pH-DEPENDENT MODULATION OF CONNEXIN-BASED GAP JUNCTIONAL UNCOUPLERS
Vytenis A. Skeberdis1,2, Lina Rimkute1,2, Aiste Skeberdyte1, Nerijus Paulauskas1,2, Feliksas F. Bukauskas2
1Lithuanian University of Health Sciences, Institute of Cardiology, 17 Sukilėlių Avenue,
Kaunas 50009, Lithuania;
2Yeshiva University, Albert Einstein College of Medicine, Dominick P. Purpura
Department of Neuroscience, 1300 Morris Park Avenue, Bronx, New York 10461, USA
Running Title: pH-dependent modulation of gap junctional uncouplers
Keywords: Gap junctions, voltage gating, pH , connexin, uncouplers i
Manuscript Word Count: 4676
Corresponding author: Feliksas Bukauskas, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, United States, e-mail:
Non-technical summary. Gap junction channels formed from connexin (Cx) proteins are expressed in a variety of tissues providing exchange of metabolites and electrical
communication between cells. We demonstrate that uncoupling effects of gap junction channel blockers such as long carbon chain alkanols (LCCA), volatile anesthetics and the antimalarial drug mefloquine can be modulated by intracellular pH in a Cx-type dependent manner. In addition, we provide data to support that the uncoupling effect of LCCAs and other uncouplers could be related to a modification of hydrogen bonding between histidine residues and uncouplers and/or inside Cx structure.
Abstract. Gap junction (GJ) channels formed from connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell communication exhibiting high sensitivity to intracellular pH (pHi). We examined pHi-dependent modulation of junctional conductance (gj) of GJs formed of Cx26, mCx30.2, Cx40, Cx43, Cx45 and Cx47 by reagents
Abbreviations: AH, coefficient characterizing the steepness of PH,o, changes as a function of voltage across the aHC; aHC, apposed hemichannel; BCECF, 2',7'-Bis(carboxyethyl)-5(6)-carboxyfluorescein; Cx, connexin; EGFP, enhanced green fluorescent protein; γopen,H and γres,H, open and residual conductances of aHC, respectively; GJ, gap junction; gj, junctional conductance; HeLa cells, Human cervix carcinoma cells; LCCA, long carbon chain alkanol; MKR, modified Krebs-Ringer; NaCH3CO2, sodium acetate; NBS,
N-Bromosuccinimide; NF, number of functional channels; NH4Cl, ammonium chloride; PH,o, open probability of aHC; ROI, region of interest; uHC, unapposed hemichannel; Vj,
transjunctional voltage; Vo,H, voltage across an aHC at which its open probability, PH,o=0.5.
Introduction
Gap junction (GJ) channels are composed of two apposed hemichannels in contiguous cells and provide a direct pathway for electrical and metabolic cell-to-cell communication (Paul, 1986; Bennett, 1994; Elfgang et al., 1995; Goodenough et al., 1996; Rackauskas et al., 2010). Six connexin (Cx) subunits oligomerize into a connexon, which after insertion into the surface plasma membrane is called a hemichannel. The family of connexin genes consists of 20 genes in the mouse and 21 genes in the human genome. Connexins are expressed in all tissues except differentiated skeletal muscle, erythrocytes and mature sperm cells. Various tissues express more than one type of connexin, and therefore homotypic, heterotypic and heteromeric GJ channels may form between cells. Gating and permeability properties of GJ channels are regulated largely by transjunctional voltage (Vj), intracellular calcium, pH and phosphorylation (Rackauskas et al., 2010). For studies of functional properties of GJs and for the development of new therapeutic approaches involving regulation of gap junctional coupling, high affinity uncouplers, especially those which affect channels in a connexin-type specific manner, are required. Current
and peptides targeting extracellular loops of connexins (reviewed in (Rozental et al., 2001; Srinivas, 2009)). Even though some of these agents inhibit channels in a Cx isoform-specific manner (Spray et al., 2002), the mechanisms of their action remain elusive. Moreover, the potency and efficacy of uncouplers may depend on composition of the extracellular or intracellular environment. For instance, arylaminobenzoates (Srinivas & Spray, 2003) or local anesthetics and antimalarial drugs (Srinivas et al., 2001) at
physiological pH exist in both charged and uncharged forms. Uncharged forms of the drugs are more lipid-soluble. In such form, they cross the membrane and then reach their blocking site on the receptor after protonation in the aqueous environment of the cytoplasm (Hille, 2001). Relatively little is known about the impact of intracellular pH (pHi) on blocking capacity of GJ uncoupling agents and how this effect depends on Cx isoform. This is important in understanding the mechanisms of modulation of junctional communication by uncouplers under ischemic or other pathological conditions leading to pHi changes.
