A novel small-molecule selective activator of homomeric GIRK4 channels

Abstract

G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity. G protein–sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2-(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylmethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the transmembrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5-bisphosphate binding site, and show that it causes channel activation by strengthening channel–phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity. G protein–gated inwardly rectifying potassium (GIRK) channels mediate the inhibitory effects of various neurotransmitters acting through G protein–coupled receptors and G proteins present in excitable cells, such as in the heart and nervous system (1Cui M. Cantwell L. Zorn A. Logothetis D.E. Kir channel molecular physiology, pharmacology, and therapeutic implications.Handb. Exp. Pharmacol. 2021; 267: 277-356Crossref PubMed Scopus (17) Google Scholar). Four GIRK channel family members (GIRK1–GIRK4) have been identified, but each is expressed either in homotetramers (GIRK2 and GIRK4) or in heterotetramers with the nonfunctional homomeric subunits (GIRK1 and GIRK3) (1Cui M. Cantwell L. Zorn A. Logothetis D.E. Kir channel molecular physiology, pharmacology, and therapeutic implications.Handb. Exp. Pharmacol. 2021; 267: 277-356Crossref PubMed Scopus (17) Google Scholar). The GIRK1 subunit, although inactive by itself, enhances heteromeric channel activity when associated with either GIRK4 or GIRK2 subunits. The cardiac GIRK channel (IKACh) is a heterotetramer composed of GIRK1 and GIRK4 subunits expressed in the atria and pacemaking cells, and its activation slows heart rate (1Cui M. Cantwell L. Zorn A. Logothetis D.E. Kir channel molecular physiology, pharmacology, and therapeutic implications.Handb. Exp. Pharmacol. 2021; 267: 277-356Crossref PubMed Scopus (17) Google Scholar). GIRK4 (or G4) homotetramers have also been reported to be expressed in atrial cells (2Wickman K. Nemec J. Gendler S.J. Clapham D.E. Abnormal heart rate regulation in GIRK4 knockout mice.Neuron. 1998; 20: 103-114Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), where vagally released acetylcholine (ACh) activates the ACh-regulated potassium current, IKACh (3Dobrev D. Friedrich A. Voigt N. Jost N. Wettwer E. Christ T. et al.The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation.Circulation. 2005; 112: 3697-3706Crossref PubMed Scopus (386) Google Scholar). It has been demonstrated that atrial fibrillation cannot be induced in GIRK4 knockout mice, suggesting the critical involvement of this current in atrial fibrillation (3Dobrev D. Friedrich A. Voigt N. Jost N. Wettwer E. Christ T. et al.The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation.Circulation. 2005; 112: 3697-3706Crossref PubMed Scopus (386) Google Scholar). Thus, GIRK1/4 inhibitors could act as potentially promising antiarrhythmic agents. Although GIRK4 is not abundantly expressed in the brain (4Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (103) Google Scholar), expression has also been detected in various neuronal populations, particularly in the pro-opiomelanocortin (POMC) and the ventromedial nucleus (VMN) neurons of the hypothalamus (5Perry C.A. Pravetoni M. Teske J.A. Aguado C. Erickson D.J. Medrano J.F. et al.Predisposition to late-onset obesity in GIRK4 knockout mice.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8148-8153Crossref PubMed Scopus (45) Google Scholar). Studies found that GIRK4 knockout mice were predisposed to late-onset obesity, by exhibiting greater food intake and a decrease in energy expenditure compared with wildtype mice. Similarly, inhibition of POMC and VMN neurons reduces energy expenditure and promotes food intake (5Perry C.A. Pravetoni M. Teske J.A. Aguado C. Erickson D.J. Medrano J.F. et al.Predisposition to late-onset obesity in GIRK4 knockout mice.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8148-8153Crossref PubMed Scopus (45) Google Scholar). Because GIRK4 channels reside in the POMC and VMN neurons, it is likely that GIRK4 is linked to changes in metabolic function and reduced satiety, leading to obesity. In addition to the heart and brain, GIRK4 homomeric channels are also expressed in the adrenal cortex. Multiple mutations of GIRK4 channel cause primary aldosteronism (PA), which is a disease characterized by hypersecretion of aldosterone. PA is the most common cause of secondary hypertension and accounts for approximately 10% of patients with newly diagnosed hypertension (6Fernandes-Rosa F.L. Boulkroun S. Zennaro M.C. Somatic and inherited mutations in primary aldosteronism.J. Mol. Endocrinol. 