pH-dependent modulation of voltage gating in connexin45 homotypic and connexin45/connexin43 heterotypic gap junctions

Abstract

Intracellular pH (pH(i)) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell-cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pH(i) affects junctional conductance (g(j)) and g(j) dependence on transjunctional voltage (V(j)). To characterize V(j) gating, we fit the g(j)-V(j) relation using a stochastic four-state model containing one V(j)-sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V(j) across it. Using the model, we estimated gating parameters characterizing sensitivity to V(j) and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH(i) changes from 7.2 to approximately 8.0 shifted g(j)-V(j) dependence of Cx45 aHCs along the V(j) axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g(j) dependence on V(j). In contrast, acidification shifted g(j)-V(j) dependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal g(j) achieved under alkaline conditions showed that only approximately 4% of channels were functional. The acid dissociation constant (pK(a)) of g(j)-pH(i) dependence of Cx45/Cx45 GJs was approximately 7. The pK(a) of heterotypic Cx45/Cx43-EGFP GJs was lower, approximately 6.7, between the pK(a)s of Cx45 and Cx43-EGFP (approximately 6.5) homotypic GJs. In summary, pH(i) significantly modulates junctional conductance of Cx45 by affecting both V(j) gating and number of functional channels.

Fig. 1.

pH_i-dependent modulation of g_j in homotypic Cx45 GJs. (A) Junctional conductance (g_j) measurements in a HeLaCx45 cell pair at different pH_is modulated by exposure to CO₂ and NH₄Cl. Repeated V_j ramps of ±10 mV in amplitude and 600 ms in duration (Inset) were used to measure g_j. (B) Normalized g_j–pH_i relations of GJs formed of Cx45 (circles) and of Cx45-EGFP (squares) with BCECF in the pipette to determine pH_i; these relations are superimposable. Triangles show g_j–pH_i dependence for experiments in which the pipette solution was buffered at different pHs and BCECF was omitted; pH_o remained constant (7.4); g_j was normalized to the maximum value at alkaline pH_i. Data for Cx45 (circles) were fit by the Hill equation (solid black curve); pK_a = 6.96 ± 0.03; Hill coefficient n = 1.43 (n = 12). Thinner dashed lines show 95% confidence interval (CI).

Fig. 2.

V_j gating during acidification of homotypic Cx45 GJs. (A) I_j measured in a HeLa Cx45 cell pair in response to repeated V_j ramps (31 s duration) from 0 to −100 mV before and during exposure to 5% CO₂. V_j steps of −10 mV were used to measure g_j between V_j ramps. (B) Experimental g_j–V_j plots (in black) were fit by the S4SM (in gray) assuming that V_j gating was symmetric around V_j = 0. (C) Relation between g_j at V_j = 0 (g_j0; normalized to control/initial conditions) and V_o,H. (black and white circles). (D) Relation between N_F normalized to control/initial conditions and V_o,H (black and white circles). Solid lines in C and D are curves fit from combined data in Fig. 2 and Fig. 3, obtained using a sigmoidal equation, y = a/(1+e^{−(x−xo)/b}) (n = 12). Dashed lines show 95% CI. White circles are from the experiment shown in A. (Ea–c) Probabilities of channels to dwell in O-O, C-O, O-C, and C-C states (P_S) depending on V_j and calculated for the three I_j records obtained during V_j ramps a–c marked with gray rectangles in A; calculated values of V_o,H are indicated.

Fig. 3.

V_j gating during alkalinization of homotypic Cx45 GJs. (A) I_j measured in a HeLaCx45 cell pair in response to repeated V_j ramps (31 s duration) from 0 to −100 mV before and during exposure to NH₄Cl alone or to NH₄Cl combined with alkaline conditions (pH_o = 8 or 8.3). V_j steps of −10 mV were used to measure g_j between V_j ramps. (B) Experimental g_j–V_j plots (in black) obtained at V_j ramps marked with gray rectangles in A were fitted to the S4SM (in gray) assuming that V_j gating was symmetric around V_j = 0. (C) Relation between g_j at V_j = 0 (g_j0; normalized to control/initial conditions) and V_o,H (black and white circles). (D) Relation between N_F normalized to control/initial conditions and V_o,H (black and white circles). Solid lines in C and D are fits to combined data from Fig. 2 and Fig. 3, obtained using a sigmoidal equation, y = a/(1+e^{−(x−xo)/b}) (n = 12). Dashed lines show 95% CI. White circles are from the experiment shown in A. (E) Probabilities of channels to dwell in O-O, C-O, O-C, and C-C states (P_S) depending on V_j and calculated for the three I_j records obtained at V_j ramps a–c marked with gray rectangles in A; calculated values of V_o,H are indicated on each graph.

