Effects of basolateral ouabain, amphotericin B, cyanide and potassium on amiloride noise during voltage clamp of Rana pipiens skin support sodium-amiloride competition

Abstract

In a previous study, the amiloride-induced corner frequency ( f c) was found to decrease as apical sodium was increased. This effect was small or absent when the basolateral surface was exposed to high potassium. It has been suggested that the apical sodium effect may be indirect, due either to increased intracellular [Na +] which repelled amiloride or to an increased potential at the apical surface which reduced amiloride affinity. High basolateral K + might then suppress the sodium effect either by preventing intracellular [Na +] from increasing or by allowing a better clamp of the apical membrane potential by reducing basolateral membrane resistance and potential. We checked the effects of basolateral [K +], of cyanide and of ouabain at concentrations known to increase intracellular [Na +]. We found only negligible effects on f c. In addition, amphotericin B added to the basolateral bathing solution either in 115 mM Na + or in 120 mM K + had no significant effect on f c. We found that relatively wide variation in clamp potential under all conditions, even with active transport severely inhibited, left f c virtually constant. Since the amiloride kinetics were independent of clamp potential, we were able to measure paracellular and transcellular conductances separately by examining the voltage dependence of clamp current (linear) and amiloride noise power (quadratic). This made possible estimation of channel density and single-channel current.