Intramolecular interactions mediate pH regulation of connexin43 channels

Abstract

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further 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.

Regulation of Intermuscular Electrical Coupling by theCaenorhabditis elegansInnexininx-6

“…A G/C to A/T transition, typical of EMS-induced lesions was identified, and caused a P353L change in the INX-6 protein near the C termini. That a point mutation in the C termini could disrupt the protein function at the restrictive temperature is consistent with a previous study in which the C terminus was shown to be very important for the normal channel function of connexins (Yeager and Nicholson, 1996;Morley et al, 1996;Wang and Peracchia, 1998). …”

Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates

“…Next, we assayed whether the pattern of cellular pH in the early chick blastoderm requires H + -V-ATPase activity. Using the pH reporting dye SNARF-1-AM (Buckler and Vaughan-Jones, 1990; Morley et al, 1996), we found that in control embryos, area pellucida cells maintain higher intracellular pH than primitive streak cells (Fig. 6G,H).…”

Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43

“…This mutation resulted in a premature stop codon leading to the expression of a Cx43 isoform that lacks the last 125 amino acid residues of the cytoplasmic C-terminal domain but still forms functional gap junctional channels when expressed in Xenopus oocytes (Morley et al, 1996) or mouse 3T3 cells (Moorby and Gherardi, 1999). Homozygous Cx43K258stop animals were born at the expected frequency, and newborns were per se viable, indicating that the mutant Cx43 surprisingly does not grossly impair normal embryonic development.…”

“…Regulation of GJIC upon acidification can be explained by an intramolecular ball-and-chain closure mechanism (Liu et al, 1993, Delmar et al, 2004, whereby amino acid residues of the cytoplasmic loop act as receptor to which the C terminus can bind (Duffy et al, 2002). Impaired channel closure due to deletion of the last 125 amino acids of the C terminus could be rescued by coexpression of the C-terminal fragment in Xenopus oocytes (Morley et al, 1996). This gating mechanism also has been observed in the regulation of Cx43 gap junctional channels by insulin/insulin-like growth factor (Homma et al, 1998), platelet-derived growth factor (Moorby and Gherardi, 1999), v-src (Zhou et al, 1999), and transjunctional voltage (Moreno et al, 2002).…”