Membrane cholesterol extraction decreases Na+transport in A6 renal epithelia

Abstract

In this study, we have investigated the dependence of Na+transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current ( Isc), transepithelial conductance ( GT), and transepithelial capacitance ( CT) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl-β-cyclodextrin (mβCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mβCD treatment. However, apical mβCD application hampered the responses of Iscand GTto hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in Iscdemonstrated that apical mβCD treatment decreased the epithelial Na+channel (ENaC) open probability ( Po) in the steady state as well as after activation of Na+transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by CTmeasurements. Na+transport activation by hypotonicity was abolished during basolateral mβCD treatment as a result of reduced Na+/K+pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na+/K+pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na+transport by reducing ENaC Po.