Role of Basolateral K ir 4.1/K ir 5.1 Channel in the Regulation of Electrolyte Balance and ENaC Activity in the Cortical Collecting Duct

Abstract

Recent genetic studies identified a critical role of basolateral inwardly rectifying K+ (Kir) channels of the distal nephron in K+ homeostasis and blood pressure control, making these channels attractive targets for hypertension treatment. The main aim of the present study was to examine the effect of established and newly discovered inhibitors of basolateral Kir channels amitriptyline, fluoxetine, and VU0134992 on the epithelial sodium channel (ENaC) activity in the cortical collecting duct (CCD). We show that inhibition of heteromeric Kir4.1/Kir5.1, but not homomeric Kir4.1 channel, substantially suppressed both amiloride-sensitive equivalent short-circuit current in cultured polarized epithelial mCCDcl1 cells and single-channel ENaC activity in principal cells of rat and human CCD tubules. Using the combination of confocal microscopy, FluoVolt™ voltage-sensitive dye, and Fluo8HT Ca2+ dye, we show that acute application of amitriptyline or VU992 in concentrations that produce an inhibitory effect on Kir4.1/Kir5.1 channel resulted in substantial depolarization of cellular membrane potential and increase in intracellular Ca2+ level in mCCDcl1 cells that can explain a significant decrease of ENaC open probability without altering channel conductance induced by these drugs. Furthermore, we evaluated the effect of nonspecific Kir inhibitor amitriptyline on electrolyte homeostasis and apical Na+ transporters' expression in the distal nephron in Dahl salt-sensitive (SS) rats. The administration of amitriptyline for three days led to a significant drop in plasma K+, an increase in Na+ and K+ excretion, and diuresis. These changes were accompanied by the compensatory overexpression of ENaC and total and phosphorylated forms of thiazide-sensitive Na+-Cl-- cotransporter. Our data uncovered the putative mechanism of the impact of basolateral Kir4.1/Kir5.1 channel inhibition on ENaC activity and further emphasized a specific role of this channel in regulation of blood K+ level and electrolyte homeostasis.