PROTEIN PHOSPHATASE 2B-DEPENDENT PATHWAY OF INSULIN ACTION ON SINGLE CL- CHANNEL CONDUCTANCE IN RENAL EPITHELIUM

The apical membrane of distal nephron epithelium (A6) has a Ca2+-depen dent outwardly rectifying Cl- channel with single channel conductances of 3 pS for outward current and 1 pS for inward current under the bas al condition. The single channel conductance for inward currents incre ased as cytosolic Ca2+ concentration ([Ca2+](c)) was elevated, while t he single channel conductance for outward currents did not change at t he range of [Ca2+](c) from 10 nM to 1 mM. Insulin (100 nM) increased t he single channel conductance for the inward current by increasing the sensitivity to cytosolic Ca2+ by 400-fold, but did not affect the sin gle channel conductance for the outward current. Further, insulin incr eased the open probability of the channel. These effects of insulin we re completely blocked by cyclosporin-A, an inhibitor of protein phosph atase type 2B (PP2B) which dephosphorylates phospho-tyrosine in additi on to phosphoserine/threonine, but not by okadaic acid, an inhibitor o f protein phosphatase type 1 and 2A. Further, these effects of insulin were also completely blocked by W7, an antagonist of calmodulin which is required for activation of PP2B. Lavendustin A, an inhibitor of pr otein tyrosine kinase (PTK), mimicked these effects of insulin; this a ction of lavendustin A required 1 hr after its application, while with in 30 min after its application lavendustin A had no significant effec ts on the single channel conductance. On the other hand, lavendustin A blocked the insulin action for a relatively short time period (i.e., within 30 min after their application). However, H89 (an inhibitor of protein kinase A) or H7 (an inhibitor of protein kinases A, C and G) d id not mimic the insulin action. Application of PP2B or protein tyrosi ne phosphatase to the cytosolic surface of the inside-out patch membra ne increased the single channel conductance and the open probability a s did insulin in cell-attached patches. The insulin-induced increases in single channel conductance and open probability were reversibly dec reased by application of PTK catalytic subunit in the presence of ATP through a decrease in the sensitivity to cytosolic Ca2+ but not by pro tein kinase A. These observations suggest that as intracellular signal ling of insulin action, PP2B-mediated dephosphorylation of phospho-tyr osine of the channel protein (or channel-associated protein) is a nove l mechanism for regulation of single channel conductance, and that at least two different types of PTKs regulate the channel characteristics .