With‐No‐Lysine kinase ‐1 increases TRPV4 calcium channel activity in the collecting duct cells

Abstract

Kidneys play a central role in regulation of potassium homeostasis and maintaining plasma K+ levels within a narrow physiological range. Dietary K+ load increases circulating levels of the mineralocorticoid aldosterone leading to stimulation of K+ secretion via the calcium-activated large conductive maxi-K+ (BK) channels in the collecting duct and kaliuresis. With-no-lysine (WNK) kinases, specifically WNK1 and WNK4 have been recognized to regulate K+ balance, in part, by orchestrating BK-dependent K+ secretion in the aldosterone sensitive renal nephron. We have recently demonstrated that Ca2+-permeable TRPV4 channel is essential for BK activation in the distal nephron. Furthermore, high K+ diet increases TRPV4 activity and expression largely in an aldosterone-dependent manner. In the current study, we aimed to test whether WNK1/4 contribute to regulation of TRPV4 by aldosterone. As expected, treatment of mpkCCDc14 cells with 1 µM aldosterone for 24 h increased TRPV4-dependent Ca2+ influx by approximately 2 folds. Inhibition of WNK1/4 with the pan-blocker of WNK, WNK463 (100 nM for 24h) decreased basal TRPV4 activity by approximately 30% and virtually abolished stimulation of TRPV4 by aldosterone. Similarly, WNK1 blockade with WNK-in-11 (400 nM, 24 h) produced comparable inhibitory effects on the basal and aldosterone-dependent TRPV4 activity as WNK463. Immunofluorescent confocal analysis revealed an overall greater TRPV4-reporting signal and plasma membrane (highlighted with WGA) TRPV4 accumulation in aldosterone-treated mpkCCDc14 cells. These effects were not observed when mpkCCDc14 were treated with aldosterone + WNK-in-11. Co-expression of TRPV4 and WNK1 into Chinese hamster ovary (CHO) cells increased the macroscopic TRPV4-dependent cation currents from 160±10 pA/pF to 250±22 pA/pF. In contrast, overexpression of TRPV4 with a dominant negative WNK1 variant (K233M) decreased the whole cell currents to 60±5 pA/pF suggesting both stimulatory and permissive roles of WNK1 in regulation of TRPV4. Overall, we show that WNK1 (and possibly WNK4) is essential for control of basal TRPV4 activity and its activation by aldosterone in collecting duct cells. We propose that this new mechanism is likely contributing to regulation of urinary K+ levels to maintain systemic homeostasis.