]i. We observed no changes in EGFP fluorescence or size of JPs during application and washout of BSA, indicating that observed gj changes were not due to variations in NT caused by de novo formation or internalization of channels in JPs (not shown). Furthermore, using the same protocol as for Fig. 2C, we examined changes of Vj-gating under application of 15 μm BSA. The averaged data from four experiments (Table 1) indicate that BSA reduced the sensitivity to Vj by enhancing Vo,H from 42 to 57 mV, which increased Po,H at Vj= 0 by 1.1-fold. In addition, NF increased from ∼377 to 582.
Figure 3C shows an accelerated gj recovery by BSA from partial uncoupling caused by 2 μm AA; gj was normalized to its initial value. Spontaneous gj recovery during washout from AA was close to linear with a steepness of ∼3% per min (n= 8), which is shown by a dashed line. On average, 2 μm AA reduced normalized gj to 0.31 ± 0.04 (n= 11), while application of BSA (15 μm) increased it to 1.27 ± 0.22 (n= 6). These experiments suggest that BSA extracts PUFAs (including AA) from the membrane; however, due to their constant production, it takes ∼11 min to reestablish the initial gj that presumably corresponds to the time necessary for reestablishing the initial AA level in the plasma membrane (Fig. 3A). Higher concentrations of AA (≥10 μm) typically caused full uncoupling (n= 20), and BSA promoted gj recovery to the control level (n= 10).
The binding of AA and other PUFAs to BSA depends upon positively charged arginine residues (Beck et al. 1998). To eliminate or significantly reduce the binding of AA to BSA, positively charged guanidine groups of the arginine residues were neutralized using CHD (for details, see Methods). Previous studies have shown that this modification significantly decreases the potency of BSA to bind PUFAs (Beck et al. 1998). Figure 3D shows that BSA–CHD caused neither gj increase under control conditions nor gj recovery after application of AA. Figure 3E shows that on average BSA–CHD did not change normalized gj (1.03 ± 0.05; n= 8), while BSA increased it to 1.6 ± 0.1 (n= 8). Even application of a threefold higher concentration (45 μm) of BSA–CHD did not evoke an increase in gj under control conditions or during gj recovery after washout from AA (n= 3, not shown). These data suggest that endogenous PUFAs could be one of the key factors in promoting a stable closed state of Cx36 GJ channels, consequently resulting in very low functional efficiency (Fig. 1C).
Modulation of endogenous AA concentration
It has been reported that among various phospholipase A2 isoforms (sPLA2: secretory PLA2; cPLA2: cytosolic PLA2; PlsEtn-PLA2: plasmalogen-selective PLA2; iPLA2: calcium-independent PLA2), cPLA2 and PlsEtn-PLA2 have greater specificity for AA production (Gijon & Leslie, 1999; Cummings et al. 2000; Leslie, 2004; Farooqui et al. 2006). In order to reduce endogenous AA production, we used MAFP to irreversibly inhibit cPLA2 and iPLA2. It has been shown that MAFP reduces endogenous AA production in HeLa cells (Huang et al. 2002). If the levels of endogenous AA, and other PUFAs derived from AA, exert an inhibitory effect on Cx36 GJ channels, then the inhibition of cPLA2 by MAFP should enhance gj by reducing the endogenous concentration of AA. We used MAFP at concentrations in the range of 0.01–0.03 μm, which, as reported in Cummings et al. (2000) and Farooqui et al. (2006), should block cPLA2. Figure 4A shows that MAFP at a concentration of 0.01 μm increased gj by ∼28% over initial gj, and this increase was further potentiated by BSA (15 μm). Figure 4C shows that MAFP at 0.01 and 0.03 μm increased gj on average up to ∼27% over initial gj.