2017; 59: R47-R63Crossref PubMed Scopus (43) Google Scholar). 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 (7Choi M. Scholl U.I. Yue P. Bjorklund P. Zhao B. Nelson-Williams C. et al.K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension.Science. 2011; 331: 768-772Crossref PubMed Scopus (814) Google Scholar). 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 (8Shalomov B. Handklo-Jamal R. Reddy H.P. Theodor N. Bera A.K. Dascal N. A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).bioRxiv. 2019; ([preprint])https://doi.org/10.1101/866202Crossref Scopus (0) Google Scholar). Therefore, GIRK4 activators could serve as a potential treatment for patients with PA. GIRK channels are gated by the Gβɣ dimer of GTP-binding proteins (9Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart.Nature. 1987; 325: 321-326Crossref PubMed Scopus (941) Google Scholar), which bind the cytoplasmic domain between the DE and LM loops (Fig. S1) of two adjacent subunits (10Mahajan R. Ha J. Zhang M. Kawano T. Kozasa T. Logothetis D.E. A computational model predicts that Gbetagamma acts at a cleft between channel subunits to activate GIRK1 channels.Sci. Signal. 2013; 6: ra69Crossref PubMed Scopus (23) Google Scholar, 11Whorton M.R. MacKinnon R. X-ray structure of the mammalian GIRK2-betagamma G-protein complex.Nature. 2013; 498: 190-197Crossref PubMed Scopus (247) Google Scholar). Another intracellular channel activator is Na+, which binds within the CD loop (Fig. S1) of GIRK2 and GIRK4 that possess a critical Asp residue (12Zhang H. He C. Yan X. Mirshahi T. Logothetis D.E. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.Nat. Cell Biol. 1999; 1: 183-188Crossref PubMed Scopus (303) Google Scholar, 13Ho I.H. Murrell-Lagnado R.D. Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels.J. Physiol. 1999; 520: 645-651Crossref PubMed Scopus (88) Google Scholar, 14Ho I.H. Murrell-Lagnado R.D. Molecular determinants for sodium-dependent activation of G protein-gated K+ channels.J. Biol. Chem. 1999; 274: 8639-8648Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Rosenhouse-Dantsker A. Sui J.L. Zhao Q. Rusinova R. Rodriguez-Menchaca A.A. Zhang Z. et al.A sodium-mediated structural switch that controls the sensitivity of Kir channels to PtdIns(4,5)P(2).Nat. Chem. Biol. 2008; 4: 624-631Crossref PubMed Scopus (49) Google Scholar), which coordinates Na+. Gβγ allosterically regulates interactions of the channel with the lipid molecule phosphatidylinositol-4,5-bisphosphate (PIP2) (12Zhang H. He C. Yan X. Mirshahi T. Logothetis D.E. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.Nat. Cell Biol. 1999; 1: 183-188Crossref PubMed Scopus (303) Google Scholar, 16Huang C.L. Feng S. Hilgemann D.W. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma.Nature. 1998; 391: 803-806Crossref PubMed Scopus (782) Google Scholar, 17Li D. Jin T. Gazgalis D. Cui M. Logothetis D.E. On the mechanism of GIRK2 channel gating by phosphatidylinositol bisphosphate, sodium, and the Gbetagamma dimer.J. Biol. Chem. 2019; 294: 18934-18948Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Na+ has also been found to work by increasing the affinity of the channel to PIP2 (12Zhang H. He C. Yan X. Mirshahi T. Logothetis D.E. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.Nat. Cell Biol. 1999; 1: 183-188Crossref PubMed Scopus (303) Google Scholar, 13Ho I.H. Murrell-Lagnado R.D. Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels.J. Physiol. 1999; 520: 645-651Crossref PubMed Scopus (88) Google Scholar, 14Ho I.H. Murrell-Lagnado R.D. Molecular determinants for sodium-dependent activation of G protein-gated K+ channels.J. Biol. Chem. 1999; 274: 8639-8648Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Direct interactions of PIP2 with the channel stabilize the GIRK channel gates, the HBC (helix bundle crossing), and the cytosolic G loop, in the open state (18Whorton M.R. MacKinnon R. Crystal structure of the mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium.Cell. 2011; 147: 199-208Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 19Niu Y. Tao X. Touhara K.K. MacKinnon R. Cryo-EM analysis of PIP2 regulation in mammalian GIRK channels.Elife. 