Abstract

G-protein gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain and heart, and are present in adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). Mutations in the pore region of GIRK4 cause loss of K+ selectivity, Na+ influx, and depolarization of zona glomerulosa cells followed by hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore. We expressed aldosteronism-linked GIRK4R52H, GIRK4E246K, and GIRK4G247R mutants in Xenopus oocytes. Whole-cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K 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 Förster Resonance Energy Transfer (FRET) analyses showed: 1) reduction of membrane abundance of all mutated channels, especially as homotetramers, 2) impaired interaction with Gβγ subunits, and 3) reduced open probability of GIRK1/4R52H. VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H and GIRK4E246K. In human adrenocortical carcinoma cell line (HAC15), VU0529331 and over-expression of heterotetrameric GIRK1/4WT, but not over-expression of GIRK1/4 mutants, reduced aldosterone secretion. Our results suggest that, contrary to pore mutants of GIRK4, non-pore mutants 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.

Abstract Figure: There are two mutations types in KCNJ5 (GIRK4) that can cause excessive secretion of aldosterone, leading to primary aldosteronism. Mutations of the first type render the channel non-selective to monovalent cations and often constitutively active, thus depolarizing the zona granulosa cells. This previously described mechanism underlies the disease-causing effects of mutations of amino acid residues located in the pore region (red color). Blockers of the channel may be useful as potential treatment to reduce aldosterone secretion. Here we show that mutations of the second type, located in the cytosolic domain remote from the pore, act by a different mechanism. They do not alter channel's ion selectivity or rectification but cause poor expression or poor activation by Gβγ, resulting in a reduction in cell's K+ conductance and depolarization. In this case, GIRK4 openers can potentially be useful to prevent the excessive aldosterone secretion.

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