Elevation of endogenous AA in the membrane could be achieved by activation of PLA2 (Balsinde et al. 1999). In order to increase endogenous AA production, we activated cPLA2 using the well-known inhibitor of the Ca2+-ATPase, thapsigargin, which causes [Ca2+]i to increase by mediating Ca2+ release from the endoplasmic reticulum (Croxtall et al. 1997). Thapsigargin has been used as an effective treatment to activate cPLA2 and increase AA concentration in many cell types, such as mouse RAW 264.7 macrophages, rat liver (C-9) cells, human colon carcinoma cells, estrogen receptor-negative A549 cells, HEL30 keratinocytes, etc. (Croxtall et al. 1997; Levine & Rabouille, 2005). Figure 4B shows that application of 0.05 μm thapsigargin reduced gj, presumably by increasing endogenous AA production. Figure 4B also shows that BSA restored gj during thapsigargin application, presumably by reducing endogenous PUFAs and AA concentration. On average, 0.05 and 0.1 μm of thapsigargin decreased normalized gj to 0.57 ± 0.03 (n= 20) and 0.34 ± 0.03 (n= 19), respectively (Fig. 4C). Figure 4D shows that BSA (15 μm) applied in the presence of thapsigargin increased gj to a value slightly above control (∼1.1-fold in both cases), i.e. the gj-enhancing effect caused by BSA alone (1.6-fold, dotted grey line) was reduced. This reduction could be explained by increased levels of AA in the membrane. These results suggest that a BSA-dependent increase in gj of Cx36 GJs could be related to a removal of endogenous AA from the plasma membrane.
Modulation of AA-dependent inhibition of Cx36 GJs by hexanol
We demonstrated that the effect of n-alkanols on gj of Cx36 GJs depends on the length of their C-chain (Fig. 2B). Here, we examined whether the gj-enhancing effect of hexanol is affected by factors that modulate the concentration of AA. Figure 5A shows that application of exogenous AA (10 μm) causes complete uncoupling, and hexanol (5 mm) applied during washout from AA increased the rate of recovery of gj; the dashed line indicates a predicted trace for spontaneous gj recovery from washout of AA (∼3% per min). The gj-enhancing effect of hexanol after pre-inhibition by 2 μm AA was reduced (1.4 ± 0.2; Fig. 5B and E; P < 0.001) as compared with an effect of hexanol alone (3.4 ± 0.3; dashed line in Fig. 5E). Presumably, at 10 μm AA, the blocking effect is too strong for hexanol to compete (Fig. 5E). After pre-inhibition of gj by 0.1 or 0.05 μm thapsigargin, hexanol increased gj 1.8 ± 0.2 (n= 8) and 2.4 ± 0.2 (n= 10) times, respectively, i.e. the increase in gj was smaller as compared with hexanol application alone (Fig. 5C and E). Thus, the gj-enhancing effect of hexanol was reduced during its combined application with thapsigargin (0.1 and 0.05 μm) or AA (2 μm). Figures 5D and 6C show that application of BSA does not reduce significantly the gj-enhancing effect of hexanol. Possible interaction of hexanol with BSA during their combined application is excluded because it was shown that n-alkanols (including hexanol) do not interact with BSA (Tan & Siebert, 2008). Similarly, we did not observe a statistically significant difference in the gj-enhancing effect of hexanol in the presence of MAFP. All these data indicate that an increase in AA levels reduces the gj-enhancing effect of hexanol, and support the hypothesis that AA and hexanol have opposing effects upon the same gating mechanism.
Effects of BSA and n-alkanols on pancreatic β-cells
It is well established that mouse pancreatic β-cells solely express Cx36 (Serre-Beinier et al. 2000; Moreno et al. 2005). To test whether n-alkanols and BSA cause similar effects in β-cells as in HeLa transfectants, we performed experiments using freshly isolated pairs of β-cells (Fig. 6A; see Methods). Typically, gj under control perfusion conditions was relatively low and varied from 0.1 and 1.2 nS; on average gj= 0.32 ± 0.09 nS (n= 16). Figure 6B shows that hexanol (5 mm) caused an increase in gj, and following BSA (15 μm) application caused an approximately similar increase in gj but with slower kinetics (fivefold). Figure 6C shows that BSA caused an increase in gj, and that hexanol can cause a further increase. In summary, we found that BSA and hexanol increased gj on average 1.8 ± 0.2-fold (n= 8) and 2.2 ± 0.6-fold (n= 7), respectively. Hexanol, when applied straight after BSA, increased gj on average 1.3 ± 0.1-fold (n= 6; normalized to BSA steady-state value; Fig. 6C). Furthermore, nonanol (0.5 mm) and decanol (0.5 mm) caused full uncoupling; an example of the uncoupling effect of decanol is shown in Fig. 6D. Thus, pancreatic β-cells expressing endogenous Cx36 show very similar modulation of gj by AA and SCCAs as shown in HeLa transfectants.
