Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl⁻ channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G _j) and its sensitivity to transjunctional voltage (V _j) by the dual voltage-clamp method. Application of forskolin induced a Cl⁻ current; increased G _j approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V _j gating, monitored by a change in the ratio between G _j steady state and G _j peak (G _jSS/G _jPK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G _j and decreased the sensitivity to V _j gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G _j and V _j changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.     

Interplay between cystic fibrosis transmembrane regulator and gap junction channels made of connexins 45, 40, 32 and 50 expressed in oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl(-) channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G (j)) and its sensitivity to transjunctional voltage (V (j)) by the dual voltage-clamp method. Application of forskolin induced a Cl(-) current; increased G (j) approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V (j ) gating, monitored by a change in the ratio between G (j) steady state and G (j) peak (G (j)SS/G (j)PK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G (j) and decreased the sensitivity to V (j) gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G (j) and V (j) changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.     

Functional Characterization of Canine Connexin45

Three gap junctional proteins have been identified in canine ventricular myocytes: connexin 43 (Cx43), connexin 45 (Cx45), and connexin 40 (Cx40). We have characterized the functional properties of canine Cx45 and examined how Cx45 functionally interacts with Cx43 in Xenopus oocyte pairs. Homotypic pairs expressing Cx45 were well coupled. Heterotypic pairs composed of Cx45 paired with either Cx43 or Cx38 also developed high levels of conductance. Junctional currents in the heterotypic pairs displayed a highly asymmetrical voltage dependence. The kinetics and steady-state voltage dependence of the heterotypic channels more closely resembled those of the Cx45 channels when the Cx45 cRNA-injected cell was relatively negative suggesting that the Cx45 connexon closes for relative negativity at the cytoplasmic end of the channel. We also show that homotypic and heterotypic channels composed of Cx45 and Cx43 exhibit differences in pH _i sensitivity. Received: 18 August 1995/Revised: 21 November 1995     

Control of Epithelial Ion Transport by Cl<Superscript>−</Superscript> and PDZ Proteins

Inhibition of epithelial Na⁺ channels (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated previously. Recent studies suggested a role of cytosolic Cl⁻ for the interaction of CFTR with ENaC, when studied in Xenopus oocytes. In the present study we demonstrate that the Na⁺/H⁺-exchanger regulator factor (NHERF) controls expression of CFTR in mouse collecting duct cells. Inhibition of NHERF largely attenuates CFTR expression, which is paralleled by enhanced Ca²⁺-dependent Cl⁻ secretion and augmented Na⁺ absorption by the ENaC. It is further demonstrated that epithelial Na⁺ absorption and ENaC are inhibited by cytosolic Cl⁻ and that stimulation by secretagogues enhances the intracellular Cl⁻ concentration. Thus, the data provide a clue to the question, how epithelial cells can operate as both absorptive and secretory units: Increase in intracellular Cl⁻ during activation of secretion will inhibit ENaC and switch epithelial transport from salt absorption to Cl⁻ secretion.This revised version was published online in August 2005 with a corrected cover date.     

Is the Voltage Gate of Connexins CO<Subscript>2</Subscript>-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 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 [Ca²⁺]_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.     

The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration

Activation of the CFTR Cl^– channel inhibits epithelial Na⁺ channels (ENaC), according to studies on epithelial cells and overexpressing recombinant cells. Here we demonstrate that ENaC is inhibited during stimulation of the cystic fibrosis transmembrance conductance regulator (CFTR) in Xenopus oocytes, independent of the experimental set-up and the magnitude of the whole-cell current. Inhibition of ENaC is augmented at higher CFTR Cl^– currents. Similar to CFTR, ClC-0 Cl^– currents also inhibit ENaC, as well as high extracellular Na⁺ and Cl^– in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and seems to be mediated by Cl^–.     

CO<Subscript>2</Subscript> Sensitivity of Voltage Gating and Gating Polarity of GapJunction Channels—Connexin40 and its COOH-Terminus-Truncated Mutant

The CO₂ 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% CO₂. The Boltzmann values of control versus CO₂-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. CO₂ 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 CO₂ 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% CO₂ was also unaffected by CT truncation. There is evidence that CO₂ 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 CO₂-induced acidification varies depending on gating polarity, possibly involving the function of the postulated V _j sensor (NH₂-terminus).This revised version was published online in June 2005 with a corrected cover date.     

