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Figure 1.
WT and G12R Cx26 gating kinetics and voltage dependence. Membrane currents were recorded from oocytes under two-electrode voltage clamp in response to depolarizing voltage steps from a holding potential of −80 mV, stepped in 10-mV increments from −60 to 50 mV, and returned to −80 mV. (a and b) Representative trace of macroscopic currents recorded in oocytes expressing WTCx26 (a) or G12R (b). (c) Normalized conductance–voltage relationship of WTCx26 and G12R hemichannels. Data obtained from the instantaneous tail currents taken at −80 mV and measured after 40-s test pulse for each voltage tested. Data symbols and error bars represent mean ± SEM; n = 20 oocytes from five different batches.
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Figure 2.
G12R impairs fast gating. Membrane currents were recorded from oocytes under two-electrode voltage clamp in response to depolarizing voltage steps from a holding potential of 30 mV, stepped in 10-mV increments to 90 mV, and returned to 30 mV. (a and b) Representative trace of macroscopic currents recorded in oocytes expressing WTCx26 (a) or G12R (b). (c) Current–voltage relationship of WTCx26 and G12R hemichannels. Data symbols and error bars represent mean ± SEM; n = 10 oocytes from three different batches.
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Figure 3.
Single-hemichannel activity recapitulates the macroscopic currents. (a and b) Representative examples of patch-clamp recordings from excised inside-out patches containing WTCx26 (a) or G12R (b) hemichannels. Traces shown were obtained in response to a 3-s test pulse at 90 mV from and returning to a holding potential of 30 mV (mimicking the fast gate macroscopic protocol). Dashed lines represent the closed state (C) and the fully open state (O; subscript numbers indicate two or more channels opening). Each trial consisted of 20 sweeps that were ensemble averaged to recapitulate the kinetics of the macroscopic currents. (c) The ensemble average (orange trace) and the macroscopic trace (black trace) obtained at 90 mV were superimposed for comparison. The time constant of the deactivation kinetics from WTCx26 hemichannel was obtained by fitting the traces to a single exponential. No significant differences were observed. τ values represent mean ± SEM; n = 15 oocytes from three different batches.
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Figure 4.
Fast deactivation kinetics in G12R hemichannels. (a and b) A pulse from a holding potential of −80 to 0 mV and returned to −80 mV was applied to analyze tail currents and their deactivation kinetics in oocytes expressing WTCx26 (a) and G12R (b). Traces show the currents elicited in oocytes bathed in extracellular solution containing 1.8 mM Ca2+ (black traces), 0.25 mM Ca2+ (red traces), or 0.01 mM Ca2+ (blue traces). (c) Time constants of the deactivation as a function of Ca2+ concentration plotted using a semilogarithmic scale. The dotted lines represent the best linear fit to the WTCx26 and G12R mutant hemichannels’ time-constant data. (d) Graph depicting normalized peak hemichannel tail currents as a function of Ca2+ concentration in WTCx26 (open circles) and G12R (red circles). Solid lines are the best fit to the data using a Hill equation for WTCx26 (black line) and G12R (red line; Eq. 1). The data points represent mean ± SEM; n = 10. (e) Representative examples of patch-clamp recordings from excised inside-out patches containing WTCx26 or G12R hemichannels. Traces shown were obtained in response to a 45-s test pulse at 0 mV from a holding potential of −80 mV and returned to −80 mV during an additional 45 s. Dashed lines represent the closed state (C) and the opening of hemichannels. Each trial consisted of 10 sweeps that were ensemble averaged to recapitulate the kinetics of the macroscopic currents. (f) The ensemble averages (black trace for WTCx26; red trace for G12R) and the macroscopic traces (red trace for WTCx26; black trace for G12R) obtained at −80 mV were superimposed for comparison. The time constant of the deactivation kinetics from WTCx26 and G12R hemichannels was obtained by fitting the traces to a single exponential. No significant differences were observed. τ values represent mean ± SEM; n = 15 oocytes from three different batches.
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Figure 5.
The G12R mutation increases open probability without modifying single-channel conductance. (a and b) Representative traces of WTCx26 (a) or G12R (b) single channels recorded at 20, 40, and 60 mV. The dashed lines represent the closed state (C), the fully open state (O), and the subconductance state (Or) of hemichannels. Note that conductance substates occur rarely and show fast kinetics in G12R hemichannels. (c) Graph depicting the plot of the current amplitude (I, pA) of the fully open state O obtained at different voltages (millivolts) for WTCx26 (black circles) and G12R (red circles). Black solid (WTCx26) and dashed (G12R) lines represent the best linear regression fit. The slope of the I-V relationship determined fully open state O slope conductance (measured at 0 mV). (d) Graph depicting the plot of the open probability (Po) of the fully open state at different voltages (millivolts) for WTCx26 (black circles) and G12R (red circles). The data points represent mean ± SEM; n = 20 oocytes from five different batches.
