A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).

Abstract

G-protein gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain, heart, and adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). According to a leading concept, mutations in the pore region of GIRK4 cause loss of K+ selectivity; the ensuing Na+ influx depolarizes zona glomerulosa cells and activates voltage gated Ca2+ channels, inducing hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore region. We expressed GIRK4R52H, GIRK4E246K, and GIRK4G247R mutants in Xenopus oocytes and human adrenocortical carcinoma cell line (HAC15). Whole-cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K (but not GIRK1/4G247R) channels were greatly reduced compared to GIRK1/4WT. Nevertheless, all heterotetrameric mutants retained full K+ selectivity and inward rectification. When expressed as homotetramers, only GIRK4WT, but none of the mutants, produced whole-cell currents. Confocal imaging, single channel and Forster Resonance Energy Transfer (FRET) analyses showed: 1) reduction of membrane abundance of all mutated channels, especially as homotetramers, 2) impaired interaction with G{beta}{gamma} subunits, and 3) reduced open probability of GIRK1/4R52H. VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H and GIRK4E246K. Our results suggest impaired gating (GIRK4R52H) and expression in plasma membrane (all mutants). We suggest that, contrary to the previously proposed mechanism, R52H and E246K mutants are loss-of-function rather than gain-of-function/selectivity-loss mutants. Hence, GIRK4 openers may be a potential course of treatment for patients with cytosolic N- and C-terminal mutations. Significance StatementMutations in KCNJ5 gene, which encodes for the GIRK4 subunit of G-protein inwardly rectifying K+ channels, are the main cause of primary aldosteronism, a major contributor to secondary hypertension. We report that three mutations in the cytosolic domain of GIRK4 cause loss-of-function, contrary to the prevailing concept that these mutations cause loss of selectivity and subsequent depolarization, i.e. essentially gain-of-function. Our findings correct the existing misconception regarding the biophysical mechanism that impairs the channel function, and may provide indications for future personalized treatment of the disease.