Chloride channel blockers activate an endogenous cationic current in oocytes of<Emphasis Type="Italic"> Bufo arenarum</Emphasis>

A two-electrode, voltage-clamp technique was used to measure the effect of the Cl^– channel blockers, 9-anthracene carboxylic acid and niflumic acid, upon the ionic currents of oocytes of the South American toad Bufo arenarum. The main results were: (1) both blockers produced a reversible increase of the outward currents on a dose-dependent manner; (2) the activated outward current was voltage dependent; (3) the 9-anthracene carboxylic acid-sensitive current was blocked with barium; and (4) the effect of 9-anthracene carboxylic acid was more pronounced in a zero-K⁺ solution than in standard (2 mmol l^–1) or high (20 mmol l^–1) K⁺ solutions, indicating that a K⁺ conductance is activated. The effect of the Cl^– channel blockers could be due to a direct interaction with endogenous cationic channels. Another possible explanation is that Cl^– that enter the cell during depolarizing steps in control solution inhibit this cationic conductance; thus, the blockade of Cl^– channels by 9-anthracene carboxylic acid and niflumic acid would remove this inhibition, allowing the cationic current to flow freely.Abbreviations 9-AC 9-anthracene carboxylic acid - E_r reversal potential - NA niflumic acid - NSC non-selective cation channel     

Simultaneous functional expression of swelling and forskolin-activated chloride currents in primary cultures of rabbit distal convoluted tubule

Ionic Cl⁻ currents induced by cell swelling and forskolin were studied in primary cultures of rabbit distal convoluted tubule (DCTb) by the whole-cell patch clamp technique. We identified a Cl⁻ conductance activated by cell swelling with an hyperosmotic pipette solution. The initial current exhibited an outwardly rectifying I-V relationship, whereas steady state current showed strong decay at depolarized membrane potentials. The ion selectivity was I⁻ > Br⁻ > Cl⁻ > > glutamate. The forskolin-activated Cl⁻ conductance demonstrated a linear I-V relationship and its ion selectivity was Br⁻ > Cl⁻ > I⁻ > glutamate. This last conductance could be related to the CFTR (cystic fibrosis transmembrane conductance regulator) previously identified in these cells. NPPB inhibited both Cl⁻ currents, and DIDS inhibited only the swelling-activated Cl⁻ current. Forskolin had no effect on the activation of the swelling-activated Cl⁻ current. In DCTb cells which exhibited swelling-activated Cl⁻ currents subsequently inhibited by DIDS, forskolin could activate CFTR related Cl⁻ currents. In the continuous presence of I⁻ which inhibited CFTR conductance, forskolin did not modify the swelling-activated current. The results suggest that both Cl⁻ conductances could be co-expressed in the same DCTb cell and that CFTR did not modulate the swelling-activated conductance.     

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl^– channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately –15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl^–. Readdition of Ca²⁺ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca²⁺ inactivated Cl^– channel (CaIC). The inhibitory potency of Ca²⁺ was a function of the external Ca²⁺ concentration with a half maximal blocker concentration of about 20 m.These channels were inhibited by the Cl^– channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4-acetamido-4-isothiocyanatostilbene-2,2-disulfonicacid (SITS), another Cl^– channel blocker, led to activation of this Cl^– channel. Like other Cl^– channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl^– current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl^– channels in Xenopus oocytes which share several features with Cl^– channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl^– channels.The microelectrode measurements are part of the PhD thesis of K. Liebold; the patch clamp contributions are part of the PhD thesis of F.W. Reifarth. This study was supported by the Deutsche Forschungsgemeinschaft (We1858/2-l) and by Sonderforschungsbereich 249.     

The Voltage Gates of Connexin Channels are Sensitive to CO2

Cx45 channel sensitivity 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_jand close preferentially by the slow gate, likely the same as the chemical gate. With CO₂-induced drop in junctional conductance (G_j), the speed of V_j-dependent inactivation of junctional current (I_j) and V_jsensitivity increased. With 40 mV V_j, the τ of single exponential I_jdecay reversibly decreased by ～40% with CO₂, and G_{j steady state}/G_{j peak}decreased multiphasically, indicating that kinetics and V_jsensitivity of chemical/slow-V_jgating are altered by changes in [H⁺]_iand/or [Ca²⁺]_i. With 15 min exposure to CO₂, G_jdropped to 0% in controls and by ～17% following CaM expression inhibition; similarly, V_jsensitivity decreased significantly. This indicates that the speed and sensitivity of V_j-dependent inactivation of Cx45 channels are increased by CO₂, and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both G_{j steady state}/G_{j peak}and τ with CO₂matched more closely that of G_{j peak}. In contrast, sensitivity and speed of V_jgating of Cx40 and Cx26 channels decreased, rather than increased, with CO₂application.     

