INTRAMOLECULAR INTERACTIONS MEDIATE PH REGULATION OF CONNEXIN43 CHANNELS

We have previously proposed that acidification-induced regulation of t he cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: th e carboxyl terminal (acting as a particle), and a region including his tidine 95 (acting as a receptor). Accordingly, intracellular acidifica tion would lead to particle-receptor binding, thus closing the channel . A premise of the model is that the particle can bind its receptor, e ven if the particle is not covalently bound to the rest of the protein . The latter hypothesis was tested in antisense-injected Xenopus oocyt e pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated a t amino acid 257 (i.e., M257) and mRNA coding for the carboxyl termina l region (residues 259-382). Intracellular pH (pH(i)) was recorded usi ng the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (G(i)) was measured with the dual volt age clamp technique. Wild-type Cx43 channels showed their characterist ic pH sensitivity. M257 channels were not pH sensitive (pH(i) tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensit ive channel (M257) was coexpressed with mRNA coding for the carboxyl t erminal. Furthermore, coexpression of the carboxyl terminal of Cx43 en hanced the pH sensitivity of an otherwise less pH-sensitive connexin ( Cx32). These data are consistent with a model of intramolecular intera ctions in which the carboxyl terminal acts as an independent domain th at, under the appropriate conditions, binds to a separate region of th e protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potass ium channels) or mediated through an intermediary molecule. The data f urther suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.