Molecular dissection of a basic COOH-terminal domain of Cx32 that inhibits gap junction gating sensitivity.
Connexin32 (Cx32) mutants were studied by double voltage clamp in Xenopus oocytes to determine the role of basic COOH-terminal residues in gap junction channel gating by CO2 and transjunctional voltage. Replacement of five arginines with N (5R/N) or T residues in the initial COOH-terminal domain (CT1) of Cx32 enhanced CO2 sensitivity. The positive charge, rather than the R residue per se, is responsible for the inhibitory role of CT1, because mutants replacing the five R residues with K (5R/K) or H (5R/H) displayed CO2 sensitivity comparable to that of wild-type Cx32. Mutants replacing R with N residues four at a time (4R/N) showed that CO2 sensitivity is strongly inhibited by R215 and mildly by R219, whereas R220, R223, and R224 may slightly increase sensitivity. Neither the 5R/N nor the 4R/N mutants differed in voltage sensitivity from wild-type Cx32. The possibility that inhibition of gating sensitivity results from electrostatic interactions between CT1 and the cytoplasmic loop is discussed as part of a model that envisions the cytoplasmic loop of Cx32 as a key element of chemical gating.
Wang
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Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642-8711, USA.
Peracchia
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D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005260:Female; D017629:Gap Junctions / Q000502:physiology*; D006639:Histidine; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis; D000097002:Gap Junction beta-1 Protein
D001120:Arginine; D017630:Connexins; D006639:Histidine; D008239:Lysine; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000097002:Gap Junction beta-1 Protein