In the present study, we examined the blocking capacity of octanol and other GJ inhibitors as a function of pHi in cells expressing Cx26, mCx30.2 (mouse ortholog of human Cx31.9), Cx40, Cx43, Cx45 and Cx47. We demonstrate for the first time that: 1) uncoupling potency of long carbon chain alkanols and other uncouplers on Cx45 GJ channels is pHi-dependent; 2) pHi-dependent modulation of uncoupling by long carbon chain alkanols is Cx-type specific; 3) octanol induced uncoupling of Cx45 GJ channels may be mediated by formation of hydrogen bonds with histidines of a Cx protein.
Materials and methods
Cells and culture conditions
atmosphere at 370C. All media and culture reagents were obtained from Life Technologies (GIBCO-BRL).
Electrophysiological measurements
For simultaneous electrophysiological and fluorescence recording, cells grown onto glass coverslips were transferred to an experimental chamber with a constant flow-through perfusion mounted on the stage of an inverted microscope Olympus IX70 (Olympus America, Melville, NY) equipped with Hamamatsu cooled digital camera and fluorescence imaging system UltraView (Perkin Elmer Life Sciences, Boston, MA). Appropriate
excitation and emission filters (Chroma technology, Brattleboro, VT) were used to image 2',7'-Bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a pH sensitive dye. Junctional conductance, gj, was measured in selected cell pairs by using a dual whole-cell patch clamp. Cell-1 and cell-2 of a cell pair were voltage clamped independently with a separate patch clamp amplifier EPC8 (HEKA Elektronik, Lambrecht, Germany) at the same holding potential, V1=V2. Voltages and currents were digitized using a MIO-163 A/D converter (National Instruments, Austin, TX) and acquired and analyzed using custom-made software (Trexler et al., 1999). By stepping the voltage in cell-1 (ΔV1) and keeping the other
Fluorescence imaging studies
Fluorescence signals were acquired using UltraVIEW software for image acquisition and analysis (Perkin Elmer Life Sciences, Boston, MA). For pHi measurement, cells were loaded with 2',7' - Bis - (2 - carboxyethyl) - 5 - (and - 6) - carboxyfluorescein (BCECF; 4 μM) introduced into the cells through the patch pipettes in whole-cell voltage clamp mode. Dye was alternately excited with 436-nm and 500-nm light and the emitted light was filtered at 540 nm and recorded. Ratios of emitted light collected at excitation light of 436 and 500 nm (background subtracted) were converted to pHi values based on calibration curve. The latter was obtained using ionophore nigericin at 20 μM concentration in the presence of 140 mM potassium to equilibrate the intracellular pH with extracellular medium of different pH (Thomas et al., 1979). All experiments were performed at room temperature. To prevent dye bleaching, imaging was performed in time-lapse mode, i.e., cells were periodically exposed (every 15 s) to a low-intensity excitation light for 500 ms. We have used similar methodology earlier (Rackauskas et al., 2007; Palacios-Prado et al., 2009; Palacios-Prado et al., 2010).
Statistical analysis
Cumulative dose response curves of octanol were obtained by testing 4 or 5 successively increasing concentrations of the compound in the control, alkaline or acidic conditions. For each individual experiment, the results of gj changes over concentration of uncouplers were fit to the 3-parameter logistic sigmoid equation, and IC50, a concentration of the compound required to produce 50% of inhibition, was derived. To evaluate gj-Vj dependence of Cx45 GJ channels, the experimental gj-Vj curves obtained during slow voltage ramps were fitted using a stochastic four state model (S4SM) (Paulauskas et al., 2009). Data are reported as means ±S.E.M. Student’s t-test was used for statistical evaluation. P<0.05 was considered significant.
