FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3134750987> ?p ?o ?g. }

• W3134750987 endingPage "100535" @default.
• W3134750987 startingPage "100535" @default.
• W3134750987 abstract "Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects. Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects. Atrial fibrillation (AF), a common cardiac arrhythmia, affects over two million Americans and is associated with increased morbidity, mortality, and healthcare costs. Antiarrhythmic drugs (AADs) used to treat AF aim to either maintain the normal sinus rhythm (SR) or control ventricular rate (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). However, conventional drugs such as sodium channel blockers (class I antiarrhythmic drugs) and potassium channel blockers (class III antiarrhythmic drugs) have limited efficacy, especially in persistent AF patients. In addition, these conventional AADs carry a risk of ventricular proarrhythmia, such as torsade de pointes (TdP) through excessive delay of ventricular repolarization (2Torp-Pedersen C. Moller M. Bloch-Thomsen P.E. Kober L. Sandoe E. Egstrup K. Agner E. Carlsen J. Videbaek J. Marchant B. Camm A.J. Dofetilide in patients with congestive heart failure and left ventricular dysfunction. Danish Investigations of Arrhythmia and Mortality on Dofetilide Study Group.N. Engl. J. Med. 1999; 341: 857-865Crossref PubMed Scopus (898) Google Scholar, 3Waldo A.L. Camm A.J. deRuyter H. Friedman P.L. MacNeil D.J. Pauls J.F. Pitt B. Pratt C.M. Schwartz P.J. Veltri E.P. Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. The SWORD Investigators. Survival with oral d-Sotalol.Lancet. 1996; 348: 7-12Abstract Full Text Full Text PDF PubMed Scopus (1176) Google Scholar). Thus, development of new antiarrhythmic agents with a more effective and safer profile is highly desirable (4Hashimoto N. Acetylcholine-activated potassium channel as a novel target for AF treatment.in: Atrial Fibrillation - Basic Research and Clinical Applications. IntechOpen Limited, London, UK2012: 321-338Crossref Google Scholar). The cardiac acetylcholine- (ACh-) activated inwardly rectifying K+ current (IKACh) consists of heterotetrameric ion channel subunits composed of Kir3.1 (GIRK1 or G1) and Kir3.4 (or GIRK4) subunits and is activated via the M2 muscarinic receptor to mediate vagal influences on heart rate and atrial repolarization. The Kir3.1/4 heterotetramers are the main contributors to the cardiac IKACh, while Kir3.4 homotetramers contribute considerably less activity. In contrast, Kir3.1 homotetramers are inactive and mostly trapped in the endoplasmic reticulum (ER) (5Ma D. Zerangue N. Raab-Graham K. Fried S.R. Jan Y.N. Jan L.Y. Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart.Neuron. 2002; 33: 715-729Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Heteromerization with the Kir3.4 traffics the heterotetramer efficiently to the plasma membrane to contribute to IKACh. Mutation of Kir3.1(F137) to the corresponding Ser residue found in Kir3.4 produces functional Kir3.1(F137S) channels although their trapping in the ER continues (6Chan 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 (98) Google Scholar, 7Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This active Kir3.1 mutant has been an invaluable tool to assessing the role of Kir3.1 subunits in the regulation of heteromeric channel activity, but in addition, it has been found to be a critical determinant of activity in the action of urea-based channel activators (8Wydeven N. Marron Fernandez de Velasco E. Du Y. Benneyworth M.A. Hearing M.C. Fischer R.A. Thomas M.J. Weaver C.D. Wickman K. Mechanisms underlying the activation of G-protein-gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10755-10760Crossref PubMed Scopus (77) Google Scholar, 9Xu Y. Cantwell L. Molosh A.I. Plant L.D. Gazgalis D. Fitz S.D. Dustrude E.T. Yang Y. Kawano T. Garai S. Noujaim S.F. Shekhar A. Logothetis D.E. Thakur G.A. The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.J. Biol. Chem. 2020; 295: 3614-3634Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Kir3 channel activity is strongly inwardly rectifying. Despite the relatively small outward conductance at physiological potentials, IKACh is an important contributor to the late AP repolarization and plays a major role in stabilizing the resting membrane potential (10Ravens U. Cerbai E. Role of potassium currents in cardiac arrhythmias.Europace. 