We have studied the role of histidine 95 (H95) on the pH gating of the cardiac gap junction protein connexin43 (Cx43). Wild through 6 for reviews on the subject). Recently, Spray and Burt2 proposed that acidification-induced uncoupling may result from the protonation of titratable histidine residues present in the cytoplasmic loop (CL) of the cardiac gap junction protein connexin43 (Cx43). Accordingly, the presence of positive charges at positions 95, 126, and/or 142 of Cx43 (positions at which histidine residues reside) should lead to channel closure, whereas the absence of positive charges in those positions may prevent acidification-induced uncoupling. This hypothesis was based on the fact that the imidazole ring of the histidine residue is the only amino acid side chain whose apparent dissociation constant from protons (pKa) falls within the physiological range,7 and it assumes that pH gating would result, at least in part, from a direct effect of the intracellular protons on the conformation of the connexin protein. A role of histidine residues on pH-mediated regulation of protein Received August 9, 1993; accepted February 24, 1994 reduced susceptibility to acidification-induced uncoupling, whereas lysine (a basic residue) was more susceptible to uncoupling than the wild-type protein. The susceptibility to acidification-induced uncoupling was enhanced for the H95Q-A94H mutant when compared with the wild-type mutant, but it was significantly reduced when histidine was placed at position 93 (H95Q-L93H). Our data indicate that a properly placed histidine residue is an important structural element for functional expression as well as for pH regulation of Cx43. The results suggest that the importance of H95 on pH gating may be associated with a possible protonation of this residue on acidification of the intracellular environment. The data are compatible with a recently proposed mechanism modeled after the "ball-and-chain" hypothesis explaining pH gating of Cx43. (Circ Res. 1994;74:1058-1064 Key Words * connexin43 * cardiac gap junctions K pHi * histidine * site-directed mutagenesis function has also been suggested for other membrane proteins (eg, References 8 through 12). More recently, Liu et al13 studied the pH sensitivity of Cx43 when expressed in Xenopus laevis oocytes and demonstrated that deletion of the last 125 amino acids from the carboxyl terminal of Cx43 causes a significant loss in pH, sensitivity, displacing pKa (measured as the value of pHi that causes 50% decrease from maximal junctional conductance [Gj]) from 6.6 in Cx43 channels to 6.1 in the mutant lacking the carboxyl tail. These results demonstrated that the carboxyl end is a necessary domain for normal pH gating of Cx43, whereas residues in the CL cannot by themselves modulate acidification-induced uncoupling. These observations, however, did not imply that the carboxyl end is the only region of the protein involved in pH gating. In fact, it is possible that the carboxyl terminal works in concert with other regions of the protein t...

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