Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannel

Abstract

Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32*43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32*43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32*43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32*43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter.

Figure 1. Current–voltage relations of Cx32*43E1 (43E1) channels

A, selected traces illustrating the change in reversal potentials of the open state of 43E1 channels obtained with inside-out patches. The concentration of the KCl in the bath solution is provided next to the current trace. B, reversal potentials obtained with outside-out patches. In all cases, the pipette solution contained 100 mm KCl. Current traces were obtained with the application of ±70 mV voltage ramps and digitally filtered at 500 Hz for presentation.

Figure 2. Plot of the absolute value of reversal potentials as a function of ionic strength

A, the absolute values of the average reversal potentials of Cx32*43E1 channels presented in Table 1 for inside-out and outside-out patches are plotted against the bath solution concentration of KCl. B, the absolute values of reversal potentials of N2E channels for inside-out and outside-out patches are plotted against the bath solution concentration of KCl. Data from Table 1 are plotted for the mean values of outside-out and inside-out patches as well as those obtained from a long-lived inside-out patch. C, the absolute values of the average reversal potentials presented in Table 1 for inside-out patches of wild-type (Cx32*43E1, abbreviated 43E1), N2E and T8D are plotted against the bath solution concentration of KCl. D, the absolute values of the average reversal potentials presented in Table 1 for outside-out patches of wild-type (Cx32*43E1, abbreviated 43E1), N2E and T8D are plotted against the bath solution concentration of KCl. E, the absolute values of the average reversal potentials presented in Table 1 for inside-out patches of Cx32*43E1 (abbreviated 43E1), G5D, G5R and N2E + G5R channels are plotted against the bath solution concentration of KCl. F, the absolute values of the average reversal potentials presented in Table 1 for outside-out patches of Cx32*43E1 (abbreviated 43E1), G5D, G5R and N2E + G5R channels are plotted against the bath solution concentration of KCl. Bath solutions concentrations are plotted logarithmically to facilitate comparisons at low and high salt concentrations.

Figure 3. Current–voltage relations of N2E channels

A, selected current–voltage traces illustrating the change in reversal potentials of the open state of a single N2E channel obtained with inside-out patches. The concentration of the KCl in the bath solution is provided next to the current trace. B, current–voltage relation of N2E channels obtained with outside-out patches. The concentration of the KCl in the bath solution is provided next to the current trace. C, the current–voltage relation of an outside-out patch containing several N2E channels in 10 mm KCl bath solution. In all cases, the pipette solution contained 100 mm KCl. Current traces were obtained with the application of ±70 mV voltage ramps and digitally filtered at 500 Hz for presentation.

Figure 4. Current–voltage relations of G5R channels

A, selected current–voltage traces illustrating the change in reversal potentials of the G5R channels obtained with inside-out patches. The concentration of the KCl in the bath solution is provided next to the current trace. B, reversal potentials obtained with outside-out patches. The concentration of the KCl in the bath solution is provided next to the current trace. In all cases, the pipette solution contained 100 mm KCl. Current traces were obtained with the application of ±70 mV voltage ramps and digitally filtered at 500 Hz for presentation.

Figure 5. The modification of an inside-out patch containing two active T8C channels by MTSEA biotin-X

Current was elicited at a holding potential of −60 mV. The duration of time at which 1 mm MTSEA biotin-X was perfused is indicated by the shaded bar. Expanded section of the record illustrates nine stepwise changes in conductance indicating modification of at least 9 of 12 T8C residues with MTSEA biotin-X.

Figure 6. Reversal potentials of model channels determined with numerical solution of PNP equations provided by Chen & Eisenberg (1993)

A, left panel, the charge distribution model of a channel containing a single region of negative charge (3e positioned at a distance from 0.75 to 0.85). This corresponds to a charge density of 1 mol l⁻¹ for a channel modelled as a right cylinder 60 Å long, 7 Å radius. Right panel, the absolute values of reversal potentials for inside-out and outside-out conditions are plotted as a logarithmic function of salt concentration. ▪ denotes inside-out patch configuration; • denotes outside-out patch configuration. The reversal potentials calculated for outside-out (O/O) and inside-out (I/O) are presented in part A of the Table for ionic conditions comparable to those used in this study. In all cases the opposing solution contained 100 mm KCl. Ionic activities used in the calculation of P_K/P_Cl ratios are those listed in Table 2 for a given salt concentration. B, left panel, a charge distribution model of a P–N junction containing a region of positive charge located near the intracellular end of the pore and a region of negative charge located near the extracellular end of the pore. Right panel, absolute values of reversal potentials of the P–N junction are plotted against the logarithm of salt concentration. ▪ denotes inside-out patch configuration; • denotes outside-out patch configuration. The reversal potentials and P_K/P_Cl ratios determined for the model channel shown in the left panel of B with the PNP equations are presented in part B of the table.

Figure 7. Reversal potentials of model channels determined with numerical solution of PNP equations provided by Chen & Eisenberg (1997)

A, a charge distribution model of a channel containing two regions of negative charge; the internal charge of 2 M/L is positioned at distance 0.1–0.2, the external charge 1 mol l⁻¹ is positioned at distance 0.8–0.9. B, the absolute values of reversal potentials for inside-out and outside-out conditions are plotted as a logarithmic function of salt concentration. ▪ denotes inside-out patch configuration; • denotes outside-out patch configuration.