2008; 10: 1133-1137Crossref PubMed Scopus (110) Google Scholar, 11Voigt N. Dobrev D. Atrial-selective potassium channel blockers.Card. Electrophysiol. Clin. 2016; 8: 411-421Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Activation of IKACh has strong atrial AP-shortening and AF-promoting effects (12Nattel S. Carlsson L. Innovative approaches to anti-arrhythmic drug therapy.Nat. Rev. Drug Discov. 2006; 5: 1034-1049Crossref PubMed Scopus (175) Google Scholar). Studies have shown that chronic AF alters IKACh properties, by causing the channel to open even in the absence of ACh, resulting in APD shortening that promotes AF (13Cha T.J. Ehrlich J.R. Chartier D. Qi X.Y. Xiao L. Nattel S. Kir3-based inward rectifier potassium current: Potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias.Circulation. 2006; 113: 1730-1737Crossref PubMed Scopus (159) Google Scholar, 14Dobrev D. Friedrich A. Voigt N. Jost N. Wettwer E. Christ T. Knaut M. Ravens U. 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 (364) Google Scholar, 15Ehrlich J.R. Cha T.J. Zhang L. Chartier D. Villeneuve L. Hebert T.E. Nattel S. Characterization of a hyperpolarization-activated time-dependent potassium current in canine cardiomyocytes from pulmonary vein myocardial sleeves and left atrium.J. Physiol. 2004; 557: 583-597Crossref PubMed Scopus (142) Google Scholar). In the heart, Kir3 channel subunits comprising IKACh are prominently expressed in the sinus and atrioventricular nodes, as well as in the atrial myocardium but are largely absent in ventricles or contribute minimally to ventricular repolarization (16Dobrzynski H. Marples D.D. Musa H. Yamanushi T.T. Henderson Z. Takagishi Y. Honjo H. Kodama I. Boyett M.R. Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.J. Histochem. Cytochem. 2001; 49: 1221-1234Crossref PubMed Scopus (87) Google Scholar, 17Lee S.W. Anderson A. Guzman P.A. Nakano A. Tolkacheva E.G. Wickman K. Atrial GIRK channels mediate the effects of vagus nerve stimulation on heart rate dynamics and arrhythmogenesis.Front. Physiol. 2018; 9: 943Crossref PubMed Scopus (16) Google Scholar). Therefore, targeting IKACh channels is expected to selectively prolong the atrial effective refractory period (ERP) and therefore terminate AF without the risk of QT prolongation or TdP (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). A number of IKACh channel inhibitors have been developed in recent years. However, no selective IKACh channel inhibitors have been approved for clinical use to date (11Voigt N. Dobrev D. Atrial-selective potassium channel blockers.Card. Electrophysiol. Clin. 2016; 8: 411-421Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 18Ehrlich J.R. Biliczki P. Hohnloser S.H. Nattel S. Atrial-selective approaches for the treatment of atrial fibrillation.J. Am. Coll. Cardiol. 2008; 51: 787-792Crossref PubMed Scopus (72) Google Scholar). Tertiapin-Q is a nonoxidizable derivative of the naturally occurring peptide toxin tertiapin from the venom of the European honey bee (Apis melifera) that selectively inhibits Kir (Kir3 and Kir1 or ROMK) channels, as well as calcium-activated large-conductance potassium channels (BK) (19Jin W. Lu Z. A novel high-affinity inhibitor for inward-rectifier K+ channels.Biochemistry. 1998; 37: 13291-13299Crossref PubMed Scopus (207) Google Scholar, 20Kanjhan R. Coulson E.J. Adams D.J. Bellingham M.C. Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner.J. Pharmacol. Exp. Ther. 2005; 314: 1353-1361Crossref PubMed Scopus (75) Google Scholar). Tertiapin-Q selectively inhibits IKACh channels in the cardiac tissue with high potency, without effect on Kir2.1 (IRK1) channels (21Jin W. Lu Z. Synthesis of a stable form of tertiapin: A high-affinity inhibitor for inward-rectifier K+ channels.Biochemistry. 1999; 38: 14286-14293Crossref PubMed Scopus (123) Google Scholar). Potential side effects of Tertiapin-Q could arise from inhibition of BK outward currents in the central nervous system (20Kanjhan R. Coulson E.J. Adams D.J. Bellingham M.C. Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner.J. Pharmacol. Exp. Ther. 2005; 314: 1353-1361Crossref PubMed Scopus (75) Google Scholar), as well as immunogenicity and plasma protein binding in humans (22Ehrlich J.R. Inward rectifier potassium currents as a target for atrial fibrillation therapy.J. Cardiovasc. Pharmacol. 2008; 52: 129-135Crossref PubMed Scopus (70) Google Scholar). NIP-142 was the first moderately selective IKACh small-molecule blocker, which inhibited heterologously expressed Kir3.1/Kir3.4 and KV1.5 currents at low micromolar concentrations (23Matsuda T. Masumiya H. Tanaka N. Yamashita T. Tsuruzoe N. Tanaka Y. Tanaka H. Shigenoba K. Inhibition by a novel anti-arrhythmic agent, NIP-142, of cloned human cardiac K+ channel Kv1.5 current.Life Sci. 2001; 68: 2017-2024Crossref PubMed Scopus (42) Google Scholar, 24Matsuda T. Takeda K. Ito M. Yamagishi R. Tamura M. Nakamura H. Tsuruoka N. Saito T. Masumiya H. Suzuki T. Iida-Tanaka N. Itokawa-Matsuda M. Yamashita T. Tsuruzoe N. Tanaka H. et al.Atria selective prolongation by NIP-142, an antiarrhythmic agent, of refractory period and action potential duration in Guinea pig myocardium.J. Pharmacol. Sci. 2005; 98: 33-40Crossref PubMed Scopus (34) Google Scholar). It terminates both AF in the canine vagal stimulation-induced AF model and atrial flutter in the canine Y-shaped incision-induced AF model (25Nagasawa H. Fujiki A. Fujikura N. Matsuda T. Yamashita T. Inoue H. Effects of a novel class III antiarrhythmic agent, NIP-142, on canine atrial fibrillation and flutter.Circ. J. 2002; 66: 185-191Crossref PubMed Scopus (43) Google Scholar). The follow-up compound NIP-151 has higher potency for IKACh and selectivity over hERG channels, also effectively terminates AF in both vagal nerve stimulation-induced AF and aconitine-induced AF models. NIP-151 significantly prolonged the canine atrial ERP without significant effects on the ventricular ERP (26Hashimoto N. Yamashita T. Tsuruzoe N. Characterization of in vivo and in vitro electrophysiological and antiarrhythmic effects of a novel IKACh blocker, NIP-151: A comparison with an IKr-blocker dofetilide.J. Cardiovasc. Pharmacol. 2008; 51: 162-169Crossref PubMed Scopus (49) Google Scholar). NTC-801, a substituted benzopyran, was the first “selective” Kir3.1/Kir3.4 inhibitor investigated in clinical studies (27Machida T. Hashimoto N. Kuwahara I. Ogino Y. Matsuura J. Yamamoto W. Itano Y. Zamma A. Matsumoto R. Kamon J. Kobayashi T. Ishiwata N. Yamashita T. Ogura T. Nakaya H. Effects of a highly selective acetylcholine-activated K+ channel blocker on experimental atrial fibrillation.Circ. Arrhythm. Electrophysiol. 2011; 4: 94-102Crossref PubMed Scopus (74) Google Scholar). It significantly prolonged the canine atrial ERP without affecting the ventricular ERP under vagal nerve stimulation. It effectively converted AF to normal sinus rhythm in both vagal nerve simulation-induced and the aconitine-induced AF model in dog and terminated and prevented the induction of AF in an atrial tachycardia-AF dog model of persistent AF (27Machida T. Hashimoto N. Kuwahara I. Ogino Y. Matsuura J. Yamamoto W. Itano Y. Zamma A. Matsumoto R. Kamon J. Kobayashi T. Ishiwata N. Yamashita T. Ogura T. Nakaya H. Effects of a highly selective acetylcholine-activated K+ channel blocker on experimental atrial fibrillation.Circ. Arrhythm. Electrophysiol. 2011; 4: 94-102Crossref PubMed Scopus (74) Google Scholar, 28Yamamoto W. Hashimoto N. Matsuura J. Machida T. Ogino Y. Kobayashi T. Yamanaka Y. Ishiwata N. Yamashita T. Tanimoto K. Miyoshi S. Fukuda K. Nakaya H. Ogawa S. Effects of the selective KACh channel blocker NTC-801 on atrial fibrillation in a canine model of atrial tachypacing: Comparison with class Ic and III drugs.J. Cardiovasc. Pharmacol. 