Results
Uncoupling of Cx45 GJs by long carbon chain alkanols depends on pHi
smooth muscles of blood vessels and neurons (Kreuzberg et al., 2006; Rackauskas et al., 2010). Lately, it was reported that gj of Cx45 GJs is regulated by pHi through modulation of the sensitivity to Vj of the fast gate and open probability of the slow gate (Palacios-Prado et
al., 2010). To study gj dependence on pHi, we used NH4Cl (Swietach & Vaughan-Jones,
2005) to increase pHi measured by loading cells with pH sensitive dye, BCECF (4 μM), through the recording patch-pipettes. pHi was measured in the regions of interest, ROI-1 and ROI-2, positioned on cell-1 and cell-2 (Fig. 1A). To measure gj and Vj-gating, we used Vj protocol composed of repeated 30 s long Vj ramps changing from 0 to -100 mV and five short steps of +10 mV (upper trace in Fig. 1B). gj was evaluated during the 5th Vj step followed by Vj ramp allowing maximal recovery of gj after Vj-gating caused by long Vj ramps. Homotypic GJs typically demonstrate symmetric gj-Vj dependence; therefore, we measured Ij (lower trace in Fig. 1B) only in response to one polarity of Vj. As it is
demonstrated in Fig. 1C, application of octanol (1 mM), blocked gj fully (n=8) with roughly no effect on pHi (ΔpHi≈-0.1). Then, addition of NH4Cl (15 mM) to the external solution in the continuous presence of octanol increased pHi to 8.2±0.1 (n=4) and unexpectedly increased gj very quickly. Moreover, gj exceeded the control level ~2-fold (195±2.4%; n=4), remained unchanged after washout of octanol and reached control level only under return to control conditions. Thus, alkalization in the presence of octanol not only fully abolished the uncoupling effect of octanol, but gj significantly exceeded its control value resulting in the gj observed with application of NH4Cl alone (Palacios-Prado et al., 2010). Octanol applied on cells already exposed to NH4Cl did not cause any detectable effect on gj until washout of NH4Cl (Fig. 1D).
Further, we measured the dependence of gj of Cx45 GJ channels on octanol
concentration under control, acidic and alkaline conditions. To measure gj and Vj-gating, we used a similar protocol as described above (Fig. 1B).
As shown in the Fig. 2A, octanol produced dose dependent inhibition of gj. A
pHi (8.2±0.1) by exposing cells to NH4Cl (15 mM; n=5; triangles), resulted in an increase of IC50 to 2.68 mM. At reduced pHi (6.9±0.11) when cells were exposed to NaCH3CO2 (10 mM; n=5; squares), IC50 decreased to 0.1 mM.
Fig. 2C shows gj-Vj plots measured at times indicated by numbers (1-5) on gj trace in Fig. 2A. To estimate changes of gating parameters of GJ channels, gj-Vj plots were fitted to a stochastic 4-state model (S4SM) (Paulauskas et al., 2009) which assumes that the voltage across each apposed hemichannel (aHC) in the GJ channel depends on the state of the hemichannel in series (“contingent gating” (Harris et al., 1981)). We assumed that each (aHC) of GJ channel contains a Vj-sensitive gate exhibiting γopen,H when the gate/aHC is fully open and γres,H (residual conductance) when the gate is closed. It was reported that a single Cx45 open unapposed hemichannel (uHC) conductance rectifies depending on transmembrane voltage, and in our evaluations γopen,H=62 pS (Valiunas, 2002) that is ~2-fold higher than the conductance of a single Cx45 GJ channel (32 pS, (Bukauskas et al., 2002)). Experimental evaluation of a unitary conductance of the residual state of uHC is complicated therefore, in our evaluations, γres,H was left as an independent parameter and its value was estimated during the fitting process. Another reason for doing so was because of the limitations of using S4SM, which accounts for only one gate per hemichannel, although indeed there are fast and slow gates per hemichannel (Bukauskas & Verselis, 2004).
Formally, for the slow gate the γres,H=0 while for the fast gate γres,H>0, therefore S4SM accounts for some averaged value of γres,H which depends on the ratio of gated fast and slow gates. The S4SM allowed us to define parameters characterizing Vj-gating, namely Vo,H (voltage across an aHC at which its open probability, PH,o=0.5) and AH (coefficient characterizing the steepness of PH,o, changes as a function of voltage across the aHC). In addition, the S4SM allows us to define the number of Cx45 GJ channels (NF) that are functional at any given time thereby providing the actual gj at Vj=0. Thus, during the fitting process γres,H, Vo,H, AH and NF were left as free parameters. Summary of data from 5
experiments similar to that shown in Fig. 2C are shown in Table 1.
