Gap junction channels formed from connexin (Cx) proteins are expressed in a variety of tissues providing exchange of metabolites and electrical communication between cells. We demonstrate that the uncoupling effects of gap junction channel blockers such as long carbon chain alkanols (LCCAs), volatile anaesthetics and the antimalarial drug mefloquine can be modulated by intracellular pH in a Cx-type-dependent manner. In addition, we provide data to support the idea that the uncoupling effect of LCCAs and other uncouplers could be related to a modification of hydrogen bonding between histidine residues and uncouplers, and/or inside the Cx structure. Abstract Gap junction (GJ) channels formed from connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell-cell communication exhibiting high sensitivity to intracellular pH (pH i). We examined pH i-dependent modulation of junctional conductance (g j) of GJs formed of Cx26, mCx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 and Cx50 by reagents representing several distinct groups of uncouplers, such as long carbon chain alkanols (LCCAs), arachidonic acid, carbenoxolone, isoflurane, flufenamic acid and mefloquine. We demonstrate that alkalization by NH 4Cl to pH ~8 increased g j in cells expressing mCx30.2 and Cx45, yet did not affect g j of Cx26, Cx40, Cx46, Cx47 and Cx50 and decreased it in Cx43 and Cx36 GJs. Unexpectedly, cells expressing Cx45, but not other Cxs, exhibited full coupling recovery after alkalization with NH 4Cl under the continuous presence of LCCAs, isoflurane and mefloquine. There was no coupling recovery by alkalization in the presence of arachidonic acid, carbenoxolone and flufenamic acid. In cells expressing Cx45, IC 50 for octanol was 0.1, 0.25 and 2.68 mm at pH i values of 6.9, 7.2 and 8.1, respectively. Histidine modification of Cx45 protein by N-bromosuccinimide reduced the coupling-promoting effect of NH 4Cl as well as the uncoupling effect of octanol. This suggests that LCCAs and some other uncouplers may act through the formation of hydrogen bonds with the as-of-yet unidentified histidine/s of the Cx45 GJ channel protein.
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