2020; 9e60552Crossref Google Scholar). The simultaneous presence of Gβɣ/Na+ shows synergism for channel activation via PIP2 (20Petit-Jacques J. Sui J.L. Logothetis D.E. Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.J. Gen. Physiol. 1999; 114: 673-684Crossref PubMed Scopus (82) Google Scholar) as Gβɣ controls predominantly the HBC gate, whereas Na+ controls the cytosolic G-loop D. Jin T. Gazgalis D. Cui M. Logothetis D.E. On the mechanism of GIRK2 channel gating by phosphatidylinositol bisphosphate, sodium, and the Gbetagamma dimer.J. Biol. Chem. 2019; 294: 18934-18948Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). activators of GIRK channels have been such as the and the and GAT1508, of which have been found to activate GIRK channels in a G but P. H. S. A in GIRK channel PubMed Scopus Google Scholar, K. Molecular mechanism activation of inwardly rectifying potassium Natl. Acad. Sci. U. S. A. 2013; Scopus Google Scholar, N. E. Y. M.C. et the activation of inwardly rectifying K+ (GIRK) channels by the Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. Cantwell L. Gazgalis D. et al.The molecule activates GIRK1/2 channel and in Biol. Chem. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). the act as such as the which with the activator for a site E. N. M. et activates inwardly rectifying potassium channels at binding site to J. Pharmacol. 2011; PubMed Scopus Google Scholar), or as such as the (e.g., by with the mechanism of Gβɣ activation K. Molecular mechanism activation of inwardly rectifying potassium Natl. Acad. Sci. U. S. A. 2013; Scopus Google Scholar, Mirshahi C. L. Jin T. Logothetis D.E. et gating inhibition to channels.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with the of GAT1508, which shows for GIRK1/2 of the a GIRK heteromeric or homomeric A activator of a in the nervous system could be for late-onset obesity, whereas in the activator be for PA and for Here, we report a selective activator, 3hi2one-G4 that the for GIRK4 homomeric channel over GIRK homotetramers or The binding site of 3hi2one-G4 in the GIRK4 channel was and 3hi2one-G4 could serve as a useful pharmaceutical probe to GIRK4 channel function as as drug for diseases such as PA and late-onset obesity. The 3hi2one-G4 (Fig. was discovered through of the for modulators of the homomeric GIRK4 Using and we found that 3hi2one-G4 activates GIRK4 channels expressed in and shows that 3hi2one-G4 activates expressed GIRK4 homomeric channels at a of 3hi2one-G4 GIRK4 in a with of in in (Fig. and in in cells, (Fig. The of 3hi2one-G4 was of its The selectivity of 3hi2one-G4 was GIRK1/2, GIRK1/4, of GIRK1 and for constitutively active G potassium Full Text Full Text PDF PubMed Scopus Google Scholar), (4Chan K.W. Sui J.L. Vivaudou M. Logothetis D.E. Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14193-14198Crossref PubMed Scopus (103) Google Scholar), GIRK2, and GIRK4 channels. The and channels were in studies than corresponding wildtype GIRK homomeric channels and are for GIRK homomeric channel function studies in the shows that 3hi2one-G4 GIRK4, and GIRK1/4 channels, but it not activate GIRK2, and GIRK1/2 channels. the activation of GIRK1 and GIRK4 subunits could have resulted of GIRK4 homomeric channel, we linked to effects GIRK4 as this heteromeric channels. 3hi2one-G4 at to activate heteromeric channel, suggesting that the GIRK1/4 activation was likely of activation of GIRK4 Thus, 3hi2one-G4 to be a highly selective activator of GIRK4 homomeric channels. 3hi2one-G4 activation of GIRK4 is PIP2 we a system that to P. of Mol. Biol. PubMed Scopus Google Scholar). In this system the N-terminal is in the whereas the is to with and to the A PIP2 lipid is linked to the C-terminal of that activation it is to the to to We this the shows a decrease in GIRK4 current suggesting that PIP2 by the resulted in of the of the but in the presence of 3hi2one-G4 GIRK4 current inhibition and a greater current (Fig. of the to activation to not in the current but also in the of inhibition activation (Fig. of cells that 3hi2one-G4 interactions with PIP2 such that the the current (Fig. and with (Fig. to the binding site of 3hi2one-G4 and its to the PIP2 binding In to effects of 3hi2one-G4 the we Using we of at the activation of we of as as as by the decrease in of (Fig. show that the at the rate of the presence of that 3hi2one-G4 