Sodium reabsorbing epithelia, such as renal distal and collecting tubules, have as their major function the control of whole-body sodium balance. These epithelia contain apical membrane sodium channels that are inhibited by the diuretic amiloride. It is at the level of these channels that the primary feedback control mechanisms necessary for the maintenance of sodium homeostasis occur. The biochemical and molecular characteristics of these important ion channels, the mechanisms involved in hormonal modulation of these channels, and their involvement in pathophysiological processes, such as Liddle's hypertension, are poorly understood. The long-term goal of this project remains to understand at the molecular level the mechanisms responsible for the regulation of ion flow through these conductive sodium entry pathways. During the previous grant period, two novel observations were made that form the basis of this continuation application. First, short actin filaments interact with cloned epithelial sodium channels (ENaCs) producing major alterations in channel conductance, cation permeability, gating kinetics, and protein kinase A sensitivity. Second, small peptides comprising the terminal beta- and gamma-ENaC tails can act as channel blocking particles. Therefore, we propose (1) to test the hypothesis that the effects of the cytoskeletal protein actin on the gating, cation selectivity, and conductance properties of ENaC are due to a direct interaction with the alpha-subunit of ENaC, and are mediated by calcium; and (2) to test the hypothesis that the terminal cytoplasmic tails of the beta- and gamma-ENaC subunits act as intrinsic regulatory elements by serving as inactivation peptides that bind to a conserved blocking domain within ENaC. Important elements of this study are that unique biochemical, physiological, and molecular biological characteristics of ENaCs will be elucidated, regions of protein sequence that participate in specific channel functions will be identified, and a picture of the gross molecular architecture of a functional ENaC will be developed. These results will offer new insights into the nature of amiloride- sensitive sodium channels, the way they are modified by interaction with associated proteins, and the basis for understanding how alterations in these regulatory pathways contribute directly to the genetic basis of certain forms of essential hypertension such as Liddle's Syndrome.
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