Chloride conductance and mitochondria-rich cell density in isolated skin of Rana catesbeiana acclimated to various environments

The Cl⁻ conductance in isolated skin of frogs (Rana catesbeiana) acclimated to 30 mM solutions of NaCl, Na₂SO₄, MgCl₂ and distilled water (DW) was studied. Transepithelial potential difference (PD_trans), short-circuit current (I_SC) and total conductance (G_t) were measured under conditions such that there was Cl⁻ flux in the presence and absence of Na⁺ transport. The Cl⁻ content of the mucosal solution was acutely replaced with SO₄²⁻ or gluconate to evaluate the effect of removal of Cl⁻ conductance on electrophysiological parameters. Mitochondria-rich cell density (D_MRC) was also measured. Skins from frogs acclimated to NaCl and Na₂SO₄ showed the lowest and the highest D_MRC, respectively, but no difference could be found between the skins from frogs acclimated to DW and MgCl₂ indicating that D_MRC is not unconditionally dependent on environmental Cl⁻ in this species. Frogs acclimated to NaCl showed marked differences when compared to the other groups: the highest G_t, probably represented by a higher paracellular conductance; the lowest transepithelial electrical potential difference which remained invariant after replacement of mucosal Cl⁻ with SO₄²⁻ or replacement of mucosal Cl⁻ with gluconate and an inwardly oriented positive current in the absence of bilateral Na⁺.     

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

Gap junction channels formed by connexin 50 (Cx50) are critical for maintenance of lens transparency. Because the C-terminus of Cx50 can be cleaved post-translationally, we hypothesized that channels formed by the truncated Cx50 exhibit altered properties or regulation. We used the dual whole-cell patch-clamp technique to investigate the macroscopic and single-channel properties of gap junctional channels formed by wild-type human Cx50 and a truncation mutant (Cx50A294stop) after transfection of N2A cells. Our results show that wild-type Cx50 formed functional gap junctional channels. The macroscopic Gjss-Vj relationship was well described by a Boltzmann equation with A of 0.10, V0 of 43.8 mV and Gjmin of 0.23. The single-channel conductance was 212 +/- 5 pS. Multiple long-lasting substates were observed with conductances ranging between 31 and 80 pS. Wild-type Cx50 gap junctional channels were reversibly blocked when pHi was reduced to 6.3. Truncating the C-terminus at amino acid 294 caused a loss of pHi sensitivity, but there were no significant changes in single-channel current amplitude or Gjss-Vj relationship. These results suggest that the C-terminus of human Cx50 is involved in pHi sensitivity, but has little influence over single-channel conductance, voltage dependence, or gating kinetics.     

CFTR mediated chloride secretion in the avian renal proximal tubule

In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl⁻-dependent short circuit current (I_SC) response, consistent with net transepithelial Cl⁻ secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl⁻ secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I_SC responses in filter grown cultured monolayers. Bumetanide (10 μM), a specific blocker of NKCC, inhibited forskolin activated I_SC by about 40%, suggesting that basolateral uptake of Cl⁻ is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl⁻ conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl⁻ gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl⁻ current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl⁻ channel to mediate cAMP-activated Cl⁻ secretion.     

The voltage gates of connexin channels are sensitive to CO(2)

Cx45 channel sensitivity to CO(2), 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 preferentially by the slow gate, likely the same as the chemical gate. With CO(2)-induced drop in junctional conductance (G(j)), the speed of V(j)-dependent inactivation of junctional current (I(j)) and V(j) sensitivity increased. With 40 mV V(j), the tau of single exponential I(j) decay reversibly decreased by approximately 40% with CO(2), and G(j steady state)/G(j peak) decreased multiphasically, indicating that kinetics and V(j) sensitivity of chemical/slow-V(j) gating are altered by changes in [H(+)](i) and/or [Ca(2+)](i). With 15 min exposure to CO(2), G(j) dropped to 0% in controls and by approximately 17% following CaM expression inhibition; similarly, V(j) sensitivity decreased significantly. This indicates that the speed and sensitivity of V(j)-dependent inactivation of Cx45 channels are increased by CO(2), and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both G(j steady state)/G(j peak) and tau with CO(2) matched more closely that of G(j peak). In contrast, sensitivity and speed of V(j) gating of Cx40 and Cx26 channels decreased, rather than increased, with CO(2) application.     

