Andersen-Tawil syndrome (ATS) type-1 is associated with loss-of-function mutations in KCNJ2 gene. KCNJ2 encodes the tetrameric inward-rectifier potassium channel Kir2.1, important to the resting phase of the cardiac action potential. Kir-channels' activity requires interaction with the agonist phosphatidylinositol-4,5-bisphosphate (PIP 2). Two mutations were identified in ATS patients, V77E in the cytosolic N-terminal "slide helix" and M307V in the C-terminal cytoplasmic gate structure "G-loop." Current recordings in Kir2.1-expressing HEK cells showed that each of the two mutations caused Kir2.1 lossof-function. Biotinylation and immunostaining showed that protein expression and trafficking of Kir2.1 to the plasma membrane were not affected by the mutations. To test the functional effect of the mutants in a heterozygote set, Kir2.1 dimers were prepared. Each dimer was composed of two Kir2.1 subunits joined with a flexible linker (i.e. WT-WT, WT dimer; WT-V77E and WT-M307V, mutant dimer). A tetrameric assembly of Kir2.1 is expected to include two dimers. The protein expression and the current density of WT dimer were equally reduced to~25% of the WT monomer. Measurements from HEK cells and Xenopus oocytes showed that the expression of either WT-V77E or WT-M307V yielded currents of only about 20% compared to the WT dimer, supporting a dominant-negative effect of the mutants. Kir2.1 sensitivity to PIP 2 was examined by activating the PIP 2 specific voltage-sensitive phosphatase (VSP) that induced PIP 2 depletion during current recordings, in HEK cells and Xenopus oocytes. PIP 2 depletion induced a stronger and faster decay in Kir2.1 mutant dimers current compared to the WT dimer. BGP-15, a drug that has been demonstrated to have an anti-arrhythmic effect in mice, stabilized the Kir2.1 current amplitude following VSP-induced PIP 2 depletion in cells expressing WT or mutant dimers. This study underlines the implication of mutations in cytoplasmic regions of Kir2.1. A newly developed calibrated VSP activation protocol enabled a quantitative assessment of changes in PIP 2 regulation caused by the mutations. The results suggest an impaired function and a dominant-negative effect of the Kir2.

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.

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 GIRK4R52H, GIRK4E246K and GIRK4G247R mutants in Xenopus oocytes. Whole‐cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K channels were greatly reduced compared with 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 or GIRK4E246K. In the human adrenocortical carcinoma cell line (HAC15), VU0529331 and overexpression of heterotetrameric GIRK1/4WT, 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 GIRK1/4WT, 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.

The G protein-activated Inwardly Rectifying K +-channel (GIRK) modulates heart rate and neuronal excitability. Following G-Protein Coupled Receptor (GPCR)-mediated activation of heterotrimeric G proteins (Gabg), opening of the channel is obtained by direct binding of Gbg subunits. Interestingly, GIRKs are solely activated by Gbg subunits released from Ga i/ocoupled GPCRs, despite the fact that all receptor types, for instance Ga q-coupled, are also able to provide Gbg subunits. It is proposed that this specificity and fast kinetics of activation stem from pre-coupling (or pre-assembly) of proteins within this signaling cascade. However, many studies, including our own, point towards a diffusion-limited mechanism, namely collision coupling. Here, we set out to address this long-standing question by combining electrophysiology, imaging, and mathematical modeling. Muscarinic-2 receptors (M2R) and neuronal GIRK1/2 channels were coexpressed in Xenopus laevis oocytes, where we monitored protein surface expression, current amplitude, and activation kinetics. Densities of expressed M2R were assessed using a fluorescently labeled GIRK channel as a molecular ruler. We then incorporated our results, along with available kinetic data reported for the G-protein cycle and for GIRK1/2 activation, to generate a comprehensive mathematical model for the M2R-G-protein-GIRK1/2 signaling cascade. We find that, without assuming any irreversible interactions, our collision coupling kinetic model faithfully reproduces the rate of channel activation, the changes in agonist-evoked currents and the acceleration of channel activation by increased receptor densities.