The chemical gating of single-gap junction channels was studied by the dual whole-cell voltage-clamp method in HeLa cells transfected with connexin43 (HeLa43) and in fibroblasts from sciatic nerves. Junctional current (I(j)), single-channel conductance, and I(j) kinetics were studied in cell pairs during CO2 uncoupling and recoupling at small transjunctional voltages (V(j) < 35 mV: V(j) gating absent) and at high V(j) (V(j) > 40 mV: V(j) gating strongly activated). In the absence of V(j) gating, CO2 exclusively caused I(j) slow transitions from open to closed channel states (mean transition time: ~ 10 ms), corresponding to a single-channel conductance of ~120 pS. At V(j) > 40 mV, V(j) gating induced fast I(j) flickering between open, γ(j)(main state), and residual, γ(j)(residual), states (transition time: ~2 ms). The ratio γ(j)(main state)/γ(j)(residual) was ~4-5. No obvious correlation between I(j) fast flickering and CO2 treatment was noticed. At high V(j), in addition to slow I(j) transitions between open and closed states, CO2 induced slow transitions between residual and closed states. During recoupling, each channel reopened by a slow transition (mean transition time: ~10 ms) from closed to open state (rarely from closed to residual state). Fast I(j) flickering between open and residual states followed. The data are in agreement with the hypothesis that gap junction channels possess two gating mechanisms, and indicate that CO2 induces channel gating exclusively by the slow gating mechanism.
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