2014; 63: 421-427Crossref PubMed Scopus (16) Google Scholar). However, a phase II study failed to meet the primary endpoint to reduce the AF burden in patients with paroxysmal AF. It is speculated that this was due to the relatively low dosing, which was necessary to avoid central nervous system side effects (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). In addition, IKACh inhibitors, AZD2927 and A7071, did not affect atrial repolarization in atrial flutter patients (29Walfridsson H. Anfinsen O.G. Berggren A. Frison L. Jensen S. Linhardt G. Nordkam A.C. Sundqvist M. Carlsson L. Is the acetylcholine-regulated inwardly rectifying potassium current a viable antiarrhythmic target? Translational discrepancies of AZD2927 and A7071 in dogs and humans.Europace. 2015; 17: 473-482Crossref PubMed Scopus (24) Google Scholar). However, the lack of desirable effects of these compounds does not diminish the value of IKACh channel inhibitors for persistent AF (1El-Haou S. Ford J.W. Milnes J.T. Novel K+ channel targets in atrial fibrillation drug development--where are we?.J. Cardiovasc. Pharmacol. 2015; 66: 412-431Crossref PubMed Scopus (21) Google Scholar). Therefore, the need remains to develop new drug candidates selectively targeting IKACh channels. A group of benzopyran derivatives with triple rings was discovered having a prolongation effect on the atrial ERP, which can be used for treatment of arrhythmias. Conventional antiarrhythmic agents that use as their main mechanism of action prolongation of the refractory period (presumably due to prolongation of the action potential in ventricular muscle) present therapeutic problems; they induce highly dangerous arrhythmias leading to sudden death, such as torsades de pointes among others. In contrast, tricyclic benzopyran compounds selectively prolong the dog atrial ERP period without any influence on the ventricular ERP (30Ohrai K. Shigeta Y. Uesugi O. Okada T. Matsuda T. Tricyclic Benzopyrane Compound as Anti-arrhythmic Agents. United States. United States Patent Office, Alexandria, VA2008Google Scholar). However, the mechanism of their antiarrhythmic activity has been unclear. From this chemical series a selection patent (31Takada Y. Kamon M. Kawahara S. Umeda Y. Novel Crystal Forms of Tricyclic Benzopyran Compound and Processes for Producing Same. European Patent Office, Munich, Germany2010Google Scholar) exemplifies a crystal form of a single 4-(aralkylamino)-2,2-dimethyl-3,4-dihydro-2H-benzopyran-3-ol compound. An undisclosed substitution of this compound is inferred to be the clinical candidate NTC-801 (27Machida T. Hashimoto N. Kuwahara I. Ogino Y. Matsuura J. Yamamoto W. Itano Y. Zamma A. Matsumoto R. Kamon J. Kobayashi T. Ishiwata N. Yamashita T. Ogura T. Nakaya H. Effects of a highly selective acetylcholine-activated K+ channel blocker on experimental atrial fibrillation.Circ. Arrhythm. Electrophysiol. 2011; 4: 94-102Crossref PubMed Scopus (74) Google Scholar). We synthesized the patented (31Takada Y. Kamon M. Kawahara S. Umeda Y. Novel Crystal Forms of Tricyclic Benzopyran Compound and Processes for Producing Same. European Patent Office, Munich, Germany2010Google Scholar) tricyclic benzopyran compounds (enantiomers), (3R,4S)-7-(hydroxymethyl)-2,2,9-trimethyl-4-(2-phenylethylamino)-3,4-dihydropyrano[2,3-g]quinolin-3-ol and found one, BP-G1 (Fig. 1A) to selectively and potently inhibit heteromeric Kir3.1/4 or Kir3.1/2 channels (IC50s ∼ 10–30 nM) over other cardiac channels, including homomeric Kir3 and Kir2 channels. The compound can be used as a useful pharmaceutical tool to explore the mechanism of action of tricyclic benzopyran compounds through their interactions with the IKACh/Kir3.x channels, as well as their selectivity over other ion channels. In this work, we characterized the molecular mechanism of BP-G1 action and its interaction with the Kir3.1/4 channel, using a combination of computational and experimental approaches. A group of benzopyran derivatives with triple rings was discovered to have a prolongation effect on the refractory period of atrial muscle without any influence on the refractory period and action potential of ventricular muscle (30Ohrai K. Shigeta Y. Uesugi O. Okada T. Matsuda T. Tricyclic Benzopyrane Compound as Anti-arrhythmic Agents. United States. United States Patent Office, Alexandria, VA2008Google Scholar). However, the mechanism of the antiarrhythmic activity of these drugs has been unclear. We tested whether benzopyran derivatives