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Figure 6.
G12R mutation causes the N terminus to move toward the cytoplasm and interact with residues in the intracellular loop. (a) Structure of the WTCx26 hemichannel showing the N terminus (pink), TM1 (red), parahelix (orange), ELs (green), and intracellular loop (light blue). (b) Graph depicting the mean pore radius variation in function of pore axis during simulation. The region around the N terminus in G12R hemichannels is slightly displaced toward the cytoplasm. The narrowing of the pore in the parahelix regions does not change in G12R versus WTCx26. Data points represent mean ± SEM; n = 12. (c) Superimposition of representative monomers of WTCx26 (black) and G12R (red). Insets show the distances between residue 12 and the R99 in both WTCx26 (13.7 Å) and G12R (4.8 Å).
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Figure 7.
Disrupting the interaction between R12 and R99 rescues the fast gating activation. Membrane currents recorded from oocytes under two-electrode voltage clamp in response to depolarizing voltage steps from a holding potential of −80 mV, stepped in 20-mV increments from −60 to 60 mV, and returned to −80 mV or representative patch-clamp single-channel recordings in oocytes from a holding potential of 0 mV and stepped to 40 mV. (a–f) Representative traces of macroscopic currents (left) and single-hemichannel recordings (right) from oocytes expressing WTCx26 (a), G12R (b), G12R/R99D (c), G12R/R99K (d), G12R/R99A (e), and R99A (f). Dashed lines in right panel indicate different current levels (O, fully open; Or, subconductance; C, closed). (g) Graph depicting the macroscopic current–voltage relationship of hemichannels calculated from the instantaneous tail currents. Data points represent mean ± SEM; n = 3.
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Figure 8.
Molecular model of the gain of function caused by G12R hemichannels. (a) Cartoon depicting a macroscopic trace of WTCx26 hemichannel showing the reduction of current amplitude at 40 mV. At the single-channel level, this reduction in the current amplitude could be explained by the transition from the fully open state (red dashed line) to a subconductance state (green dashed line). (b) Cartoon depicting a macroscopic trace of G12R hemichannels illustrating the lack of current reduction observed in WTCx26. At the single-channel level, this difference could be caused by the lack of fast gating activation resulting from the absence of transitions to the subconductance state. This altered functioning would be caused by interactions between R12 and R99, which trap the N terminus in a rigid position, preventing its regular function and the activation of the fast gate.
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Figure S1. Position of the N terminus in WT and mutant Cx26 hemichannels. Graph depicting the position of the N terminus in WT and mutant Cx26
hemichannels obtained by measuring the geometrical distance between the centers of mass of the N terminus with respect to the center of mass of the
whole structure. Data are presented as mean ± SEM; n = 12.
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Figure S2. Local bending angles in TM2 (residues 75–105) for WTCx26 (black line) and G12R (red line). The peaks at residue P87 in both WTCx26 and
G12R reveal the presence of a kink. Shadow represents SD; n = 4. The percentages of the total simulation time in which the formation of a hydrogen bond between T86 and I82 can be observed are summarized in Table 2. No differences were observed between WTCx26 and G12R hemichannels.
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Figure S3. Root mean square fluctuation (RMSF) of simulated systems as a function of residue number. Three regions (N terminus region, K41 residue, and intracellular loop region) can be identified that show a different behavior in WTCx26 versus the G12R mutant. In general, the G12R mutant protein
shows decreased mobility with respect to the WTCx26 in the indicated regions, especially in the intracellular loop. Data are presented as mean ± SD; n = 4.
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Figure S4. Correlation between N terminus displacement and the side chain angle of R12 (in G12R) in relation to the z axis. Different colors indicate different replicas (n = 4).
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Figure S5. Single-hemichannel activity recapitulates the macroscopic currents in G12R/R99A. (a–c) Representative examples of patch-clamp recordings from inside-out patches containing WTCx26 (a), G12R (b), and G12R/R99A (c) hemichannels. Traces shown were obtained in response to a 3-s test pulse
at 90 mV from and returning to a holding potential of 30 mV (mimicking the fast gate macroscopic protocol). Dashed lines represent the closed state (C) and
the fully open state (O; subscript numbers indicate two or more channels opening). Each trial consisted of 20 sweeps that were ensemble averaged to recapitulate the kinetics of the macroscopic currents. The ensemble average (orange trace for WTCx26 and G12R; light blue trace for G12R/R99A) and the macroscopic trace (black trace) obtained at 90 mV were superimposed for comparison. The time constants of the deactivation kinetics were obtained by fitting the traces to a single exponential. τ values represent mean ± SEM; n = 15 oocytes from three different batches.
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