Hexanol and nonanol alone did not change pHi (not shown). Fig. 3A-B shows that uncoupling caused by hexanol (5 mM) and nonanol (0.2 mM) was fully recovered by exposing cells to NH4Cl in a continuous presence of alkanols, suggesting that pHi modulates the blocking potency of LCCAs independently on the length of their carbon chain. Interestingly, recovery of coupling by wash in of NH4Cl in the presence of hexanol, octanol or nonanol completes in 10-15 seconds (see Fig. 1C, the inset in Fig. 3A and the gj changes in Fig. 3B indicated by dashed arrow), which is comparable with the time constant of gj increase during application of NH4Cl alone (Fig. 1D). Time constants of gj changes during application or washout of LCCAs are several times longer (~100 s; Figs. 1C and
3A-B). This may be explained by a higher mobility of H+ ions than LCCAs and suggests a
possible mechanism of interaction discussed below.
pHi–dependent modulation of gj by octanol varies among Cx isoforms
Numerous reported data show that GJ channels and hemichannels composed of different Cx isoforms can be fully blocked by LCCAs and at lower concentrations for alkanols with longer carbon chain (Srinivas, 2009). To examine whether the uncoupling potency of octanol can be also modulated by pHi in GJs formed of other Cxs rather than Cx45, we examined HeLa cells stably transfected with Cx26, mCx30.2, Cx40 and Cx47 and Novikoff cells expressing endogenously Cx43 by using the same protocol as shown in Figs. 1 and 3. These Cxs are expressed abundantly in many different tissues, including cardiovascular and nervous systems, lungs, skin, etc. (reviewed in (Harris, 2001; Rackauskas et al., 2010)). Fig. 4A-E shows that octanol (1 mM) rapidly induced full uncoupling in cells expressing Cx26, mCx30.2, Cx40, Cx43 and Cx47 that did not recover during combined application of octanol and NH4Cl. Thus, the observed modulation of octanol induced uncoupling by NH4Cl is Cx-type specific.
To examine whether Cx45 differs from other examined Cxs in its reaction to
alkalization, we examined changes of gj under application of NH4Cl in cells expressing five different Cxs. Fig. 4F shows that intracellular alkalization with NH4Cl (10 mM;
pHi-dependent modulation of Cx45 GJs by different uncouplers
We examined whether pHi modulates Cx45 mediated cell-cell coupling by other well established GJ uncouplers such as: 1) forane (isoflurane), a general anesthetic that exhibits uncoupling effect at concentrations used for inhalation anesthesia (Burt & Spray, 1989); 2) flufenamic acid, an anti-inflammatory drug (Harks et al., 2001; Srinivas & Spray, 2003); 3) mefloquine, antimalarial drug, which inhibits GJs in connexin-type specific manner
(Srinivas et al., 2001; Cruikshank et al., 2004); 4) carbenoxolone, a derivative of glycyrrhetinic acid used to treat oesophageal ulceration and inflammation (Davidson & Baumgarten, 1988); and 5) arachidonic acid, a precursor of prostaglandines and
leukotrienes, one of the essential fatty acids, freed from a phospholipid molecule by the enzyme phospholipase A2 (Rozental et al., 2001). We have tested all these compounds for their effect on gj when applied alone or in combination with NH4Cl by performing at least four experiments with each of them.
During application of forane (3 mM) for ~500 s, gj gradually approached zero (Fig. 5A), while pHi remained unchanged. NH4Cl (15 mM) applied in a presence of forane resulted in full gj recovery. Thus, the uncoupling effect of forane is pHi–dependent similar to the effect of LCCAs (Figs 1 and 3).
It was reported that mefloquine inhibits function of Cx26, Cx32, Cx43 and Cx46 GJs with a 10-20 - fold higher potency than function of Cx35 and Cx50 GJs (Srinivas et al., 2001). Mefloquine (10 μM) caused full uncoupling of Cx45 GJs (Fig. 5B). Since
mefloquine is a weak base, a small initial increase in gj was probably caused by small transient increase in pHi. NH4Cl (15 mM) applied in the presence of mefloquine induced a rapid rise in pHi and complete removal of the mefloquine blocking effect. Washout of NH4Cl returned the pHi and gj to their control values.