Fig. 4.

pH_i-dependent modulation of g_j in heterotypic Cx45/Cx43-EGFP GJs. (A) Fluorescence image of a HeLa cell pair forming heterotypic Cx45/Cx43-EGFP GJs are enlarged in Inset. (B) pH_i and g_j measured in a Cx45/Cx43-EGFP cell pair exposed to CO₂ at the indicated percentages and at increasing concentrations of NH₄Cl alone or combined with alkaline MKR solution (pH_o = 8.3) and finally with 3% CO₂. (C) Junctional conductance (g_j)–pH_i dependence of Cx45/Cx43-EGFP GJs (white circles are from the experiment shown in B and gray circles are from other five experiments). The red line shows fit of the Hill equation to the data; pK_a = 6.70 ± 0.03, Hill coefficient n = 1.6 (n = 6). Dashed lines show 95% CI. (D) P_o–pH_i dependence of Cx45 (green) and Cx43-EGFP (blue) homotypic GJs calculated from data shown Fig. 1 and Fig. S4, respectively. Dashed green and blue lines are square roots of P_o–pH_i dependence of Cx45 and Cx43-EGFP homotypic GJs, respectively. (E) P_o–pH_i dependence of Cx45 (green), Cx43-EGFP (blue), and Cx45/Cx43-EGFP (red) GJs. The black line shows the product of P_o,_Cx45–pH_i and P_o,Cx43–pH_i plots shown in D (predicted). (F) Junctional conductance (g_j)–V_j plots (black lines) calculated by applying pairs of V_j ramps (35 s duration) from 0 to +100 and to −100 mV at the numbered times shown on the g_j trace in B. Gray lines show calculated g_j–V_j plots fit by the S4SM to the experimental data. (G) Probabilities of channels being in the O-O state at times of ramps #1, #3, and #6 obtained from fits to g_j–V_j plots with indicated pH_is and numbers corresponding to those shown in F. (H) Relation between g_j normalized to g_j at V_j = 0 under control conditions (g_j0, norm.) and V_{o, Cx45}. (I) Relation between N_F normalized to N_F at pH = 7.2 and V_o,Cx45. Acidic conditions are shown in light pink, and alkaline conditions are shown in light blue relative to pH_i = 7.2. Solid lines in H and I are fits of a sigmoidal equation, y = a/(1+e^{−(x−xo)/b}). Dashed lines show 95% CI.

Fig. 5.

V_j gating during acidification of heterotypic Cx45/Cx43-EGFP GJs. (A) I_j measured in a Cx45/Cx43-EGFP cell pair in response to repeated V_j ramps (25 s duration) from 0 to −100 mV and to +100 mV, and short (500-ms) steps of 20 mV during application of 5% CO₂. (B) Junctional conductance (g_j)–V_j data (black lines), calculated from V_j and I_j records marked with numbers from 1 to 8 shown in A, were fit by the S4SM (in gray). (C) Probability that GJs dwell in the O-O state as a function of V_j; attached numbers correspond to those shown in A and B. (D) Relation between g_j normalized to its value under control conditions at V_j = 0 (g_j0, norm.) and V_o,Cx45; numbers correspond to those shown in A and B. (E) Relation between N_F normalized to N_F at pH = 7.2 and V_o,Cx45. Solid lines in D and E are fit to the data using a linear regression of the second order. Dashed lines show 95% CI.

Fig. 6.

Functional efficiency of HeLaCx45-EGFP GJs. (A) Fluorescence image of a region where two cells overlap and form a JP oriented parallel to the focal plane. The area encircled by the dashed line is the region of interest (ROI) in which fluorescence intensity could be measured without edge effects because of the large size of the JP. (B) Image of a HeLaCx45 cell pair forming a JP enclosed in an ROI. Solid lines indicate cell borders. (C) The relation between N_Fmax estimated from g_j measurements and N_T. Linear regression (solid line) shows that under alkaline conditions N_Fmax was linearly related to N_T with slope of 0.039 ± 0.03. Thus, only ~4% of GJ channels are functional.