Molecular analysis of gap junction voltage dependence
Rubin, Joshua Bennett
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Gap junctions are aggregates of intercellular channels that allow for the passage of molecules up to 1 KD in size. By this means, cells of nearly all types are electrically and metabolically coupled. Gap junction channels are constructed from two hemichannels, donated by each of two coupled cells. Each hemichannel is a hexamer of a subunit protein called the connexin. The connexins are a multigene family. They are physiologically divergent yet share a common membrane topology and regions of extensive amino acid sequence identity. Sequence divergence tends to occur in topologically defined regions of the molecule and therefore it is reasonable to predict that the differences in physiology are determined by the activities of topologically defined domains. In order to understand the molecular basis of voltage dependent gating in gap junctions a number of chimeric connexins were constructed in which topologically defined domains expected to be involved in voltage dependence were exchanged between two closely related connexins, Cx32 and Cx26. Chimeras were constructed by a novel PCR based mutagenesis technique. Voltage dependence of wild type and chimeric connexins was determined by double voltage clamping Xenopus oocytes injected with RNA transcribed in vitro from wild type and chimeric clones. The pairing of two oocytes injected with the same or two oocytes injected with different RNAs allowed us to characterize both homotypic gap junctions, those composed of identical hemichannels, and heterotypic gap junctions, those composed of different hemichannels. In this way the relative contributions of hemichannel interactions and hemichannel independence to voltage dependence was assessed. Exchange of the most divergent sequences between Cx32 and Cx26 did not convert Cx32 into Cx26 suggesting that connexin character may require global interactions that can only be reproduced with combinatorial exchanges. The replacement of the first extracellular loop produced characterizable mutants whose behavior has made it possible to deduce the polarity of slow transjunctional voltage dependence and demonstrate that the fast inside-outside voltage dependence requires hemichannel interactions for its expression while fast transjunctional voltage dependence appears to be hemichannel property.
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