Interestingly, the recovery of gj during washout of the flufenamic acid is as slow as gj uncoupling (not shown). However, as it follows from the presented record, the recovery of gj during washout of flufenamic acid in the presence of NH4Cl was greatly accelerated.
The mechanism of action of carbenoxolone is largely unexplored. There are no reports indicating sensible differences in its uncoupling efficacy among different Cx isoforms. Carbenoxolone (1 mM), like flufenamic acid, caused a decrease in pHi by ~0.2 units, which was too small to explain the observed uncoupling (Fig. 5D). Then, an increase in pHi by NH4Cl (15 mM) did not cause the recovery of gj. Similarly, arachidonic acid (10 μM) completely blocked gj and NH4Cl (15 mM) failed to reestablish the cell-cell coupling (Fig. 5E). Thus, the pHi-dependent modulation of the blocking capacity of GJ channel inhibitors is apparently inhibitor-type specific.
To test the hypothesis that inhibition of gj by octanol is mediated by forming hydrogen bonds with histidine residues, which are likely involved in pH-dependent effects (Spray & Burt, 1990), the influence of 0.3 mM octanol was compared in a control condition and after pre-incubation with N-Bromosuccinimide (NBS), which reduces histidine’s ability to form hydrogen bonds by modifying its imidazole ring. Such an experimental protocol was recently used to demonstrate a Zn2+ interaction with histidines of Cxs in the horizontal cells of retina (Sun et al., 2009). After pre-incubation of HeLa cells expressing Cx45 in the solution containing 1 mM NBS for 40 min, a degree of uncoupling by octanol (0.3 mM) was reduced from 0.66±0.05 (n=5) to 0.24±0.03 (n=6) (p<0.001). In addition, the coupling-promoting effect of NH4Cl (10 mM) was reduced from 203±18% (n=5) (control) to 157±5% (n=6) (p<0.05) (Fig. 6). The correlation coefficient was 1.09 indicating that the action of H+ and octanol may take place at the same histidine residue/s. NBS (1 mM) did not affect gj in HeLaCx45 cells exposed to 1 mM NBS during electrophysiological experiments lasting ~30 min.
Discussion
This study was stimulated by the highly unexpected observation that, in cells expressing Cx45, the uncoupling effect of octanol and other LCCAs can be fully eliminated by
carbenoxolone and mefloquine and maybe some other but not yet examined uncouplers, have the potential to reduce neurological tremors (Bushara et al., 2004; Martin & Handforth, 2006). More detailed studies revealed that IC50 for an uncoupling effect of octanol increased from 0.1 to 0.25 and 2.68 mM with an increase in pHi from 6.9 to 7.2 and 8.1, respectively. Analysis of gj-Vj plots (Fig. 2C) using a stochastic 4-state model
(Paulauskas et al., 2009) revealed that an increase of Vj-sensitive gating of GJs under application of octanol is caused by a decrease of Vo,H ~2.6-fold and NF ~6-fold (Table 1) that leads to uncoupling by LCCAs. Interestingly, Cx45 GJs exhibited similar changes under acidification (Palacios-Prado et al., 2010), which resulted in a decrease of Vo,H and NF without substantial changes in AH. This suggests that pHi- and octanol-induced blocking effects of Cx45 GJs share similar mechanisms. The blocking effect of chemical uncouplers is realized mainly through the slow gate that correlates with full but not partial uncoupling. This may explain data showing a reduction of γres,H supporting the notion that, at the final state of uncoupling, most channels gate by the slow gates between the open and fully closed states instead by the fast gate between open and the residual/substate states.
Cells expressing other examined Cxs (Cx26, mCx30.2, Cx40, Cx43 and Cx47), however, did not exhibit coupling recovery by exposing them to NH4Cl under a continuous presence of octanol. In cells expressing Cx45, alkalization eliminated uncoupling effect of isoflurane and mefloquine. On the contrary, uncoupling caused by arachidonic acid, carbenoxolone and flufenamic acid could not be rescued by alkalization.
