Functional interaction between CFTR and Cx45 gap junction channels expressed in oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl(-) current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl(-) current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G(j)). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.

CO(2) sensitivity of voltage gating and gating polarity of gapjunction channels--connexin40 and its COOH-terminus-truncated mutant.

The CO(2) sensitivity of transjunctional voltage ( V(j)) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR). V(j) sensitivity, determined by a standard V(j) protocol (20 mV V(j) steps, 120 mV maximal), decreased significantly with exposure to 30% CO(2). The Boltzmann values of control versus CO(2)-treated oocytes were: V(0) = 36.3 and 48.7 mV, n = 5.4 and 3.7, and G(j min) = 0.21 and 0.31, respectively. CO(2) also affected the kinetics of V(j)-dependent inactivation of junctional current ( I(j)); the time constants of two-term exponential I(j) decay, measured at V(j) = 60 mV, increased significantly with CO(2) application. Similar results were obtained with Cx40-TR, suggesting that CT does not play a role in this phenomenon. The sensitivity of Cx40 channels to 100% CO(2) was also unaffected by CT truncation. There is evidence that CO(2) decreases the V(j) sensitivity of Cx26, Cx50 and Cx37 as well, whereas it increases that of Cx45 and Cx32 channels. Since Cx40, Cx26, Cx50 and Cx37 gate at the positive side of V(j), whereas Cx45 and Cx32 gate at negative V(j), it is likely that V(j) behavior with respect to CO(2)-induced acidification varies depending on gating polarity, possibly involving the function of the postulated V(j) sensor (NH(2)-terminus).

Opposite Cx32 and Cx26 voltage-gating response to CO2 reflects opposite voltage-gating polarity.

Previous studies have shown that the V(j)-dependent gating behavior of gap junction channels is altered by CO(2) exposure. V(j)-dependent channel closure is increased by CO(2) in some connexin channels and decreased in others. Since the former type of channels gate on the relatively negative side by V(j) (negative gaters) and the latter at the positive side (positive gaters), it has been hypothesized that gating polarity determines the way CO(2) affects V(j) closure. To test this hypothesis, we have studied the CO(2)-mediated changes in V(j) gating in channels made of Cx32, Cx26, or a Cx32 mutant (Cx32-N2D) in which asparagine (N) at position 2 was replaced with aspartate (D). With exposure to CO(2), Cx32 channels (negative gaters) show increased V(j)-dependent closure, whereas Cx26 channels (positive gaters) respond in the opposite way to V(j). Additionally, Cx32-N2D channels (positive gaters) show decreased V(j) closure with exposure to CO(2). The reciprocal Cx26 mutant, Cx26-D2N (negative gater), could not be tested because it did not express functional homotypic channels. The data support the hypothesis that polarity of fast V(j) gating determines whether CO(2) increases or decreases the V(j) dependent closure of gap junction channels.

Is the voltage gate of connexins CO2-sensitive? Cx45 channels and inhibition of calmodulin expression.

The sensitivity of Cx45 channels to CO2, transjunctional voltage ( V(j)) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage clamp. Cx45 channels are very sensitive to V(j) and close with V(j) preferentially by the slow gate, likely to be the same as the chemical gate. With a CO2-induced drop in junctional conductance ( G(j)), both the speed of V(j)-dependent inactivation of junctional current ( I(j)) and V(j) sensitivity increased. With 40-mV V(j)-pulses, the tau of single exponential I(j) decay reversibly decreased by;40% during CO2 application, and G(j steady state)/G(j peak) decreased multiphasically, indicating that both kinetics and V(j) sensitivity of chemical/slow V(j) gating are altered by changes in [H(+)](i) and/or [Ca(2+)](i). CaM expression was inhibited with oligonucleotides antisense to CaM mRNA. With 15 min CO2, relative junctional conductance ( G(jt)/ G(jt0)) dropped to 0% in controls, but only by;17% in CaM-antisense oocytes. Similarly, V(j) sensitivity was significantly lessened in CaM-antisense oocytes. The data indicate that both the speed and sensitivity of V(j)-dependent inactivation of the junctional current of Cx45 channels are affected by CO2 application, and that CaM plays a key role in channel gating.

Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating.

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).

Slow gating of gap junction channels and calmodulin.

Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V(j)) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G(j)) slowly increased by severalfold or decreases to nearly zero with V(j) positive or negative, respectively, at mutant side, and V(j) positive at mutant side reversed CO(2)-induced uncoupling. This suggested that the CO(2)-sensitive gate might be a V(j)-sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V(j) and CO(2) sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating.

Calmodulin directly gates gap junction channels.

Cytosolic changes control gap junction channel gating via poorly understood mechanisms. In the past two decades calmodulin participation in gating has been suggested, but compelling evidence for it has been lacking. Here we show that calmodulin indeed is associated with gap junctions and plays a direct role in chemical gating. Expression of a calmodulin mutant with the N-terminal EF hand pair replaced by a copy of the C-terminal pair dramatically increases the chemical gating sensitivity of gap junction channels composed of connexin 32 and decreases their sensitivity to transjunctional voltage. The increased chemical gating sensitivity, most likely because of the higher overall Ca(2+) binding affinity of this mutant as compared with native calmodulin, and the decreased voltage sensitivity are only observed when the mutant is expressed before connexin 32. This indicates that the mutant, and by extension native calmodulin, must interact with connexin 32 before gap junctions are formed. Immunofluorescence data suggest further that this interaction leads to incorporation of native or mutant calmodulin into the connexon as an integral regulatory subunit.

Chemical gating of gap junction channels.

Chemical gating of gap junction channels is a complex phenomenon that may involve intra- and intermolecular interactions among connexin domains and a cytosolic molecule (calmodulin?) that may function as channel plug. This article focuses on the methodology we have employed for studying the molecular basis of chemical gating by lowered cytosolic pH. Our approach has combined molecular genetics and biophysics, using exposure to 100% CO(2) for assaying chemical gating efficiency. Chimeras of connexin 32 (Cx32) and connexin 38 (Cx38) and Cx32 mutants modified at residues of the cytoplasmic loop, the initial C-terminus domain, or both have been expressed in Xenopus oocytes, and channel expression and gating have been tested electrophysiologically by double voltage clamp. In addition, various channel forms, including homotypic, heterotypic, and heteromeric channel combinations, have been evaluated for chemical gating sensitivity.

Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels.

Connexin channels are gated by transjunctional voltage (Vj) or CO2 via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT1) of connexin32 (Cx32) were shown to determine CO2 sensitivity, and a gating mechanism involving CL-CT1 association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT1 interactions, display atypical Vj and CO2 sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH2-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT1 arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to Vj polarity, the existence of a "slow gate" distinct from the conventional Vj gate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO2 sensitive than homotypic Cx32 channels, and CO2-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting Vj sensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.

Molecular dissection of a basic COOH-terminal domain of Cx32 that inhibits gap junction gating sensitivity.

Connexin32 (Cx32) mutants were studied by double voltage clamp in Xenopus oocytes to determine the role of basic COOH-terminal residues in gap junction channel gating by CO2 and transjunctional voltage. Replacement of five arginines with N (5R/N) or T residues in the initial COOH-terminal domain (CT1) of Cx32 enhanced CO2 sensitivity. The positive charge, rather than the R residue per se, is responsible for the inhibitory role of CT1, because mutants replacing the five R residues with K (5R/K) or H (5R/H) displayed CO2 sensitivity comparable to that of wild-type Cx32. Mutants replacing R with N residues four at a time (4R/N) showed that CO2 sensitivity is strongly inhibited by R215 and mildly by R219, whereas R220, R223, and R224 may slightly increase sensitivity. Neither the 5R/N nor the 4R/N mutants differed in voltage sensitivity from wild-type Cx32. The possibility that inhibition of gating sensitivity results from electrostatic interactions between CT1 and the cytoplasmic loop is discussed as part of a model that envisions the cytoplasmic loop of Cx32 as a key element of chemical gating.

Chemical gating of heteromeric and heterotypic gap junction channels.

