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 and heart, and are present in the 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 GIRK4_R52H , GIRK4_E246K and GIRK4_G247R mutants in Xenopus oocytes. Whole-cell currents of heterotetrameric GIRK1/4_R52H and GIRK1/4_E246K channels were greatly reduced compared with GIRK1/4_WT . Nevertheless, all heterotetrameric mutants retained full K⁺ selectivity and inward rectification. When expressed as homotetramers, only GIRK4_WT , 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 GIRK1/4_R52H . VU0529331, a GIRK4 opener, activated homotetrameric GIRK4_G247R channels, but not GIRK4_R52H or GIRK4_E246K . In the human adrenocortical carcinoma cell line (HAC15), VU0529331 and overexpression of heterotetrameric GIRK1/4_WT , but not overexpression 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. KEY POINTS: Mutations in GIRK4 (KCNJ5) G protein-gated channels cause primary aldosteronism, a major cause of secondary hypertension. The primary mechanism is believed to be loss of K⁺ selectivity. R52H and E246K, aldosteronism-causing mutations in cytosolic N- and C- termini of GIRK4, were reported to cause loss of K⁺ selectivity. We show that R52H, E246K and G247R mutations render homotetrameric GIRK channels non-functional. In heterotetrameric context with GIRK1, these mutations impair membrane expression, interaction with Gβγ and open probability, but do not alter K⁺ selectivity or inward rectification. In the human aldosterone-secreting cell line, a GIRK4 opener and overexpression of heterotetrameric GIRK1/4_WT , but not overexpression of GIRK1/4 mutants, reduced aldosterone secretion. Aldosteronism-causing mutations in the cytosolic domain of GIRK4 are loss-of-function mutations rather than gain-of-function, selectivity-loss mutations. Deciphering of exact biophysical mechanism that impairs the channel is crucial for setting the course of treatment.