- The epithelial Na channel (ENaC) consists of at least three structurally related subunits (alpha beta, and gamma). Each subunit has two predicted membrane-spanning domains separated by large extracellular loops. The function of these ectodomains in channel activity is not known. The structural determinants of the pore are als unknown. The proposed studies are based on the hypotheses that selected domain within the extracellular loops of ENaC subunits participate in subunit-subunit interactions required for functional assembly of ENaCs, and that selected residues immediately preceding and within the second membrane-spanning region of the three subunits line the pore. The first hypothesis will be addressed by studies to identify regions within ENaC hydrophilic ectodomains which participate in subunit-subunit interactions. The underlying hypothesis is that ectodomains bind to each other producing functional multimeric channels. They will inject Xenopus oocytes with wild-type channel subunits and an excess of ectodomains or fractions of ectodomains to disrupt interactions between fully formed subunits. The functional assay will be development of amiloride-sensitive current in the oocytes. They have demonstrated that such co-injection abolished amiloride-sensitive currents in preliminary studies and that the ectodomains appear to interact with full-length ENaC subunits with a dominant-negative role in functional expression. This inhibition of functional channel expression may be the result of faulty assembly of channels in which some subunits lack membrane-spanning and intracellular domains and/or of inhibition of trafficking to the cell surface. Deletion and chimera construct experiments are proposed with co-immunoprecipitation to confirm subunit-subuni interactions. For the second part of the studies, identification of residues within the pore region of the channel will be based on analyses of channels with single amino acid substitutions within and preceding the second membrane-spanning domains. Mutations of residues which line the channel pore will alter selected functional properties of the channel, including single-channel conductance and/or cation selectivity.
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