where gmax and gmin are maximal and minimal con-ductanes obtained at lowest and highest Vj, V0 is the voltage at which the voltage-sensitive component of gj (gmax - gmin) is reduced by 50%, and A is a slope factor from which the equivalent number of gating changes, n, can be calculated (Spray et al., 1981a). Evaluation of voltage sensitivity after the exogenous expression of connexins in mammalian cells or in weakly endoge-nously coupled cell pairs has indicated that for most connexins, the steady-state conductance is symmetric around 0 mV and is well fit to Boltzmann relationships (Fig. 7). Moreover, Boltzmann parameters are now known to be distinct for gap junction channels formed of each connexin subtype, as is described in more detail in Section D. For most of the gap junction channels that have been studied, the relaxation of junctional current from its initial to steady-state levels is well fit by a single exponential decay function for each voltage. This implies that a first-order process underlies channel voltage-dependent transitions.
Measurement of single gap junction channels from poorly coupled cells provided additional insight into the gating of gap junction channels (Fig. 8). At the microscopic level, junctional currents of most connexins exhibit direct, interconverting transitions between the fully open state and the voltage-insensitive or residual conductance substate (Bukauskas et al., 1995; Moreno et al., 1994a). The ratio of unitary conductances of the main open state and the subconductance state has in all cases been found to be similar to the gmin/gmax ratio, thus indicating that the residual conductance gmin seen at high Vj arises from channel transitions occurring from the main state to the residual subconductance state (Moreno et al., 1994a). Single channel open probability measurements further indicate that the voltage sensitivity of the macroscopic conductance is due to the ensemble activity of identical and independent channels (Srini-vas et al., 1999; Bukauskas et al., 1995).
Domains involved in voltage-dependent gating have not been explicitly identified. Unlike other voltage-gated channels, connexins do not contain a highly charged helical motif upon which the voltage gradient is likely to act. Thus, it is conceivable that voltage dependence may arise from interactions between several regions of the channel macromolecule. For example, mutation of Pro87 in M2 of Cx26 reverses the sign of voltage sensitivity when the mutant is paired with wild-type Cx26 in oocyte expression experiments (Suchyna et al., 1993). More detailed mutagenesis experiments on Cx32 and Cx26 have further implicated charged residues in the amino terminus and at the M1-E1 margin of these connexins (Verselis et al., 1994). Nevertheless, conceptual understanding of just which residues are the voltage sensors and how sensing of the voltage field is transduced in vivo into conformational change resulting in channel closure is almost completely lacking.
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