does not the activity of the Thus, the strengthening of interactions are to the of 3hi2one-G4 GIRK4 and not the The binding site of 3hi2one-G4 was to be near the PIP2 binding site in the GIRK4 channel (Fig. A and molecular the 3hi2one-G4 to with the GIRK4 through transmembrane domains and and and the slide helix (Fig. A and It formed interactions with and interactions with and interactions with and with (Fig. and the 3hi2one-G4 binding site in the GIRK4 channel, we studies and characterized to 3hi2one-G4 of to GIRK4 wildtype expressed in shows the of the 3hi2one-G4 effects over of the the of the were to (Fig. GIRK4 to with 3hi2one-G4 the of the to with the of of residue to not yield a in the of 3hi2one-G4 to the GIRK4 whereas a but the the and channel and reduced for the 3hi2one-G4 binding between GIRK2 and GIRK4, are two slide helix in the 3hi2one-G4 binding site, and I82 for GIRK2, and the corresponding and L77 for GIRK4 channels (Fig. Therefore, two could to the selectivity of for GIRK4 over GIRK2 channels. we found than reduced the channel to 3hi2one-G4 (Fig. we with the corresponding GIRK2 and could be to and but not could be by 3hi2one-G4 at a of (Fig. the as a selectivity critical residue for 3hi2one-G4 probe the molecular of the 3hi2one-G4 activation of GIRK4 homomeric channel, we molecular the GIRK4 channel in the presence and of the the and the of the two major channel gates, the HBC and G shows the of each of the two channel as a function of the the GIRK4 and 3hi2one-G4 the of HBC and G-loop In we a K+ through the GIRK4 channel to 3hi2one-G4 and the at K+ the HBC and G-loop within the binding site residue interactions with we molecular binding energy and residue binding energy shows the binding energy each residue in the binding to of the major were L77 and the GIRK selectivity residue (Fig. with 3hi2one-G4 through interactions. The between residue L77 and 3hi2one-G4 as a function of was the (Fig. GIRK channels be through G that by the Gβγ through G protein–coupled activation (9Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart.Nature. 1987; 325: 321-326Crossref PubMed Scopus (941) Google Scholar). 3hi2one-G4 activates GIRK4 channels in a G we the with the GIRK4 channel in for shows GIRK4 channel activation by the and by 3hi2one-G4 of the two activators is with the that 3hi2one-G4 activates the GIRK4 channel in a of G 3hi2one-G4 could activate GIRK4 channel in and we GIRK4 channel expressed in and not suggesting that are not two also to be by In the which is was by 3hi2one-G4 (Fig. the of 3hi2one-G4 in cells, we in cells (Fig. cells GIRK4 channels we the to 3hi2one-G4 in and cells (Fig. We found that 3hi2one-G4 inwardly rectifying GIRK which were by and current of cells was to the (Fig. 3hi2one-G4 current in cells compared with (Fig. we cells with and the of 3hi2one-G4 (Fig. In this the of cells that was GIRK4 current, to and cells (Fig. is a number of GIRK channel modulators was discovered through a as the first activator, a selectivity in for activation of GIRK1/2 over GIRK1/4 channels K. E. C. A. L. et the first and selective activator of the GIRK potassium channel, in Chem. 2013; 4: PubMed Scopus Google Scholar). A.K. S. K.K. et and of as GIRK1/2 potassium channel Chem. 2017; PubMed Scopus Google Scholar) was discovered as a GIRK1 activator, which shows selectivity to for GIRK1/2 over GIRK1/4 channels. a and was also shown to be a GIRK channel activator, selectivity for GIRK1/2 over GIRK1/4 channels M. Y. M. Y. activates GIRK channels in a PIP2 Gbetagamma and amino acid residue at the slide helix the Physiol. 2017; PubMed Scopus Google Scholar). was discovered as a the GIRK1/2 but not the GIRK1/4 channel Y. Cantwell L. Gazgalis D. et al.The molecule activates GIRK1/2 channel and in Biol. Chem. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). GIRK channel was discovered as the first GIRK channel GIRK channels the GIRK1 subunit, such as GIRK1/2 and GIRK1/4 channels M. J. J. et of neuronal activity a GIRK channel Sci. PubMed Google Scholar). Y. A. et of a activator of GIRK 2020; Full Text Full Text PDF PubMed Scopus (17) Google Scholar), activator, was as a GIRK1/2 channel selective activator to act the the first small-molecule activator of a homomeric GIRK channel, selectivity of activation not for GIRK2 over GIRK4 but also for GIRK1/2 over GIRK1/4 channels Y. S. et and of a small-molecule activator of homomeric G inwardly potassium (GIRK) Chem. 2019; PubMed Scopus Google Scholar). the of GIRK channel modulators has been a GIRK4 homomeric activator not been reported to the present Here, we report the GIRK4 homomeric channel activator, which homomeric GIRK4 and not homomeric GIRK2 or heteromeric GIRK1/2 or GIRK1/4 channel 3hi2one-G4 activates the GIRK4 channel in a of G We characterized the potential binding site of 3hi2one-G4 in the GIRK4 by modeling, molecular and The binding site was by and The unique binding site is located near the PIP2 binding the between the of GIRK2 to and GIRK4 to channels, we show that residue L77 of GIRK4 is a major determinant of selectivity for activation by The shows of its activity by whereas the corresponding of its activity by the was reported to be a selective activator for GIRK1/2 over GIRK1/4 channels M. Y. M. Y. activates GIRK channels in a PIP2 Gbetagamma and amino acid residue at the slide helix the Physiol. 2017; PubMed Scopus Google Scholar). and studies of GIRK2 and GIRK4 channels, I82 in GIRK2 was as a major determinant for to In addition, and were shown to are located near the PIP2 binding Although the interactions between and GIRK1/2 were not it that may a binding site for GIRK2 channel activation to 3hi2one-G4 and could also a molecular mechanism of activation with the 3hi2one-G4 site in (or residue is in for activation In a is at for 3hi2one-G4 activation of computational model shows that 3hi2one-G4 binds near PIP2, which is for GIRK channel activation (12Zhang H. He C. Yan X. Mirshahi T. Logothetis D.E. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.Nat. Cell Biol. 1999; 1: 183-188Crossref PubMed Scopus (303) Google Scholar). The binding of 3hi2one-G4 enhances interactions between the channel and PIP2 and channel (Fig. The that the of HBC and G-loop were in the system compared with the and to and in the GIRK2 were to be critical with 3hi2one-G4 through and interactions (Fig. of to the 3hi2one-G4 and also with mutations of also activity of the channel Zhang H. T. Jin T. J. Logothetis D.E. in Kir interactions Full Text Full Text PDF PubMed Scopus Google Scholar). L77 was also to be a critical residue with 3hi2one-G4 through interactions. of this residue to or GIRK2 corresponding or channel (Fig. In for residue in GIRK4, corresponding to in GIRK2, the a but whereas not channel the that L77 in GIRK4 is a selectivity critical residue of the channel for It is that and and current compared with the wildtype The of mutations in GIRK4 channel activity to be GIRK4 channel loss-of-function were reported and (8Shalomov B. Handklo-Jamal R. Reddy H.P. Theodor N. Bera A.K. Dascal N. A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).bioRxiv. 2019; ([preprint])https://doi.org/10.1101/866202Crossref Scopus (0) Google Scholar). GIRK4 channel activators could serve as a potential for treatment of patients with PA. The 3hi2one-G4 activates but does not activate the nonfunctional and is with the of VU0529331 the (8Shalomov B. Handklo-Jamal R. Reddy H.P. Theodor N. Bera A.K. Dascal N. A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).bioRxiv. 2019; ([preprint])https://doi.org/10.1101/866202Crossref Scopus (0) Google Scholar). with which is not a selective homomeric GIRK channel activator, 3hi2one-G4 is a highly selective activator for GIRK4 homomeric channels, it a better drug for this The present work that optimization for 3hi2one-G4 are a In this the brain of GIRK4 in the brain (e.g., its in that could be to it the Similarly, the expression of GIRK4 homotetramers in the atria and pacemaking cardiac cells may not to activation by In the present we identify the first activator of GIRK4 homomeric channels. activates GIRK4 homomeric channels, GIRK1 or or GIRK2 3hi2one-G4 activates GIRK4 by binding at a site near the PIP2 binding site, which is formed by the and the of the The with the channel through and interactions. the of the binding between GIRK4 and GIRK2, we L77 in GIRK4, as a major molecular determinant of of the corresponding I82 to in GIRK2 it to 3hi2one-G4 that binding of 3hi2one-G4 the of GIRK4 channel the of the binding site and activation mechanism with a activator of GIRK1/2 heteromeric channels, we that 3hi2one-G4 and of action for corresponding We that 3hi2one-G4 could be a useful pharmaceutical probe to GIRK4 channel function and to for treatment of such as

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