Molecular Determinants of Electrical Rectification of Single Channel Conductance in Gap Junctions Formed by Connexins 26 and 32

Abstract

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current–voltage (I–V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current–voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I–V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405–1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I–V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I–V relations that closely resembled the observed I–V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (Vj)-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.

Gating and regulation of connexin 43 (Cx43) hemichannels

“…Truncation of the C terminus can block fast gating while leaving slow gating intact (38). Mutations in the N terminus also affect fast gating and can reverse its polarity (39)(40)(41).…”

Gap Junctions

“…Fast rectification has been shown using Cx32/Cx26 heterotypic channels (Oh et al 1999). However, neither connexin displays particularly fast homotypic voltage-dependent gating and thus the rectification observed cannot be predicted from the properties of the individual channels.…”

Expression of Connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development

“…It is also indicated by the occasional detection of Cx43 between adjacent GCNA1-positive cells. Thus, homotypic, heterotypic or heteromeric channels containing Cx43 (Bukauskas et al, 1995;Elenes et al, 1999;Elfgang et al, 1995, Oh et al, 1999 may communicate between these cell types. Cx43-mediated heterocellular contacts between Sertoli and germinal cells is consistent with recent findings of Cx43 gene transcript in germinal and Sertoli cells by in situ hybridization (Batias et al, 2000).…”