MOLECULAR MECHANISMS OF EPITHELIAL SODIUM CHANNEL REGULATION

Yale University, New Haven CT

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Abstract

Maintenance and variations in blood pressure levels are highly dependent on the total Na+ reabsorbed from the surrounding milieu. The amiloride- sensitive epithelial Na+ channel plays a fundamental role in determining the net amount of Na+ reabsorbed and thus, it is the target for many regulatory mechanisms. This proposal focuses on several aspects of the functional regulation of the amiloride-sensitive epithelial Na+ channel. The aims we are going to pursue are the following: AIM 1. Biosynthesis of Na+ channels and regulation by aldosterone. Effects of aldosterone and of salt intake in the biosynthesis of Na+ channels in the whole animal (in vivo) and in primary cultures of cortical collecting tubules (in vitro). Aldosterone effects will be examined at several time points over a time course in both the in vivo and the in vitro models. The following levels on the biosynthesis of channels will be examined:a) Transcriptional activation of the individual subunits. b)Synthesis of new channel proteins: rate of synthesis, degradation and lifetimes of the individual subunits. c)Assembly of the channel complex, distribution in different cellular compartments and cell surface expression. AIM 2. Modulation of channel activity by phosphorylation. To demonstrate biochemically protein phosphorylation and to identify the cellular pathways and kinases that mediate channel phosphorylation. a)To determine the subunit(s) that are phosphorylated and to identify the amino acids that are phosphorylated by specific kinases. b)To study the changes in channel activity induced by phosphorylation by proteinkinases (PKA, PKC, Ca2+/calmoduline, kinase, thyrosine kinases). c)Functional consequences of replacing the phosphorylated amino acids by other residues. AIM 3. Isolation of other proteins that associate with the Na+ channel. Ion channels are complex multimeric proteins with a general structure consisting of pore-forming subunit(s) and other associated proteins involved in the modulation of channel activity. Here we propose: a)To isolate and identify proteins associated to the subunits of the Na+ channel. b)To examine the functional roles of these proteins in modulating the activity of channels. c)To investigate aldosterone effects on the levels of expression of the associated proteins. d)To study the molecular determinants of the protein- protein interactions and the cellular processes that change these interaction.