Due to ionization or tautomerization processes, many drug-like molecules can be transformed to different protonation states. The solubility and membrane permeability of a drug can vary significantly depending on the protonation state, which can be affected by pH and the local surrounding environment. The predicted conformations of a drug-like
molecule, as well as the binding mode and binding affinity of ligands with proteins, are all expected to be influenced by the protonation state of a ligand (Shelley et al., 2007). Hence, the pHi-dependence of GJ channel inhibition may be explained in two ways. First,
Reported data demonstrate that the effects of uncouplers, other than LCCAs, also are sensitive to pHi. It has been shown that blocking potency of flufenamic acid (Srinivas & Spray, 2003), mefloquine (Srinivas et al., 2001) and halothane (Tamalu et al., 2001)
depends on the extracellular pH suggesting that these agents in their uncharged form may more easily cross the membrane. In all our experiments, however, extracellular pH was maintained at 7.4 and GJ uncoupling agents had no or negligible effect on it. Moreover, an elevation of pHo causes an additional increase in pHi and gj of Cx45-expressing cells (Palacios-Prado et al., 2010). The protonated state of uncouplers can also be affected by the intracellular environment when they cross the membrane or dwell in the inner site of the plasma membrane. However, LCCAs can not be protonated due to their nucleophilicity. A second possibility is that protons may mediate the interaction of some uncouplers with the receptor. It is broadly assumed that acidification-induced uncoupling is caused by
protonation of histidines of Cxs (Spray & Burt, 1990). Hydrogen bonding is one of the factors modulating the 3-dimensional structure of Cxs and therefore affects their probability to open. Hydrogen bonding occurs between amide groups in the secondary protein structure as well as between "side chains" in tertiary protein structures in a variety of amino acid combinations. Factors that can change this structure are heat, pH, alkanols, etc. Octanol due to its OH functional group is very likely to form hydrogen bond with both proton acceptors and donors (three bonds in total: one with partly positive hydrogen, the other two with two lone pairs of oxygen). Octanol can replace existing hydrogen bonds in connexins by forming the new ones with its OH group, which can lead to transformation of the protein structure. This bonding can be markedly influenced by pH. The only amino acid, which is affected by pH in the physiological range is histidine. Its imidazole ring at pH<6 is
protonated, i.e., both nitrogens carry a hydrogen. At pH>7, the molecule is neutral, and the nitrogen that does not carry a hydrogen makes a double bond instead. Therefore, at higher pHi, the ability of octanol to form hydrogen bonds with lone electron pair of oxygen is reduced and successively the uncoupling properties of octanol in Cx45 are lost.
effect of NH4Cl and the uncoupling effect of octanol in Cx45 GJs. However, a role of histidines in the regulation of GJ conductance may differ depending on Cx type. For instance, histidine modification by diethyl pyrocarbonate evoked different effects on Cx46 and Cx50 hemichannels (Beahm & Hall, 2002). This suggests that LCCAs and some other uncouplers act on Cx45 through the formation of hydrogen bonds with histidines in the Cx45 protein. It is assumed that all uncoupling agents execute their action through the slow gating mechanism, which closes GJs fully (Bukauskas & Verselis, 2004). A slow (or “loop”) gate is located most likely in the gap region of the GJ channel that includes both extracellular loops (Bukauskas & Verselis, 2004; Verselis et al., 2009). Since LCCAs and some other GJ blockers are lipophilic compounds, which probably can affect preferentially extracellular or transmembrane domains of Cxs, then three histidines present in the
extracellular loops of Cx45 are likely candidates for such specific interaction as there are no histidines in transmembrane domains. At this point, we can only speculate that histidine-204, which is present in the second extracellular loop of Cx45 but not in other examined Cxs, may be important for the observed alkalization dependent coupling recovery.
Therefore, the presented data demonstrate for the first time that uncoupling potency of long carbon chain alkanols, forane and mefloquine on GJ channels is pHi- and Cx-type dependent. Presumably, this type of modulation is mediated by formation of hydrogen bonds between uncouplers and histidines of Cx protein.
Acknowledgments: We thank Dr. Willecke, Dr. Mammano and Bargiello for kindly providing the constructs and/or HeLa cell lines stably expressing connexins used in this study. We thank Valeryia Mikalayeva and Angele Bukauskiene for excellent technical assistance. This work was supported by Lithuanian State Science and Studies Foundation Grant B-07041 to V.A.S. and National Institute of Health Grants HL084464 and NS072238 to F.F.B.
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Author contributions:
V.A.S., L.R. and F.F.B designed the study, performed, analyzed, interpreted experiments and drafted the manuscript, A.S. designed and performed experiments related to NBS involvement in pH- and LCCA-dependent regulation of junctional conductance, and N.P. analyzed experimental data related to the voltage gating of gap junctions using a S4SM. All authors read and commented on the manuscript.