Gap junction channels contain two hemichannels (connexons), each being a connexin (Cx) hexamer. In cells expressing multiple connexins, heteromeric connexons are believed to form, whereas cell pairs expressing different connexins generate heterotypic channels. To define gating behavior of heteromeric and heterotypic channels, CO2-induced gating was tested in Xenopus oocyte pairs expressing Cx32, or 5R/N (Cx32 mutant), as well as in pairs in which one oocyte (mx) expressed a 50/50 mixture of Cx32 and 5R/N and the other either the mixture (mx), Cx32 (32) or 5R/n (R/N). In 5R/N, replacement of 5 C-terminus arginines with asparagines greatly increased CO2 sensitivity. In response to 3 and 15 min CO2 exposures, junctional conductance (Gj) decreased to 85% and 47%, in 32-32 pairs, and to 7% and 0.9%, in R/N-R/N pairs, respectively. In mx-mx and mix-32 pairs, Gj decreased to similar values (33% and 35%, respectively) with 15 min CO2. The sensitivity of mx-R/N pairs was similar to that of heterotypic 32-R/N pairs, as Gj dropped to 36% and 38%, respectively, with 3 min CO2. Monoheteromeric (mx-32 and mx-R/N) and biheteromeric (mx-mx) channels behaved as if Cx32 were dominant, suggesting that hemichannel sensitivity is not an average of the sensitivities of its connexin monomers. In contrast, heterotypic channels behaved as if the two hemichannels of a cell-cell channel had no influence on each other.

Positive charges of the initial C-terminus domain of Cx32 inhibit gap junction gating sensitivity to CO2.

Gap junction channels close with CO2 exposure. To determine whether the carboxy-terminus (CT) of connexin32 (Cx32) participates in gating, the CO2 sensitivity of channels made of Cx32 or Cx32 mutants was studied by double voltage clamp. In Xanopus laevis oocytes expressing Cx32, junctional conductance (Gj) dropped to 85% and 47% of controls with 3- and 15-min CO2 exposures, respectively. In response to the 15-min exposure to CO2, pHi dropped to approximately 6.4 in 5-7 min and did not decrease further, even with 30-min exposures. CT deletion by 84% did not affect CO2 sensitivity, but replacement of five arginines (R215, R219, R220, R223, and R224) with asparagines (N) or threonines at the beginning of CT (CT1) in Cx32 or Cx32 deleted beyond residue 225 greatly enhanced CO2 sensitivity (with 3-min CO2 Gj dropped to approximately 8%). Partial R/N replacement resulted in intermediate CO2 sensitivity enhancement. R215 is a stronger inhibitor than R219-220, whereas R223-224 may diminish the inhibitory efficiency of R215 and R219-220. Therefore, positive charges of CT1 reduce the CO2 sensitivity of Cx32, whereas the rest (> 80%) of CT seems to play no role in CO2-induced gating. The role of presumed electrostatic interactions among Cx32 domains in CO2-induced gating is discussed.

Connexin domains relevant to the chemical gating of gap junction channels.

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both amino- and carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2) of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R) with asparagines (N) at the beginning of CT (C1) greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL (IL1) and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively.

Two distinct gating mechanisms in gap junction channels: CO2-sensitive and voltage-sensitive.

The chemical gating of single-gap junction channels was studied by the dual whole-cell voltage-clamp method in HeLa cells transfected with connexin43 (HeLa43) and in fibroblasts from sciatic nerves. Junctional current (Ij), single-channel conductance, and Ij kinetics were studied in cell pairs during CO2 uncoupling and recoupling at small transjunctional voltages (Vj < 35 mV: Vj gating absent) and at high Vj (Vj > 40 mV: Vj gating strongly activated). In the absence of Vj gating, CO2 exclusively caused Ij slow transitions from open to closed channel states (mean transition time: approximately 10 ms), corresponding to a single-channel conductance of approximately 120 pS. At Vj > 40 mV, Vj gating induced fast Ij flickering between open, gamma j(main state), and residual, gamma j(residual), states (transition time: approximately 2 ms). The ratio gamma j(main state)/gamma j(residual) was approximately 4-5. No obvious correlation between Ij fast flickering and CO2 treatment was noticed. At high Vj, in addition to slow Ij transitions between open and closed states, CO2 induced slow transitions between residual and closed states. During recoupling, each channel reopened by a slow transition (mean transition time: approximately 10 ms) from closed to open state (rarely from closed to residual state). Fast Ij flickering between open and residual states followed. The data are in agreement with the hypothesis that gap junction channels possess two gating mechanisms, and indicate that CO2 induces channel gating exclusively by the slow gating mechanism.

