Connexin channels are gated by transjunctional voltage (V_j) or CO₂ via distinct mechanisms. The cytoplasmic loop (CL) and arginines of a COOH-terminal domain (CT₁) of connexin32 (Cx32) were shown to determine CO₂ sensitivity, and a gating mechanism involving CL-CT₁ association-dissociation was proposed. This study reports that Cx32 mutants, tandem, 5R/E, and 5R/N, designed to weaken CL-CT₁ interactions, display atypical V_j and CO₂ sensitivities when tested heterotypically with Cx32 wild-type channels in Xenopus oocytes. In tandems, two Cx32 monomers are linked NH₂-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replace CT₁ arginines. On the basis of the intriguing sensitivity of the mutant-32 channel to V_j polarity, the existence of a "slow gate" distinct from the conventional V_j gate is proposed. To a lesser extent the slow gate manifests itself also in homotypic Cx32 channels. Mutant-32 channels are more CO₂ sensitive than homotypic Cx32 channels, and CO₂-induced chemical gating is reversed with relative depolarization of the mutant oocyte, suggesting V_j sensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.