PH REGULATION OF CONNEXIN43 - MOLECULAR ANALYSIS OF THE GATING PARTICLE

Gap junction channels allow for the passage of ions and small molecule s between neighboring cells, These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other t issues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin chann els is that they close upon acidification of the intracellular space, Previous studies have shown that truncation of the carboxyl terminal o f Cx43 impairs pH sensitivity, In the present study, we have used a co mbination of optical, electrophysiological, and molecular biological t echniques and the oocyte expression system to further localize the reg ions of the carboxyl terminal that are involved in pH regulation of Cx 43 channels. Our results show that regions 261-300 and 374-382 are ess ential components of a pH-dependent ''gating particle,'' which is resp onsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent, The function o f region 261-300 may be related to the presence of a poly-proline repe at between amino acids 274 and 285, Furthermore, site-directed mutagen esis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one a mino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for prolin e, which has been associated with some cases of cardiac congenital mal formations in humans, also disrupts the pH gating of Cx43, although de letion of amino acids 364-373 has no effect on acidification-induced u ncoupling. These results provide new insight into the molecular mechan isms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.