Connexin 32/38 chimeras suggest a role for the second half of inner loop in gap junction gating by low pH.

Gap junction channels are regulated by gates that close with cytosolic acidification and transjunctional voltage (Vj). For identifying the connexin (Cx) domain(s) involved in channel gating, CO2 and Vj sensitivities of channels made of Cx38, Cx32, Cx32/Cx38 chimeras, and Cx32 mutants were studied in Xenopus oocyte pairs. Recently, we have reported that Cx38 is more sensitive to CO2 and Vj than Cx32 because of differences in the Cx inner loop. To identify the responsible inner loop domain, chimeras of Cx32/Cx38 in which the first (I1) or the second (I2) half of the inner loop of Cx38 replaced that of Cx32 and I2 mutants of Cx32 were tested. The chimera Cx32/Cx38I2 (Cx32 with I2 of Cx38) was like Cx38 in CO2 sensitivity but like Cx32 in Vj sensitivity. Cx32/Cx38I1 (Cx32 with I1 of Cx38) did not express channels. Of the three Cx32 mutants, Cx32-VH/IR VH of Cx32 replaced with IR of Cx38) and Cx32-WW/MC WW of Cx32 replaced with MC of Cx38) were like Cx32 in both CO2 and Vj sensitivity, whereas Cx32-S*T/Q*P (S*T of Cx32 replaced with Q*P of Cx38) was closer to Cx38 in CO2 sensitivity but behaved like Cx32 in Vj gating. The data suggest that I1 and I2 contain domains relevant for Vj and CO2 gating, respectively.

Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7-hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.

Chimeric evidence for a role of the connexin cytoplasmic loop in gap junction channel gating.

Gap junction channels are regulated by gates that close upon exposure to 100% CO2, probably via an increase in intracellular Ca2+ concentration, [Ca2+]i. For defining connexin (Cx) domain(s) involved in gating, we have studied chemical and voltage gating sensitivities of channels made of Cx38, Cx32 or chimeras of the above, expressed in Xenopus oocytes. Cx38 channels are more sensitive to CO2 and voltage than those of Cx32. A 3-min exposure to 100% CO2 reduces Cx38 junctional conductance (Gj) to 0% of initial values at a maximum rate of 25%/min, whereas even a 15-min exposure to 100% CO2 reduces Cx32 Gj by approximately 50% at the slow rate of 9%/min. Of the various Cx32 mutants and Cx32/38 chimeras constructed, two chimeras (Cx32/38I and Cx32/38N) expressed functional channels. Upon exposure to CO2, channels made of Cx32/38I (Cx32 inner loop replaced with that of Cx38) reproduced precisely the uncoupling behavior of Cx38 channels in uncoupling magnitude and in both uncoupling and recoupling rates, whereas channels made of Cx32/38N (N-terminus replaced) behaved closer to Cx32 than to Cx38 channels. Cx38 channels were more voltage sensitive than those of Cx32, with V0, i.e., the transjunctional voltage at which voltage-sensitive conductance is half maximal = 35.3 and 59.5 mV, and n, i.e., equivalent gating charge = 3.3 and 2.1, respectively. Of the two chimeras, Cx32/38I channels were similar to Cx38 channels, with V0 = 40.6 mV, Gj min, i.e., the theoretical minimal normalized junctional conductance = 0.35 and n = 3.0, whereas Cx32/38 N channels displayed very low voltage sensitivity, with V0 = 84.8 mV, Gj min = 0.5 and n = 1.1. The data suggest that the inner loop plays a major role in pH and voltage gating sensitivity, but whether other domains also participate in the gating mechanism cannot be excluded.

Inhibition of calmodulin expression prevents low-pH-induced gap junction uncoupling in Xenopus oocytes.

