The goals of this proposal are to examine mechanisms by which Na+ transport across renal tissue can be controlled or regulated at a cellular level. The two tissues which will be used for this work are cells from the cortical collecting tubule of rat and cells from the amphibian distal nephron line, A6. Both of these tissues display trans-epithelial sodium transport which can be induced by the hormone, aldosterone, and blocked by the diuretic, amiloride. The primary emphasis of this proposal will be an examination of apical Na+ channels using patch-voltage clamp methods. The rationale for the experiments is that the patch clamp method allows unambiguous identification and characterization of single transport proteins uncomplicated by interaction with other processes. Also, patch-clamp allows access to the inner surface of the cell membrane which is in general inaccessible in intact tissue. Such accessibility allows an examination of the role of intracellular factors in the normal function and control of the ion transport channels.
The specific aims of the project are twofold. First, to characterize the channels which are present in the epithelial cell membranes in terms of their selectivity to various ions, their opening and closing kinetics and voltage dependence (if any). The second phase of the project will examine mechanisms by which Na+ transport may be physiologically regulated at a cellular level. In this phase, the possible regulatory roles of the physiologicical ions, Na+, Ca++, and H+, will be examined. Also the effect on single Na channels of the hormone regulators, aldosterone, ADH, and ANF will be investigated. Finally, the possibility that natural proteolytic agents, such as kallikrein, might contribute to the normal regulation of the channel will be investigated. The primary hypothesis which motivates the second phase of the proposal is that the majority of the increase in Na transport induced by aldosterone can be attributed to the conversion of non-functional Na channels which are present in the apical membrane prior to exposure to aldosterone into functional, high selectivity Na channels. ADH, on the other hand, produces its effects primarily by promoting the incorporation of new channel proteins in the membrane.
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