Connexin 32/38 chimeras suggest a role for the second half of inner loop in gap junction gating by low pH

Gap junction channels are regulated by gates that close with cytosolic acidification and transjunctional voltage (V-j). For identifying the connexin (Cx) domain(s) involved in channel gating, CO2 and V-j sensitivities of channels made of Cx38, Cx32, Cx32/Cx38 chimeras, and Cx32 mutants were studied in Xenopus oocyte pairs. Recently, we have reported that Cx38 is more sensitive to CO2 and V-j than Cx32 because of differences in the Cx inner loop. To identify the responsible inner loop domain, chimeras of Cx32/Cx38 in which the first (I-1) or the second (I-2) half of the inner loop of Cx38 replaced that of Cx32 and I-2 mutants of Cx32 were tested. The chimera Cx32/Cx38I(2) (Cx32 with I-2 of Cx38) was like Cx38 in CO2 sensitivity but like Cx32 in V-j sensitivity. Cx32/Cx38I(1) (Cx32 with I-1 of Cx38) did not express channels. Of the three Cx32 mutants, Cx32-VH/IR (VH of Cx32 replaced with IR of Cx38) and Cx32-WW/MC (WW of Cx32 replaced with MC of Cx38) were like Cx32 in both CO2 and V-j sensitivity, whereas Cx32-S*T/Q*P (S*T of Cx32 replaced with Q*P of Cx38) was closer to Cx38 in CO2 sensitivity but behaved like Cx32 in V-j gating. The data suggest that I-1 and I-2 contain domains relevant for V-j and CO2 gating, respectively.