FEEDBACK-REGULATION OF NA CHANNELS IN RAT CCT .2. EFFECTS OF INHIBITION OF NA ENTRY

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (P(o)), and single-channel currents (i) were measured at 37-degrees-C during continuous superfusion of the tubule. Addition of amiloride (10 muM) or benzamil (0.5 muM) to the superfusate resulted in a twofold increase in the mean number of open channels (NP(o)) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NP(o) reversed within 3 min after removal of amiloride. The increase in NP(o) was accounted for, at least in part, by an increase in P(o). Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NP(o). No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel P(o) by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.