Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina

Abstract

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to approximately 400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is approximately 90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH(i) from 7.2 to 7.8 increased gap junctional conductance (g(j)) of approximately 100-fold with pK(a) = 7.41. We show that this g(j) increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca(2+)](o) = approximately 0.3 mm or below, demonstrating that a moderate reduction of [Ca(2+)](o) can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH(i) with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina.

Figure 7. Permeability of Cx57-EGFP gap junction channels to dyes

A, representative images illustrating LY (a), AF³⁵⁰ (b), EtBr (c) and DAPI (d) but not PrI (e) permeate GJ channels. Images were recorded ∼10 min after opening the patch in cell-1 with pipette filled with dye (indicated by two asterisks). Dye acceptor cell is shown by one asterisk. B, Cx57-EGFP single channel permeability to AF³⁵⁰. Two consecutive applications of NH₄Cl (10 mm) were used to increase g_j. The grey bar shows CO₂ application used to measure P_p that characterizes AF³⁵⁰ permeability from cell-2 (dye recipient cell) to patch pipette-2. Repeated V_j ramps of ±12 mV were used to measure I_j (see the inset). The patch to cell-1 was open at the beginning of the recording. The vertical arrow shows the moment when the patch was open in cell-2 to measure I_j. FI₁ and FI₂ traces show dynamics of fluorescence intensities in cell-1 and cell-2, respectively. P_γ trace shows the single channel permeability.

Figure 1. Representative images showing junctional plaques in cell pairs expressing Cx57-EGFP

A–B, fluorescence images of cell pairs with small (A) and large (B) JPs (see arrows). C, fluorescence image of the region where cells overlap and form large JP oriented parallel to the focal plane. Areas encircled with dashed lines are regions of interest (ROI) where fluorescence intensity was measured.

Figure 2. Voltage gating of Cx57-EGFP gap junction channels

A–B, g_j–V_j dependence (B) measured in Cx57-EGFP cell pair by applying 60 s long V_j ramps (A). The fitting curve shown in grey (B) was obtained by using the model of contingent gating. C, g_h–V_j dependencies of left- and right-side AHCs (grey and black lines, respectively) integrating conductances of open and gated AHCs. D, probability of channels to dwell in different states (O–O, C–O, O–C and C–C) depending on V_j. Black line shows P_o-o, while grey lines show P_c-o, P_o-c, and P_c-c. E, an example of V_j-gating of Cx57-EGFP cell pair. The bottom trace shows dynamics of g_j calculated from V_j and I_j traces. g_j decayed in response to V_j step of +75 mV. Measurements of g_j during repeated V_j ramps (±15 mV) show that g_j recovery after V_j step has two distinct phases: fast and slow. Dashed lines in the inset show the fitting curves to a single exponential function of the data in periods corresponding to the fast and slow phases of g_j recovery. Time constants of the fast and the slow phases were ∼0.3 and 2.6 s, respectively.

Figure 3. Unitary gating events in HeLaCx57-EGFP cell pairs

A, an example of I_j record during V_j step of −95 mV in a cell pair with low coupling at control conditions (∼0.1 nS). Most gating transitions are between open (γ_o) and residual states (γ_res). The inset shows calculated g_j for the time window between 64 and 78 s. Frequency histogram (top-right) reveals two major peaks of 11 ± 0.85 and 57 ± 1.35 pS, which correspond to γ_res and γ_o, respectively. B–C, simulation of V_j-gating in the junction containing 200 homotypic GJ channels using the model of contingent gating. I_j trace in B was obtained in response to V_j step of −95 mV (upper trace). C, probability of channels to dwell in different states (O–O, C–O, O–C and C–C) depending on V_j. The black line shows P_o-o (see the inset), while grey lines show P_c-o, P_o-c, and P_c-c. D, I_j recording from cell pair exhibiting unitary gating events in response to three consecutive V_j ramps of ±80 mV. E, I_j–V_j plot of data showed in D. Continuous lines are drawn over I_j traces corresponding to one and two open channels, and dashed lines correspond to the residual state.

