Unusual slow gating of gap junction channels in oocytes expressing connexin32 or its COOH-terminus truncated mutant.

Gap junction channels are gated by a chemical gate and two transjunctional voltage (V (j))-sensitive gates: fast and slow. Slow V (j) gate and chemical gate are believed to be the same. The slow gate closes at the negative side of V (j) and is mostly inactive without uncouplers or connexin (Cx) mutations. In contrast, our present data indicate otherwise. Oocytes expressing Cx32 were subjected to series of -100 mV V (j) pulses (12-s duration, 30-s intervals). Both peak (PK) and steady-state (SS) junctional conductances (G (j)), measured at each pulse, decreased exponentially by 50-60% (tau = approximately 1.2 min). G (j)PK dropped more dramatically, such that G (j)SS/G (j)PK increased from 0.4 to 0.6, indicating a drop in V (j) sensitivity. Less striking effects were obtained with -60 mV pulses. During recovery, G (j), measured by applying 20 mV pulses (2-s duration, 30-s intervals), slowly returned to initial values (tau = approximately 7 min). With reversal of V (j) polarity, G (j)PK briefly increased and G (j)SS/G (j)PK decreased, suggesting that V (j)-dependent hemichannel reopening is faster than hemichannel closing. Similar yet more dramatic results were obtained with COOH-terminus truncated Cx32 (Cx32-D225), a mutant believed to lack fast V (j) gating. The data indicate that the slow gate of Cx32 is active in the absence of uncouplers or mutations and displays unusual V (j) behavior. Based on previous evidence for direct calmodulin (CaM) involvement in chemical/slow gating, this may also be CaM-mediated.