selectively inhibited Kir3.x channels that underlie cardiac IKACh that is specifically found in the atrial myocardium. We tested the inhibitory activity of the benzopyran derivative BP-G1 (Fig. 1A) on several inwardly rectifier K+ channels, including Kir2.1, Kir2.3, Kir3.1∗, Kir3.2∗, Kir3.4∗, Kir3.1/2, and Kir3.1/4 channels by two electrode voltage-clamp (TEVC) (see Experimental procedures) experiments (where Kir3.1∗: Kir3.1(F137S); Kir3.2∗: Kir3.2(E152D); Kir3.4∗: Kir3.4(S143T)). The compound showed strong inhibitory selectivity for heteromeric Kir3 channels containing Kir3.1 (Kir3.1/2 and Kir3.1/4) over other Kir channels, including homomeric Kir3 channels (Kir3.1∗, Kir3.2∗, Kir3.4∗, Kir3.1∗/Kir3.2 and Kir3.1∗/Kir3.4) (Fig. 1B). Figure 1, C and D show representative experiments of responses of Kir3.1/4 and Kir3.1∗ channels to the compound. Upon application of the BP-G1 [1 μM], the Kir3.1/3.4 (or Kir3.1/4) channel current was significantly reduced over 100 s as compared with the Kir3.1∗ channel current. BP-G1 selectively inhibited heteromeric Kir3.1/2 and Kir3.1/4 whole-cell currents in a concentration-dependent manner and with comparable potency, IC50s of 55.9 nM and 55.0 nM in TEVC oocyte experiments (Fig. 1, E and F), and 32.2 nM and 10.5 nM in whole-cell patch-clamp HEK-293 cell experiments, respectively (Fig. 1, G and H). The pore helix (F137) residue has been shown to be a critical Kir3.1 determinant for current potentiation of heteromeric Kir3 currents and also for ML297- and GAT1508-induced current potentiation (6Chan 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 (98) Google Scholar, 7Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 8Wydeven N. Marron Fernandez de Velasco E. Du Y. Benneyworth M.A. Hearing M.C. Fischer R.A. Thomas M.J. Weaver C.D. Wickman K. Mechanisms underlying the activation of G-protein-gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10755-10760Crossref PubMed Scopus (77) Google Scholar, 9Xu Y. Cantwell L. Molosh A.I. Plant L.D. Gazgalis D. Fitz S.D. Dustrude E.T. Yang Y. Kawano T. Garai S. Noujaim S.F. Shekhar A. Logothetis D.E. Thakur G.A. The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.J. Biol. Chem. 2020; 295: 3614-3634Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In addition, the M2 helix residue D173 was also shown to be critical for ML297- and GAT1508-induced current stimulation (Fig. 2A). Thus, we proceeded to test whether Kir3.1(F137) and Kir3.1(D173) were also important for BP-G1 inhibition in the background of Kir3.4 channels. We chose to mutate the S143 and N179 corresponding residues of the Kir3.4 to the F137 and D173 of the Kir3.1 channel and coexpressed them with Kir3.4 wild-type subunits (6Chan 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 (98) Google Scholar). Interestingly, while BP-G1 did not inhibit the wild-type Kir3.4 channel or the Kir3.4/4(N179D) channel mutant, it did inhibit the Kir3.4/4(S143F) channel mutant in a concentration-dependent manner (IC50 = 62.1 nM) (Fig. 2, B and C). In the background of the BP-G1 sensitive Kir3.4/4(S143F) channel, the double-mutant Kir3.4/4(S143F, N179D) showed a 20% less inhibition by BP-G1 (Fig. 2B). Because an Asp residue in position 179 in the context of the Kir3.4(S143F) did not contribute to the inhibition by BP-G1, the results of Figure 2 suggested that the pore helix Phe residue (S143F in Kir3.4 corresponding to F137 in Kir3.1 subunit) is a critical determinant for selective inhibition by BP-G1. To better understand the molecular mechanism of the BP-G1 inhibition on Kir3 channels, we built homology models for the Kir3.4 and Kir3.1/4 channels based on a crystal structure of the Kir3.2 channel (PDBID: 3SYA). Sequence alignment among Kir3.1, Kir3.4, and Kir3.2 channels was generated by the ClustalW server (http://www.genome.jp/-tools/clustalw/). The sequence identities between Kir3.2 and Kir3.4 or Kir3.2 and Kir3.1 are 72% and 53%, respectively, which makes the Kir3.2 crystal structure an excellent structural template to generate accurate homology models for the Kir3.4, Kir3.1/2, and Kir3.1/4 channels (32Cui M. Cantwell L. Zorn A. Logothetis D.E. Kir channel molecular