Maintenance of extracellular fluid volume homeostasis is essential for hemodynamic stability, and abnormalities of renal sodium handling have been linked to cardiovascular disease and hypertension. Ultimate regulation of sodium excretion in the kidney occurs in the distal nephron and is modulated by the mineralocorticoid aldosterone. The long-term goal of this research are to understand the cellular mechanisms by which aldosterone regulates sodium reabsorption. The initial physiologic event in aldosterone action is an increase in apical membrane permeability due to an increase in number and open probability of conductive sodium channels. This study proposes to study the biochemical mechanisms underlying this channel activation. Carboxymethylation of proteins is known to be stimulated by aldosterone but the exact site is in dispute. This study will characterize the aldosterone dependent carboxymethylation of specific subunits of the sodium channel and employ the techniques of deletion and site specific mutagenesis of in vitro transcribed peptides to determine the specific site of the gating carboxymethylation. These experiments will be paralleled by reconstitution studies in lipid bilayers to validate the physiologic relevance of the methylation site. Studies of the regulation of the methyltransferase and its specific substrate will be carried out to determine the nature of the aldosterone regulated step. Since channel subunits are apparent final effector or substrate for aldosterone regulation, studies will be carried out to define the time course and nature of aldosterone regulation of subunit composition in the apical membrane. Finally, studies will be initiated to examine the transcriptional activation by mineralocorticoids and glucocorticoids both in terms of receptor translocation, transport activation and biochemical targets.
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