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 (Vj). For identifying the connexin (Cx) domain(s) involved in channel gating, CO2 and Vj 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 Vj 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 (I1) or the second (I2) half of the inner loop of Cx38 replaced that of Cx32 and I2 mutants of Cx32 were tested. The chimera Cx32/Cx38I2 (Cx32 with I2 of Cx38) was like Cx38 in CO2 sensitivity but like Cx32 in Vj sensitivity. Cx32/Cx38I1 (Cx32 with I1 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 Vj 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 Vj gating. The data suggest that I1 and I2 contain domains relevant for Vj and CO2 gating, respectively.
Wang
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Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, New York 14642-8642, USA.
Peracchia
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017630:Connexins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D005260:Female; D017629:Gap Junctions / Q000502:physiology*; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014981:Xenopus; D000097002:Gap Junction beta-1 Protein
D017630:Connexins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D000097002:Gap Junction beta-1 Protein