Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels

Abstract

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 CO2sensitivity, 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-CT1interactions, display atypical V jand 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 jpolarity, the existence of a “slow gate” distinct from the conventional V jgate 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 jsensitivity of chemical gating. A hypothetical pore-plugging model involving an acidic cytosolic protein (possibly calmodulin) is discussed.

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

“…This is interesting, because gating to full channel closure is believed to reflect the behavior of the slow V j gate (5,17,20). The function of the slow V j gate is usually hidden, in the absence of chemical uncouplers, in all connexin channels except in Cx45 channels (6), but it manifests itself in various disparate channels made of connexin mutants (17,20,23,31).…”

“…If indeed histidine protonation does not play a role, an alternative mechanism could involve an interplay between the V j sensor and chemical gate. Because the chemical gate is likely to be negatively charged (17,20), if this gate were to come in proximity of the V j sensor, before closing the channel completely, it could potentially hinder or facilitate the V jinduced displacement of the sensor from the channel lumen in positive or negative gaters, respectively. Significantly, the absence of a DEPC effect on CO 2 induced uncoupling efficiency seriously questions the potential role of histidine protonation in the uncoupling mechanism as well.…”

“…On the basis of their behavior at the single channel level, fast V j gate and chemical gate are believed to be distinct: the former closes rapidly (Ͻ1 ms) but incompletely, leaving a 20 -30% residual conductance, whereas the latter closes slowly (8 -10 ms) but completely (5). In contrast, slow V j gate and chemical gate are likely to be the same (5,17,20). Slow and fast V j gates are in series, and each hemichannel appears to have both gates.…”

“…In the early 1990s, CO 2 application was reported to increase the V j sensitivity of Cx32 channels (38). More recently chemical gating induced by CO 2 was shown to be reversed by V j gradients positive at the mutant side of heterotypic channels between Cx32 and various Cx32 mutants (17,20), and chemical gating was reversed in insect cells by bilateral hyperpolarization (37). These observations suggest that chemical and voltage gates may be sensitive to V j and CO 2 , respectively.…”

Interactions of connexins with other membrane channels and transporters

“…At least two voltage sensitive gates have been identified: fast and slow. The fast gate and chemical gate are believed to be distinct, whereas slow gate and chemical gate are likely to be the same (Bukauskas and Peracchia, 1997;Peracchia et al, 1999Peracchia et al, , 2000. Slow and fast V j gates are in series and each hemichannel appears to have both gates.…”