Kir5.1 Is Associated with NEDD4‐2 And Is Required for High K+ Intake Induced Inhibition of Kir4.1 and NCC

Abstract

Dietary K+ intake plays an important role in regulating NCC such that a high K+ intake (HK) decreases while a low K+ intake increases NCC. Our previous study has demonstrated that Kir4.1 plays an important role in mediating the effect of dietary K+ intake on NCC expression and activity since the deletion of Kir4.1 not only caused K+ wasting but also abolished the effect of dietary K+ intake on NCC. While Kir4.1 forms the heterotetramer with Kir5.1 and is responsible for providing K+ conductance of the basolateral membrane in the DCT, the function of Kir5.1 in the heterotetramer is not completely understood. GST‐pull‐down and immunoprecipitation have shown that Kir5.1 is associated with the Nedd4‐2 with its second WW domain on phosphothreonine‐binding motif (TPVT) of Kir5.1 at its c‐terminus (AA249–252). Ubiquitin assay and patch‐clamp experiments demonstrate that co‐expression of Nedd4‐2 decreases the expression of Kir4.1 and its currents in HEK293 cells transfected with Kir4.1/5.1 but not in the absence of Kir5.1. We now test the hypothesis that Kir5.1 is required for the effect of HK intake on the basolateral K+ channel activity of the DCT and apical NCC function. Immunostaining and western blot show that Kir4.1 expression is upregulated in Kir5.1−/− mice. Moreover, the patch‐clamp experiments demonstrate that the deletion of Kir5.1 increases the basolateral K+ conductance of the DCT and increased the negativity of DCT membrane potential (hyperpolarization). Renal clearance study has also demonstrated that thiazide‐induced natriuretic effect in Kir5.1−/− mice is significantly larger than in WT mice, indicating the upregulation of NCC in Kir5.1−/− mice. HK intake significantly decreases the basolateral K+ conductance in the DCT, depolarized the DCT membrane and inhibited NCC activity in WT mice. However, HK intake fails to decrease the basolateral K+ conductance in the DCT in Kir5.1−/− mice. Moreover, renal clearance experiments demonstrate that HK intake attenuates thiazide‐induced natriuresis only in WT mice but not in Kir5.1 knockout mice, suggesting that Kir5.1 is important in mediating HK intake‐induced inhibition of NCC. We conclude that Kir5.1 is a binding partner of Nedd4‐2 which plays a role in mediating the effect of HK intake on the basolateral Kir4.1/5.1 activity in the DCT. We speculate that HK intake may stimulate the Kir5.1/Nedd4‐2‐dependent down‐regulation of the basolateral Kir4.1 in the DCT thereby facilitating the down‐regulation of NCC.Support or Funding InformationNIH‐DK115366This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.