Do Cx isoforms differ in sensitivity to BSA and n-alkanols?
To answer this question, we tested the effect of BSA and hexanol on gj of GJs formed by mCx30.2, Cx36, Cx45 and Cx47 expressed in HeLa cells, and Cx43 endogenously expressed in Novikoff cells (rat hepatoma cells). All these Cxs are expressed in the nervous system: Cxs m30.2, 36 and 45 in neurons; Cx43 in astrocytes; and Cx47 in oligodendrocytes (Eugenin et al. 2012). BSA (15 μm) was applied 10 min after reaching a steady-state after establishing a dual whole-cell recording.
The summarized data presented in Fig. 7 show that BSA (15 μm) promoted coupling only in Cx36- and Cx47-expressing cells, while it had no effect in cells expressing mCx30.2, Cx43 and Cx45. These results indicate that endogenous PUFAs inhibit Cx36 and Cx47 GJ channels at resting conditions. If the mechanism by which PUFAs and SCCAs exert opposite effects on gj is through competition for a conserved lipophilic binding domain, then hexanol should increase gj in Cx47 GJs – as shown for Cx36 – by competing with endogenous PUFAs. However, we found that hexanol (5 mm) reduces gj in Cx47 GJs to 0.07 ± 0.02 (n= 6; Fig. 7), suggesting that there are different binding domains or sensorial elements for PUFAs and SCCAs in Cx47. GJs formed by other Cxs were blocked by hexanol as well (Fig. 7).
Combined fluorescence imaging and electrophysiological studies showed that the ratio between the number of open Cx36 GJ channels at Vj= 0 and the total number of channels assembled in JPs is extremely low, K= 0.008 (Fig. 1). For example, in a cell pair exhibiting gj= 1 nS, only ∼167 GJ channels with γ= 6 pS are open, while NT is ∼21,410 GJ channels (167/0.0078). Furthermore, because we estimated an open probability of functional channels equal to ∼0.61 (see Table 1), then ∼274 (167/0.61 ≈ 274) are functional. Of these functional channels, only 167 are open at any given time on average. We assume that most channels in JPs are permanently closed, and presumably channels that were inhibited by AA could be transferred to a functional state by SCCAs, BSA or lowering the activity of different PLA2 isoforms. Such a small fraction of functional channels is not unique to Cx36, and it has been demonstrated that other Cxs also show low functional efficiency (KCx43=∼0.1; KCx45=∼0.04; KCx57=∼0.01; Bukauskas et al. 2000; Palacios-Prado et al. 2009, 2010). Recently, it was shown that in electrical synapses between neurons in the mesencephalic trigeminal nucleus only ∼0.1% of Cx36 GJs are functional (Curti et al. 2012). Also, our unpublished data (F.F.B.) demonstrate the value of K in the range of 0.005–0.05 for Cx46 and Cx50. Accumulating data suggest that GJs formed by all Cxs will have only a small fraction of functional channels under control conditions. Different factors may influence channel transitions between functional and non-functional populations, such as time needed for maturation after docking of two HCs, interaction with other proteins and components of cytoskeleton, phosphorylation, etc. However, we found no changes in the fluorescence of JPs or their size during hexanol, BSA or AA application. The kinetics of changes in gj during an exposure to hexanol, BSA or AA, and their washout, are too fast to be explained by de novo formation or internalization of GJ channels. This property of GJs may allow having a high number of GJ channels ready to be recruited to function on a short time scale.
