ANALYSIS OF BASOLATERAL ION CHANNELS IN SKIN

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Despite much study little is known about the basolateral conductance in sodium transporting epithelia and of the factors regulating it in response to alterations in the rate of sodium transport. Although it is clearly established that most of the basolateral conductance in frog skin, a widely used model for sodium absorbing epithelia, is due to potassium, the nature of the potassium and other ion channels and their possible modulation by intracellular factors are unknown. Recent technical advances have made it possible to record single ion channel currents flowing through a small patch of cell membrane and to characterize unambiguously the components of complex macroscopic currents in isolated cells. Preliminary experiments have shown the feasibility of applying these "patch-clamp" techniques to enzymatically isolated viable epithelial cells from the frog skin. Experiments are proposed in intact isolated epithelia and dispersed cells to characterize the elementary properties (i.e. single-channels conductance, voltage- and time- dependent gating kinetics, ionic selectivity) of the ion channels that constitute the conductance pathways of the basolateral membrane. The effects on channel activity of possible intracellular regulating agents (pH, free Ca and Na concentrations, ATP) as well as the role of protein phosphorylation (by cAMP-dependent protein kinase) will be analyzed. Other experiments in intact epithelia treated with ionophores will characterize the macroscopic K current so that it will be possible to identify the channels responsible for the basolateral membrane K conductance under different conditions. Patch-clamp techniques will be used also to investigate the response of basolateral channels in isolated cells and intact epithelia to modifiers of Na transport (vasopressin, isoproterenol, etc.). The results of these experiments will help elucidate the mechanisms of ion transport and their regulation at the basolateral membrane of Na absorbing epithelia and will lead to a better understanding of the interactions between transepithelial transport and cell homeostasis.