The ojective of the proposed study is to identify and characterize the molecular events involved in the physiological regulation of Na+ channels in toad bladder epithelium, a model system for the mammalian distal nephron. It is based on our recent success in developing a vesicle system which exhibits large amiloride blockable 22Na+ fluxes, reconstituting Na+ channels in liposomes, and identifying a Ca dependent process which down regulates Na+ channels by inducing a covalent modification of the apical membrane. The experiments suggested will test the possibilities that Na+ channels can be regulated by means of phosphorylation, methylation, membrane cytoskeleton interactions, and electrostatic interactions with H+, Ca2+ or Na+. The possible involvement of these processes in the activation of channels by aldosterone, ADH, and cellular metabolism will be assessed as well. In addition, we shall further improve the reconstitution system for use as an assay in the fractionation of apical proteins and partial purification of the Na+ channels. It is expected that the above experiments will clarify some of the events mediating the hormonal and intracellular regulation of Na+ channels in tight epithelia, and provide the basis for further molecular studies.