Numerous studies have shown that n-alkanols, including pentanol and hexanol, caused full uncoupling in cells expressing Cx26, Cx30.2, Cx32, Cx37, Cx40, Cx43, Cx45, Cx46 and Cx57 GJs (Burt et al. 1991; Moreno et al. 1991; Veenstra et al. 1994; Harris, 2001; Spray et al. 2002; Skeberdis et al. 2011). Other studies have shown that SCCAs (including hexanol) have no effect on electrical coupling between pancreatic acinar cells, cultured human fibroblasts or only slightly inhibit cell coupling in cumulus cells (Chanson et al. 1989; Fagbohun & Downs, 1991), which do not express Cx36. However, we found that SCCAs produce a marked gj increase in HeLaCx36 cells (Fig. 2) and pancreatic β-cells (Fig. 6). Fitting of gj–Vj plots with a S4SM (Fig. 2C and D; Table 1) revealed that hexanol increased Vo,H, which in turn enhanced open probability of gates and could explain, at least in part, an increase in gj. Therefore, to explain a ∼fourfold increase of functional efficiency of Cx36 GJs (Fig. 2B), it should be assumed that in parallel with an increase in the open probability there was also an increase in NF. According to Ebihara & Steiner (1993) and Oh et al. (1999), the gates could be in two or more closed states. Our unpublished data support these notions, and we assume that gates could be in the initial closed (c1) state from which gates can operate between closed and open states, and that there is one or more deep closed (c2) states from which gates can transit only to the initial closed state but not directly to the open state. We speculate that, for example, hexanol facilitates c2→c1 transitions.
Besides AA, many other PUFAs and monounsaturated fatty acids are known to block GJ channels when externally applied. For example, oleic (18:1), linoleic (18:2), linolenic (18:3), palmitoleic (16:1) and myristoleic (14:1) acids and their derivatives oleamide and anandamide have all been shown to reduce cell–cell coupling (Giaume et al. 1989; Malewicz et al. 1990; Burt et al. 1991; Hirschi et al. 1993; Hii et al. 1995; Lavado et al. 1997; Boger et al. 1998; Harris, 2001). Here, we show that BSA at a concentration of 15 μm increased gj∼1.6-fold in Cx36- and ∼1.5-fold in Cx47-expressing cells, but had no effect on Cx30.2-, Cx43 and Cx45-expressing cells (Figs 3 and 7). Presumably, this difference could be influenced by AA-binding affinity, i.e. the stronger the binding of AA to Cx, the lower the ability of BSA to affect coupling. There could be many other reasons for Cx-type dependent differences in gj changes under an effect of BSA and n-alkanols. We assume that the gj-enhancing effect of BSA is mainly achieved by extracting PUFAs (including AA) from the plasma membrane in HeLa cells (Figs 3 and 7) and pancreatic β-cells (Fig. 6). This assumption is strongly supported by an absence of gj-enhancing effect of BSA–CHD (Fig. 3D and E). Changes of gating properties under application of BSA were similar to those obtained under application of hexanol (Table 1), which in both cases resulted in a substantial increase in gj. The simplest explanation for the observed changes in AH and Vo, which revealed an increase of both Po,H and NF, is that the dominant effect of hexanol and BSA on the channels is to stabilize an open state of the gate relative to the closed state, as reflected in the large shifts in Vo,H and relatively small changes in AH.
The observed changes in gj during reduction or elevation of endogenous PUFA levels with MAFP or thapsigargin (Fig. 4), respectively, support the notion that Cx36 GJs are normally inhibited by endogenous PUFAs and the gj-enhancing effect of BSA depends on reduction of endogenous PUFA levels in the plasma membrane (Fig. 4). It has been shown that the effect of thapsigargin on [Ca2+]i is transient, and normal [Ca2+]i is reestablished after ∼10 min of thapsigargin exposure in HeLa cells (Peppiatt et al. 2004). However, the observed effect of thapsigargin on gj was stable and persistent. Thus, it is unlikely that direct Ca2+-dependent uncoupling caused gj reduction.