The relationship among intracellular pH (pHi), -log10 intracellular Ca2+ concentration (pCai) and gap junctional conductance, the participation of Ca2+ stores, and the role of calmodulin in channel regulation have been studied in Xenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO2. The time courses of pHi [measured with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorscein (BCECF)], pCai (measured with the membrane-associated fura-C18) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with pCai than with pHi. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on CO2-induced uncoupling, coupled to negative results with Ca2+-free external solutions, point to a low-pHi -induced Ca2+ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO2 within 72 h. The effect of CO2 on junctional conductance was reduced by approximately 60% in 24 h, by approximately 76% in 48 h and by approximately 93% in 72 h. Oocytes that had lost gating sensitivity to CO2 partially recovered gating competency following calmodulin injection. The data suggest that lowered pHi uncouples gap junctions by a Ca2+- calmodulin-mediated mechanism.

Ca-mediated and independent effects of arachidonic acid on gap junctions and Ca-independent effects of oleic acid and halothane.

In Novikoff hepatoma cell pairs studied by double perforated patch clamp (DPPC), brief (20 s) exposure to 20 microM arachidonic acid (AA) induced a rapid and reversible uncoupling. In pairs studied by double whole-cell clamp (DWCC), uncoupling was completely prevented by effective buffering of Cai2+ with BAPTA. Similarly, AA (20 s) had no effect on coupling in cells perfused with solutions containing no added Ca2+ (SES-no-Ca) and studied by DPPC, suggesting that Ca2+ influx plays an important role. Parallel experiments monitoring [Ca2+]i with fura-2 showed that [Ca2+]i increases with AA to 0.7-1.5 microM in normal [Ca2+]o, and to approximately 400 nM in SES-no-Ca solutions. The rate of [Ca2+]i increase matched that of Gj decrease, but [Ca2+]i recovery was faster. In cells studied by DWCC with 2 mM BAPTA in the pipette solution and superfused with SES-no-Ca, long exposure (1 min) to 20 microM AA caused a slow and virtually irreversible uncoupling. This result suggests that AA has a dual mechanism of uncoupling: one dominant, fast, reversible, and Ca(2+)-dependent, the other slow, poorly reversible, and Ca(2+)-independent. In contrast, uncoupling by oleic acid (OA) or halothane was insensitive to internal buffering with BAPTA, suggesting a Ca(2+)-independent mechanism only.

Gap junction gating sensitivity to physiological internal calcium regardless of pH in Novikoff hepatoma cells.

Gap junction conductance (Gj) and channel gating sensitivity to voltage, Ca2+, H+, and heptanol were studied by double whole-cell clamp in Novikoff hepatoma cell pairs. Channel gating was observed at transjunctional voltages (Vj) > +/- 50 mV. The cells readily uncoupled with 1 mM 1-heptanol. With heptanol, single (gap junctional) channel events with unitary conductances (gamma j) of 46 and 97 pS were detected. Both Ca(2+)-loading (EGTA.Ca) and acidifying (100% CO2) solutions caused uncoupling. However, CO2 was effective when Ca2+i was buffered with EGTA (a H(+)-sensitive Ca-buffer) but not with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (a H(+)-insensitive Ca-buffer), suggesting a Ca(2+)-mediated H+ effect on gap junctions. This was tested by monitoring the Gj decay at different pCai values (9, 6.9, 6.3, 6, and 5.5; 1 mM BAPTA) and pHi values (7.2 or 6.1, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-(N-morpholino)ethansulphonic acid, respectively). With pCai > or = 6.9 (pH 7.2 or 6.1), Gj decreased to 10-70% of initial values in approximately 40 min, following single exponential decays (tau = approximately 28 min). With pCai 6-6.3 (pH 7.2 or 6.1), Gj decreased to 10-25% of initial values in 15 min (tau = approximately 5 min); the Student t gave a P = 0.0178. With pCa 5.5 the cells uncoupled in less than 1 min (tau = approximately 20 s). Low pHi affected neither time course nor shape of Gj decay at any pCai tested. The data indicate that these gap junctions are sensitive to [Ca2+]i in the physiological range (< or = 500 nM) and that low pHi, without an increase in [Ca2+]i, neither decreases Gj nor increases channel sensitivity to Ca2+.