Role of the sodium pump and the background K+ channel in passive K+(Rb+) uptake by isolated cardiac sarcolemmal vesicles

Abstract

A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of 86Rb+ by the preparation was measured in the presence of an opposing K+ gradient using a rapid ion exchange technique. At low extravesicular Rb+ concentrations, at least 50% of the uptake was blocked by addition of 1 mM ouabain to the assay medium. Orthovanadate (50 microM), ADP (2.5 mM) or Mg (1 mM) were also partial inhibitors of Rb+ uptake under these conditions, and produced a complete block of Rb+ influx in the presence of 1 mM ouabain. When 86Rb+ was used as a tracer of extravesicular K+ (Rb+0 less than or equal to 40 microM, K+0 = 0.1-5 mM) a distinct uptake pathway emerged, as detected by its inhibition by 1 mM Ba2+ (K0.5 = 20 microM). At a constant internal K+ concentration (K+in = 50 mM), the magnitude of the Ba2+-sensitive K+ uptake was found to depend on K+0 in a manner that closely resembles the K+ concentration dependence of the background K+ conductance (IK1) observed electrophysiologically in intact cardiac cells. We conclude that K+ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.