Biophysical characterization of connexin 32 and its association with human disease

Abstract

Gap junctions are intercellular channels that allow the direct transmission of ions, second messengers, and cellular metabolites between coupled cells. Connexin (Cx) genes encode gap junction proteins. The precise molecular mechanisms underlying voltage dependence of gap junction are largely unknown. Using electrophysiological measurements of wild type and mutants a more detailed picture for structure-function relations of gap junctions is provided. The rectification of the initial conductance-voltage relation of Cx32/Cx26 junctions corresponded to the rectification of single channel conductance. Charged amino acid residues located in the amino terminus and first extracellular loop were major determinants of the rectification of the Cx32/Cx26 channel. The current-voltage relations of wild type and mutant channels could be modeled with an electro-diffusive model (Poisson-Nemst-Plank equations). The rectification of the Cx32/26 channel results from the asymmetry in number and position of charged residues in the two connexins. Cx32 and Cx26 have opposite voltage-gating polarities due to a difference in the charge of the second amino acid. Negative charge substitutions of the second amino acid residue in Cx32 reverse the gating polarity of Cx32 hemichannels. To determine the stoichiometry of Vj-gating polarity reversal by negative charge substitution, a chimeric connexin was used. Heteromeric hemichannels comprised of wild-type and negatively charged mutant subunits display bipolar V j gating. The frequency of bipolar hemichannels observed correlates with the fraction of hemichannels that are expected to contain at least one oppositely charged connexin subunit, indicating that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. Cx32 mutations have been shown to be responsible for X-linked form of Charcot-Marie-Tooth (CMTX) disease. To examine the molecular basis for loss-of-function mutations, nine CMTX mutations were tested. Mutations formed intercellular channel when expressed in Xenopus-oocytes. Single-channel studies demonstrate reduced junctional permeability caused by a decrease in either pore size (S26L) or open channel probability (M34T). It is proposed that the attenuation of cAMP signaling resulting from a decrease in permeation through the reflexive pathway formed by mutant channels may impair normal glial-neuronal interactions and thereby cause demyelination and axonal degeneration.