Connexin32 gap junction channels in stably transfected cells. Equilibrium and kinetic properties.

摘要

Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (gj) to transjunctional voltages (Vjs) of either polarity, with the ratio of minimal to maximal gj (gmin/gmax) being approximately 0.1 at the highest Vjs. Steady-state gj values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which gj was reduced by 50%, was approximately 25-30 mV; A, the Boltzmann parameter describing voltage dependence, was approximately 0.06 (corresponding to an energy difference between states of approximately 1 kCal/mol and to approximately 2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (tau greater than 5 s at 20-40 mV, tau = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (alpha) was approximately 30% lower than that of the closing rate constant (beta) over the Vj range 0-70 mV; at higher voltages, voltage sensitivity of alpha and beta saturated. The kinetic response of gj to a paradigm in which gj was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state.