Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigaed the dependence of Na+ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I-SC), transepithelial conductance (G(T)), 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-beta-cyclodextrin (m beta CD) 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 m beta CD treatment. However, apical m beta CD application hampered the responses of I-SC and G(T) to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I-SC demonstrated that apical m beta CD treatment decreased the epithelial Na+ channel (ENaC) open probability (P-O) 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 CT measurements. Na+ transport activation by hypotonicity was abolished during basolateral m beta CD 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 P-O.