Altered conductance and permeability of Cx40 mutations associated with atrial fibrillation

Gap junctions ensure the rapid propagation of the action potential throughout the myocardium. Three mutant forms of connexin40 (Cx40; A96S, M163V, and G38D), the primary component of the atrial gap junction channel, are associated with atrial fibrillation and retain the ability to form functional channels. We determined the biophysical properties of these mutant gap junctions in transiently transfected HeLa and N2A cells. All three mutants showed macroscopic junctional conductances over the range of 0.5 to 40 nS, and voltage dependences comparable to those of wild-type (WT) Cx40. However, the unitary conductance of G38D channels was ∼1.6-fold higher than that of WT Cx40 channels (∼220 vs. ∼135 pS), whereas the unitary conductances of the A96S and M163V mutants were similar to that of WT Cx40. Furthermore, the M163V and G38D channels exhibited approximately two- and approximately fivefold higher permeability to the anionic dye Lucifer yellow (LY) relative to K⁺ (LY/K⁺) compared with that of WT Cx40, whereas A96S LY transfer was similar to that of WT (G38D > M163V > A96S ≈ Cx40WT). In contrast, G38D channels were almost impermeable to cationic ethidium bromide (EtBr), suggesting that G38D alters channel selectivity. Conversely, A96S and M163V channels showed enhanced EtBr permeability relative to WT Cx40, with the following permeability order: M163V > A96S > Cx40WT > G38D. Altered conductive and permeability properties of mutant channels suggest an essential role for Cx40-mediated biochemical and electrical coupling in cardiac tissues. The altered properties of the three single-base substitution mutants may play a role in mechanisms of reentry arrhythmias.     

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

The effect of CO₂-induced acidification on transjunctional voltage (V_j) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO₂ application greatly decreased the V_j sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO₂ also affected the kinetics of V_j dependent inactivation of junctional current (I_j), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO₂ sensitivity of chemical gating such that even CO₂ concentrations as low as 2.5% significantly lowered junctional conductance (G_j). With Cx50 channels G_j dropped by 78% with a drop in intracellular pH (pH_i) to 6.83, whereas with Cx50-D3N channels G_j dropped by 95% with a drop in pH_i to just 7.19. We have previously hypothesized that the way in which V_j gating reacts to CO₂ might be related to connexin’s gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO₂ on V_j gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes     

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.     

Evidence for a simple Cl conductance pathway in nutrient membrane of frog stomach

A decrease in nutrient Cl⁻ results in an increased negativity of the nutrient relative to the secretory side. The possibility emerged that Cl⁻ transport could be attributed to a neutral mechanism involving Cl⁻ in the nutrient membrane coupled to a simple Cl⁻ conductance pathway in the secretory membrane. The decrease in PD (potential difference) with a decrease in nutrient Cl⁻ could arise from a decrease in cellular Cl⁻ so that the ratio of Cl⁻ in cell to Cl⁻ in secretory solution was decreased. Experiments were designed to determine whether there was a need to assume a simple Cl⁻ conductance pathway. A 10-fold decrease in Cl⁻ gave in HCO₃⁻-containing nutrient solutions a PD decrease of 20 mV, in HCO₃⁻-free nutrient solutions, a PD decrease of 13.5 mV, and in HCO₃⁻-free and Na⁺-free solutions, a PD decrease of 6.7 mV. The decrease of 6.7 mV could not be attributed to a neutral Cl⁻HCO₃⁻ exchanger or a NaCl symport. Also there was no evidence for a KCl symport from changes in Cl⁻ in presence and absence of K⁺. It followed that the decrease of 6.7 mV provided evidence for a simple Cl⁻ conductance pathway in the nutrient membrane.     

Inhibition of epithelial Na currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated Cl⁻ conductance in parallel with an enhanced amiloride sensitive Na⁺ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na⁺ currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-1-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTR-dependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na⁺ transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.