Hydrogen ions are recognized as one of the major regulators of biochemical reactions. As such, functional processes in living cells can be drastically altered by changes in intracellular pH. In the case of the heart, intracellular acidification is known to alter the function of various channel membrane proteins, including gap junctional channels. In fact, it has been proposed that acidification-induced uncoupling in the heart may play an important role in the development of malignant ventricular arrhythmias following myocardial infarction. We propose to characterize the molecular mechanisms involved in pH gating of the cardiac gap junction protein, connexin43 (Cx43). Recent results from our laboratory using a combination of molecular biological and electrophysiological techniques have shown the that pH sensitivity of Cx43 can be altered either by truncation of the carboxyl tail at amino acid 257, or by replacing histidine 95 with amino acids of different charge and polarity. Our data have therefore shown that there are at least two regions of th Cx43 protein that are involved in pH gating. The data further suggest that neither one of these two regions is solely responsible for channel closure, since mutation of one is enough to partially prevent pH-induced uncoupling despite the presence of the other. We will use a combination of optical, electrophysiological and molecular biological techniques to carry out structure function studies of pH gating of Cx43 channels expressed in Xenopus laevis oocytes.
The specific aims are; 1) To localize and establish the precise limits of the region of the carboxyl tail of Cx43 that is involved in pH gating of junctional conductance, and determine whether specific residues in that region play any ole in acidification- induced uncoupling; 2) to characterize the role played by histidine 95 in the pH gating of Cx43, nd identify the role that other residues of the cytoplasmic loop may play in acidification-induced uncoupling; 3) to determine whether or not the carboxyl tail region responsible for pH gating can function as a separate domain, independent of the connexin protein; and 4) to characterize the kinetic of the ph gating reaction, and identify the possible role of calcium as the rate-limiting step in the development of acidification-induced uncoupling of Cx43 channels. Overall, these experiments should provide a better understanding of the molecular mechanisms mediating acidification-induced closure of Cx43 channels. Ultimately, our results should help to elucidate the molecular bases for the pH regulation of intercellular communication in the heart.
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