Gap19, a Cx43 Hemichannel Inhibitor, Acts as a Gating Modifier That Decreases Main State Opening While Increasing Substate Gating

Abstract

Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element.

Keywords: automated analysis; channel gating; graphic user interface; transition analysis.

Figure 1

Flowchart of semi-automated analysis of unitary hemichannel currents.

Figure 2

Validation of HemiGUI analysis by comparing results obtained from user intervention analysis, fully automated analysis and analysis by Clampfit software. Experiments were performed on a dataset consisting of 20 current traces randomly selected from HeLa-Cx43 whole-cell recordings involving voltage steps from −30 mV to +70 mV (N = 3; N = 4; n = 20). (A) All conduction transition histograms were obtained by semi-automated analysis by four independent observers. (B) Automated analysis and the effect of different median filter time window (T_MF) sizes. A T_MF of 300 ms value based on the slowest transition time observed in the dataset gave a distribution most close to the one observed with semi-automated user intervention-based analysis. (C) All-point histogram generated from baseline-corrected traces generated by Clampfit. (D) Summary graph of nominal open probability (NPo) data demonstrating no statistically significant differences between Clampfit and the two modes of HemiGUI analysis (one-way ANOVA, Bonferroni post-test).

Figure 3

Unitary currents in HeLa-WT and HeLa-Cx43 cells. (A) Representative current traces in response to stepping positive membrane potentials (Vm) from −30 mV to potentials in the range of +40 to +70 mV. Unitary event activity was only observed in HeLa-Cx43. (B) Average unitary conductance and time constant of the transitions in HeLa-Cx43 obtained by two observers (N = 4; N = 15; n = 132, 33 traces per Vm step). There were no differences in conductance or time constants recorded at different voltages (one-way ANOVA with Bonferroni post-test).

Figure 4

Distribution of transition conductance and the effect of CT-tagged EGFP and CT-interacting Gap19 peptide. (A) Histogram analysis of transitions expressed as conductance illustrating non-significant event activity in Hela-WT cells (N = 3; N = 9; n = 65, 16 traces per + 40, + 50, and + 60 mV steps and 17 traces per + 70 mV step). (B) HeLa-Cx43 cells showed significant event activity with a unitary conductance of the transitions centered around 217 pS (66 pS SD of the distribution) (N = 4; N = 15; n = 132, 33 traces per Vm step). (C) Transitions were similarly distributed in HeLa-Cx43-EGFP cells with a main peak at 211 pS (N = 3; N = 6; n = 39, 9 traces per + 40 mV step and 10 traces per + 50, + 60, and +70 mV steps). (D) The distribution was completely altered by exposure of HeLa-Cx43 cells to 100 µM Gap19 (N = 3; N = 8; n = 50, 12 traces per + 40 and + 50 mV steps and 13 traces per + 60 and + 70 mV steps), demonstrating a smaller main peak and the appearance of a second peak centered at 80 pS (red curve). The dashed red curve in panel (B) is an event frequency-adjusted scaled version of the 80 pS curve. The number of transitions were normalized to maximum observed in each cluster of the experiment. The main peaks (fully open state) were compared between the different groups by one-way ANOVA and Bonferroni post-test, resulting in no difference in conductance. (E) Ratios of the number of substate transitions (AUC_sub) relative to all transitions (AUC_full + AUC_sub). (F) All transition event counts expressed per 30 s trace for the different conditions shown.

Figure 5

Distribution of transition kinetics and effect of CT-tagged EGFP and CT-interacting Gap19 peptide. Mono- and biexponential fits are displayed in blue and red, respectively. (A) In HeLa-Cx43 cells, tc_trans was distributed along a monoexponentially decreasing function characterized by a τ of 5.2 msec (N = 4; N = 15; n = 132). (B) For HeLa-Cx43-EGFP, τ was more than twice as large compared to HeLa-Cx43 (N = 3; N = 6; n = 39). (C) HeLa-Cx43 treated with Gap19 showed a second longer τ compared to the control (N = 3; N = 8; n = 50). (D) Selective analysis of tc_trans for transitions in the subconductance and main state demonstrated that Gap19 significantly slowed down transitions to the subconductance state while leaving main state transitions unaffected (Mann–Whitney U test, #### p ≤ 0.0001). Number of events: HeLa-Cx43 80 pS (129 events), HeLa-Cx43 217 pS (1656 events), HeLa-Cx43 + Gap19 80 pS (193 events), HeLa-Cx43 + Gap19 217 pS (453 events).