title: Functional requirement for a highly conserved charged residue at position 75 in the gap junction protein connexin 32.
abstract: Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg(75) position. This residue is highly conserved among members of the connexin family, and disease-causing mutations have been identified at this (or the corresponding) position in Cx26, Cx43, and Cx46. Thus, a better understanding of the effects of mutations of this position in Cx32 may have relevance to pathogenesis of a number of different human diseases. All three mutants associated with CMT1X (R75P, R75Q, and R75W) showed very low levels of coupling similar to those of the cells transfected with vector alone. Heterotypic pairing with Cx32 WT showed that the absence of coupling for these mutants in the homotypic configuration could be explained by shifts in their hemichannel G(j)-V(j) relations. Examination of the expression levels and gating characteristics of seven additional mutants (R75A, R75D, R75E, R75H, R75K, R75L, and R75V) at this position suggest that the positive charge at position 75 in Cx32 is required for normal channel function but not for gap junction assembly. Our studies also suggest that disease treatment strategies for CMT1X, which correct trafficking abnormalities in Cx32, may be ineffective for the group of mutations also conferring changes in gating properties of Cx32 channels.
authors.affiliation: Department of Neurology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, USA. charles.abrams@downstate.edu
authors.forename: Charles K
authors.initials: CK
authors.lastname: Abrams
authors.forename: Mahee
authors.initials: M
authors.lastname: Islam
authors.forename: Rola
authors.initials: R
authors.lastname: Mahmoud
authors.forename: Taekyung
authors.initials: T
authors.lastname: Kwon
authors.forename: Thaddeus A
authors.initials: TA
authors.lastname: Bargiello
authors.forename: Mona M
authors.initials: MM
authors.lastname: Freidin
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics; D000072259:Connexin 26; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D055672:Static Electricity; D013329:Structure-Activity Relationship; D000097002:Gap Junction beta-1 Protein
chemical_list: D001120:Arginine; D000072259:Connexin 26; D017630:Connexins; D050505:Mutant Proteins; D000097002:Gap Junction beta-1 Protein; C000607042:GJB2 protein, human