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

Abstract

ClassificationBiological Sciences: Physiology. AbstractG-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 Ca 2+ 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 wholecell 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. 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-offunction 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 gainof-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.

How do Kir3.4 mutations cause hereditary hyperaldosteronism?

“…Shalomov et al . (2022) confirmed expression of the Kir3.4 protein in a human adrenocortical carcinoma cell line (HAC15), and showed that the overexpression of Kir3.4 wt, but not of the Kir3.4 R52H mutant, reduced aldosterone secretion. This is good supporting evidence.…”

“…They observed that G247R responded well to VU0529331, a Kir3.4 channel opener, proposing a potential new therapeutic approach. Taken together, they concluded that the pathophysiological mechanism of Kir3.4 mutants R52H, E246K and G247R in the cytosolic region is due not to loss of ion selectivity, but to reduced function/expression (Shalomov et al 2022).…”

“…Taken together, they concluded that the pathophysiological mechanism of Kir3.4 mutants R52H, E246K and G247R in the cytosolic region is due not to loss of ion selectivity, but to reduced function/expression (Shalomov et al . 2022).…”

“…(2011) and the present study by Shalomov et al . (2022), it is now clear that there are two types of pathophysiological mechanisms for hereditary hyperaldosteronism associated with Kir3.4 mutations, i.e. loss of ion selectivity caused by pore mutations and reduced function/expression caused by cytosolic mutations.…”

A novel small-molecule selective activator of homomeric GIRK4 channels

“…Loss of selectivity mutations, such as GIRK4 G151R, T158A, and L168R, which are located at or near the selectivity filter, were reported to yield K + /Na + nonselective GIRK4 and GIRK1/4 channels ( 7 ). GIRK4 mutations R52H, E246K, and G247R, which are located in the cytosolic N-terminal and C-terminal domains, resulted in a loss-of-function phenotype ( 8 ). Therefore, GIRK4 activators could serve as a potential treatment for patients with PA.…”