Abstract
Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.
Highlights
The epithelial sodium channel (ENaC)1 provides the ratelimiting step in the absorption of sodium ions in the distal nephron and functions to maintain Naϩ ion homeostasis [1, 2]
We show that syntaxins 1A and 3 interact with and functionally regulate rat ENaC complexes when they are co-expressed in Xenopus oocytes
Effect of Syntaxins 1A and 3 on Amiloride-sensitive Currents—To test the hypothesis that syntaxins functionally regulate ENaC, amiloride-sensitive currents were assayed in Xenopus oocytes expressing ENaC with or without co-expression of syntaxin 1A and 3 (Fig. 1)
Summary
The epithelial sodium channel (ENaC)1 provides the ratelimiting step in the absorption of sodium ions in the distal nephron and functions to maintain Naϩ ion homeostasis [1, 2]. We show that syntaxins 1A and 3 interact with and functionally regulate rat ENaC complexes when they are co-expressed in Xenopus oocytes. Similar functional results were observed when syntaxin 1A was co-expressed with an ENaC complex containing a COOHterminal truncated  subunit (as occurs in Liddle’s syndrome [7]).
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