Cellular and Functional Defects in Aldosteronism-linked Cytosolic Domain Mutations in GIRK4 (KCNJ5)

Abstract

GIRK4 (KCNJ5) is one of four subunits of the G-protein gated inwardly rectifying potassium channels (GIRK1-4). GIRK4 subunits are abundant in heart and adrenal cortex, and form homo- or heterotetramers with GIRK1. GIRK channels mediate inhibitory transmission via Gi/o-protein coupled receptors, with Gβγ subunits as their main gating factor. KCNJ5 mutations were causally linked to primary aldosteronism (PA). The leading concept for PA-related mutations in KCNJ5 is that mutations in the pore region cause loss of K+ selectivity, which increases Na+ conductance and membrane depolarization followed by Ca2+ influx and aldosterone secretion. This mechanism later was extended to mutations in cytosolic N- and C-terminal domains far from the selectivity filter: GIRK4R52H, GIRK4E246K, and GIRK4G247R. Previously, using Xenopus laevis oocytes, we have shown that the activity of heterotetrameric GIRK1/4R52H and GIRK1/4E246K channels was reduced. We also showed that the mutations did not affect the selectivity or inward rectification. Here, we show that the activity of GIRK1/4G247R was not different from GIRK1/4WT. We corroborated these results in human adrenal cortex cell line (HAC15). Confocal imaging revealed that abundance in plasma membrane of mutated homo- or heterotetrameric channels was significantly reduced. Moreover, mutated homotetrameric channels were non-functional; however, VU0529331, an opener of GIRK4 homotetramers, rescued the activity of GIRK4G247R. Using Fluorescence Resonance Energy Transfer (FRET), we demonstrate that interaction of Gβγ with GIRK1/4R52H and GIRK1/4E246K is significantly impaired in a wide range of acceptor/donor molar ratios. Interaction with GIRK1/4G247R was only slightly impaired. Our results suggest an impairment of both Gβγ interaction and expression for GIRK4R52H and GIRK4E246K, but mainly a reduction in expression for homotetrameric (and not heterotetrameric) GIRK4G247R. Furthermore, these results rule out a previously proposed mechanism for R52H and E246K mutants, and indicate potential ways of clinical treatment.