The content of AA varies depending on cell type: ∼1 μm in human leukocytes (Chilton et al. 1996), ∼5 μm in human platelets (Neufeld & Majerus, 1983), ∼13 μm in human normal skin tissue (Hammarstrom et al. 1975) and ∼15 μm in isolated islets of Langerhans from rat (Ramanadham et al. 1992). We presume that normal levels of AA in β-cells (15 μm) keep Cx36 GJs inhibited (Serre-Beinier et al. 2009), which correlates with low intrinsic coupling between isolated pairs of β-cells found in this study; gj varied between 0.1 and 1.2 nS. Other groups also reported low cell–cell coupling between pancreatic β-cells from rodents; gj varied in the range of ∼0.17–0.95 nS (Moreno et al. 2005; Zhang et al. 2008; Benninger et al. 2011). Cx36 knockout mice exhibit coupling deficiency and asynchronous Ca2+ transients between pancreatic β-cells, leading to irregular dynamics and insufficient insulin secretion (Ravier et al. 2005; Head et al. 2012). Furthermore, specific deletion of Cx36 in β-cells leads to major alterations in glucose-induced insulin secretion (Wellershaus et al. 2008). Thus, Cx36-mediated coupling between β-cells plays an important role in insulin secretion and the development of diabetes. Our data show that in β-cells, gj changes in response to BSA and n-alkanols (Fig. 6) are very similar to gj changes induced in HeLaCx36 cells. There are some differences in the magnitude and kinetics of gj changes between HeLa and β-cells that can be attributed to differences in levels of expression of Cx36, composition of lipids of the plasma membrane, etc. Nonetheless, collected data support the notion that in β-cells under control conditions most of the functional Cx36 GJ channels are affected by endogenous levels of PUFA derivatives (including AA). PUFA-dependent inhibition of coupling between β-cells and its modulation by relatively small lipophilic molecules, such as SCCAs, may play an important role in insulin secretion and highlights new avenues for regulation of β-cell function. The gj-enhancing effects of SCCAs and BSA are not a unique property found in HeLa cells expressing Cx36, but are also found in pancreatic β-cells and possibly neurons expressing endogenous Cx36.
In summary, our data show that the gj-enhancing effect of SCCAs is related to their ability to interfere with AA-dependent inhibition of Cx36. Furthermore, elevation of AA concentration by thapsigargin or exogenous application of AA (2 μm) reduced the potentiating effect of hexanol on gj. Conversely, MAFP and BSA, which tend to reduce the levels of PUFAs (including AA) in the plasma membrane, did not reduce the gj-potentiating effect of hexanol (Fig. 5D and E) or did they enhance it, which may suggest that at relatively low levels of AA the ability of hexanol to lessen an effect of AA is enhanced. Collected data allow us to assume that Cx36 contains a unique structural domain, which results in gj increase under an exposure to SCCAs, while all other Cxs isoforms examined in this study and reported by others show uncoupling (Burt et al. 1991; Moreno et al. 1991; Veenstra et al. 1994; Harris, 2001; Spray et al. 2002; Skeberdis et al. 2011).
A.M., N.P-P. and F.F.B.: conception, design of experiments, collection, analysis and interpretation of data, drafting of manuscript; L.R. and V.A.S.: collection and analysis of data, critically revising the manuscript. All authors approved the final version for publication.
The authors thank Dr. Michael V.L. Bennett for reviewing the manuscript; Dr. Teresa DiLorenzo and Dr. Gayatri Mukherjee for providing isolated pancreatic islet of Langerhans; and Nerijus Paulauskas, Angelė Bukauskienė and Valeryia Mikalayeva for excellent technical assistance. Nicolás Palacios-Prado is a Howard Hughes Medical Institute International Student Research Fellow. This work was supported by National Institute of Health Grants HL084464 and NS072238 to F.F.B. A.M. thanks the Lithuanian Research Council for supporting her visit to F.F.B. laboratory (fellowship No. SDS-2010-027).