Figure 4. pH_i-dependent modulation of junctional conductance

A, dynamics of I_j and g_j measured by applying repeated V_j ramps of ±20 mV (enlarged I_j responses to V_j ramps are shown in the inset) in response to applications of NH₄Cl (10 mm). B, g_j and pH_i measurements in one cell pair after application of 1, 3, 7 and 15 mm of NH₄Cl. C, summarized g_j–pH_i relationship in which the continuous line is a fitting curve to the Hill equation; pK_a= 7.41 ± 0.02 and the Hill coefficient of 5.9 ± 0.5 (n= 6). Filled circles are from the experiment shown in B.

Figure 5. Dynamics of V_j-gating during intracellular alkalization in HeLaCx57-EGFP cell pairs

A, I_j recording in response to repeated V_j ramps from 0 to −110 mV before and during application of NH₄Cl (1 and 6 mm). Voltage steps of −20 mV were used to measure g_j in between voltage ramps. The insets show I_j responses on an enlarged scale to voltage ramps before and after application of 1 mm NH₄Cl. B, g_j–V_j dependencies calculated from V_j and I_j records shown in A. Grey lines show fitting curves obtained by using the model of contingent gating for each experimental g_j–V_j plot. Grey numbers in B–D attached to different plots correspond to numbers of V_j ramps shown in A. C, P_o-o dependence on V_j for different g_j–V_j plots. D, changes of V_h,o (open circles) and N_f (filled circles) over time during recordings shown in (A). Continuous lines show regression curves of the second order (continuous lines).

Figure 6. Voltage gating of Cx57-EGFP/Cx45 heterotypic gap junction channels

A, I_j response in cells expressing Cx57-EGFP evoked by V_j ramps applied to the cell expressing Cx45 shows I_j/V_j asymmetry. Application of NH₄Cl (2 mm) increased g_j∼2-fold. B, an averaged g_j–V_j dependence (black dots) that was obtained by overlapping normalized g_j–V_j plots measured under control conditions. The white line inside experimental points is a fitting curve obtained from the model of contingent gating assuming that the gating polarity of Cx57 is negative, i.e. the same as for Cx45. The black line shows the simulated g_j–V_j plot with the same parameters of the model for white line but assuming that the gating polarity of Cx57 is positive. C, I_j recordings in cell expressing Cx57-EGFP that exhibit unitary gating events in response to positive and negative V_j steps applied to the cell expressing Cx45, and the histogram of g_j data obtained from the I_j/V_j ratio, suggesting that unitary conductance of Cx45–Cx57 heterotypic junction is ∼49 pS.

Figure 8. Dye uptake in co-cultured HeLaCx57-EGFP and HeLa parental cells

A–C, fluorescence images of cells recorded 60 min after exposure to DAPI (5 μm) and EtBr (0.5 μm) added to DMEM. There was no noticeable difference in uptake of DAPI (B) and EtBr (C) between two cells expressing (shown in green) and not expressing Cx57-EGFP. D–E, cells expressing Cx57-EGFP (green) but not HeLa parental cells demonstrate enhanced uptake of DAPI (blue) and become round in shape ∼25 min after exposure to a Ca²⁺-free (no added Ca²⁺) MKR solution containing 5 mm of NH₄Cl. F–G, dynamics of DAPI (F) and EtBr (G) uptake at [Ca²⁺]_o= 100 μm in MKR solution; FI was measured in arbitrary units (a.u.) by subtracting background intensity.

Figure 9. pH-dependent modulation of g_j in HeLa cells expressing wild-type Cx57 and untagged EGFP

A, intracellular alkalization with NH₄Cl (5 mm) increased g_j from ∼0.2 nS to ∼8 nS. MKR solution containing 2 mm of CaCl₂ was used in this experiment. g_j was measured by applying repeated V_j ramps of ±20 mV. The inset shows I_j responses to V_j ramps during an initial period of NH₄Cl application. I_j responses to positive and negative ∼70 mV voltage steps show moderate (∼25%) decay. B, I_j trace measured in response to 60 s long V_j ramps from 0 to −100 mV shows well expressed V_j-gating under control conditions at pH_{pipette−sol}= 7.3, while under exposure to NH₄Cl, the I_j–V_j relationship is almost linear indicating almost no V_j-gating.