9509222 Middleton The primary structure of a family of proteins has been identified as components of the rat colon epithelial sodium channel. The coexpression of three homologous subunits, a, B, and g, in Xenopus laevis oocytes results in the expression of a sodium current that is like that described in the sodium transporting epithelia. Partial clones of each subunit from Xenopus kidney mRNA have been obtained. The objectives of this research are (1) to determine if the three clones encode renal sodium channels, and (2) to determine if the clones are sufficient to produce sodium channel activity, and whether all three are necessary for proper channel activity. Specifically, antisense oligonucleotides will be used to suppress sodium channel expression in A6 cells, a cell line derived from Xenopus kidney. Subunit specific antisense oligonucleotides have been generated from the sequences of the partial clones. The effectiveness of the oligonucleotides at suppressing protein expression will be assessed by looking at target mRNA and protein levels with Northern and Western analysis, respectively. Protein levels will be ascertained by Western analysis, and the suppression of sodium channel activity will be determined electrophysiologically. %%% The importance of epithelial sodium transport in maintaining proper organismic sodium levels is very clear. Understanding the structural correlates of function of the epithelial sodium channel will lead to a better understanding of sodium transport and its regulation. This research will determine whether a family of proteins encoded by three Xenopus clones represents the sodium channel responsible for regulated sodium transport in the distal nephron. It will be a first step in the investigation of structure-function relationships of the epithelial sodium channel. ***