Figure legends:
Figure 1. Intracellular alkalization attenuates the blocking effect of octanol on Cx45 GJ channels. A, Fluorescence image of Cx45 expressing HeLa cell pair loaded with pH sensitive fluorescent dye BCECF (4 μM). B, Vj-gating and gj was measured in response to
squares) under control conditions and during application of octanol (1 mM) and/or NH4Cl (15 mM). C, Octanol causes full uncoupling without essential changes in pHi, while NH4Cl quickly increases gj exceeding its control value almost twice. D, gj increase caused by application of NH4Cl is not affected by octanol
Figure 2. The inhibition of Cx45 GJ channels by octanol is pHi-dependent. A, gj
measured in response to Vj steps applied in between voltage ramps. During indicated periods the cells were exposed to four concentrations of octanol. B, Concentration-response curves of the inhibitory effect of octanol on gj in control, alkaline and acidic conditions. The points show the mean and SEM. Continuous lines were derived from a non-linear least-mean-squares regression of the means to the logistic sigmoid equation. IC50 of octanol in control (pHi=7.2; circles), alkaline (pHi=8; triangles) and acidic (pHi=6.9; squares) conditions was 0.25 mM (n=5), 2.68 mM (n=5) and 0.1 mM (n=5), respectively. C, gj-Vj dependencies recorded at the times indicated by corresponding numbers in A in response to voltage ramps from 0 to -100 mV. Experimental gj-Vj plots were fitted to S4SM assuming that Vj-gating was symmetric around Vj = 0
Figure 3. pHi modulates the blocking potency of LCCAs independently on the length
of their carbon chain in Cx45 expressing HeLa cells. A, Hexanol (5 mM) reduced gj to 23.2±5.6% (n=4) while combined application of hexanol and NH4Cl (15 mM) increased gj to 158±6.9% (n=4) of the control value. B, Nonanol (0.2 mM) reduced gj to 1.8±1.6% (n=4), while combined application of nonanol and NH4Cl (15 mM) increased gj to 137±4.7% (n=4) of the control value. gj increase during application of NH4Cl was much faster (see the inset in A and dashed arrow in B) than gj changes during application or washout of alkanols alone
Figure 4. Modulation of gj and octanol-induced uncoupling by NH4Cl is connexin-type
alkalinization is Cx-type specific. After application of NH4Cl (10 mM), gjs measured in cells expressing Cx26, mCx30.2, Cx40, Cx43, Cx45 and Cx47 was 1.06±0.08 (n=5), 1.35±0.04 (n=5), 0.92±0.03 (n=4), 0.15±0.09 (n=4), 2.12±0.16 (n=5) and 1.0±0.04 (n=4), respectively, relative to their control values
Figure 5. pHi-dependent modulation of Cx45 GJs by different uncouplers. pHi in the
cell pair was measured concomitantly with gj under control conditions, and during application of GJ blockers alone and together with NH4Cl. A, gj in response to forane (3 mM) decreased approaching zero. Combined application of forane and NH4Cl (15 mM) increased gj exceeding its control value by 87±9% (n=5). Forane alone had no effect on pHi. B, Mefloquine slightly and transiently increased pHi which was followed by a transient increase in gj; however, later it caused the complete block of gj which fully recovered under application of NH4Cl. gj in response to mefloquine (10 μM) and mefloquine together with
NH4Cl (15 mM) was 12.1±2.1% (n=4) and 150±13.9% (n=4) of control conditions, respectively. C, Flufenamic acid alone slightly reduced pHi (~0.2 units) and caused a virtually complete block of gj that was not recovered by NH4Cl; the same was observed in all four experiments. D-E, Uncoupling effects of carbenoxolone (D) and arachidonic acid (E) were not affected by NH4Cl
Figure 6. Modulation of gj by pHi and LCCAs depends on histidine’s ability to form
Table 1. Gating parameters of Cx45 GJs derived during fitting of experimental gj-Vj plots, similar to those shown in Fig. 2C, using S4SM (n=5). γopen,H is conductance of fully open aHC; γres,H is residual conductance of the closed aHC; Vo,H is a voltage across an aHC at which its open probability, PH,o, is equal 0.5; AH is a coefficient characterizing the
steepness of P H,o changes as a function of voltage across the aHC; NF is a number of Cx45 GJ channels that are functional at any given time.
