Inhibition of K(+) Transport through Na(+), K(+)-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

Capsazepine (CPZ) inhibits Na(+),K(+)-ATPase-mediated K(+)-dependent ATP hydrolysis with no effect on Na(+)-ATPase activity. In this study we have investigated the functional effects of CPZ on Na(+),K(+)-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na(+),K(+)-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na(+),K(+)-ATPase current in the presence of extracellular K(+). In contrast, CPZ stimulated pump current in the absence of extracellular K(+). Similar conclusions were attained using HEK293 cells loaded with the Na(+) sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na(+),K(+)-ATPase indicated that CPZ stabilizes ion interaction with the K(+) sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na(+) congener at the Na(+) specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na(+),K(+)-ATPase. CPZ increased oxonol VI fluorescence in the absence of K(+), reflecting increased Na(+) efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K(+), likely indicating opening of an intracellular pathway selective for K(+). As revealed by the recent crystal structure of the E(1).AlF(4) (-).ADP.3Na(+) form of the pig kidney Na(+),K(+)-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K(+) sites in Na(+),K(+)-ATPase.