physiology, pharmacology and therapeutic implications.in: Handbook of Experimental Pharmacology. Springer, Berlin, Germany2021Google Scholar). We used the MODELLER program (33Sali A. Blundell T.L. Comparative protein modelling by satisfaction of spatial restraints.J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10512) Google Scholar) to generate ten initial homology models for Kir3.4 and Kir3.1/4, respectively, based on the Kir3.2 structural template and selected the one with the best internal DOPE (Discrete Optimized Protein Energy) score for modeling the compound and channel interactions. Based on the homology model of Kir3.1/4 channel, we performed molecular docking simulations to explore the potential binding site in the channel. Since our experimental results showed that the Kir3.1(F137) residue [corresponding to the Kir3.4(S143)] is critical for the inhibitory activity of BP-G1, we selected these residues together with the HBC residues F181(Kir3.1) and F187(Kir3.4) to define the grid box for docking simulations. The box covered the entire central cavity of the channel. We performed grid-based rigid protein and flexible docking using Glide and followed by Induced Fit Docking (IFD, Schrödinger, Inc), which takes into account the induced fit effects between the protein and ligand interactions. The pose with the lowest XP score from the IFD docking was selected as the predicted binding pose of the ligand (Fig. 3A). The detailed interactions between the BP-G1 and the channel were analyzed using the LIGPLOT program (34Wallace A.C. Laskowski R.A. Thornton J.M. LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions.Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4333) Google Scholar). Figure 3, B and C show predicted interactions between the BP-G1 and Kir3.1/4 channel. Three predicted interactions stood out: two between the nitrogen of ring A of the compound and a conserved residue in Kir3 channels (Kir3.1: E141 in the pore helix) and one between the hydroxyl of ring C of the compound and D173 that is unique to Kir3.1 compared with Asn residues found in the other Kir3 channels (32Cui M. Cantwell L. Zorn A. Logothetis D.E. Kir channel molecular physiology, pharmacology and therapeutic implications.in: Handbook of Experimental Pharmacology. Springer, Berlin, Germany2021Google Scholar). Interestingly, the critical Kir3.1(F137) that is required for BP-G1 inhibition (Figs. 1B and 2, B and C) was not predicted by the model to interact directly with the compound. Instead, the model suggested that F137 influenced residue Kir3.1(E141), which directly interacted with the BP-G1 compound. Figure 3D shows the relationship of F137 to E141 relative to the compound. The bulky F137 side chain appears to enable sterically the salt bridge between E141 and the compound. To test the role of the Kir3.1(E141) residue in the BP-G1 inhibition, we mutated it to Ala. The Kir3.1(E141A)/4 showed only a 25% current block compared with the 75 to 80% block seen with the wild-type Kir3.1/4 (Figs. 1B and 4, A and C). In contrast, the neighboring conserved Kir3.1(T143A)/4 showed no significant reduction in BP-G1 block (Fig. 2A). We next probed the role of the Kir3.1 unique residue D173. The Kir3.1(D173A) mutant also showed a ∼25% current block by 1 μM BP-G1 (Fig. 4, A and C). Moreover, the double mutant Kir3.1(E141A/D173A) abolished any current block by BP-G1 (Fig. 4, A and C). As we saw in Figure 2, although BP-G1 showed no blo" @default.
• W3134750987 created "2021-03-15" @default.
• W3134750987 creator A5005028004 @default.
• W3134750987 creator A5007736608 @default.
• W3134750987 creator A5013997590 @default.
• W3134750987 creator A5021819560 @default.
• W3134750987 creator A5033419461 @default.
• W3134750987 creator A5034740703 @default.
• W3134750987 creator A5049876521 @default.
• W3134750987 creator A5053106007 @default.
• W3134750987 creator A5065023289 @default.
• W3134750987 creator A5070927098 @default.
• W3134750987 creator A5074434206 @default.
• W3134750987 creator A5082964334 @default.
• W3134750987 creator A5084438338 @default.
• W3134750987 creator A5085443086 @default.
• W3134750987 creator A5087424945 @default.
• W3134750987 date "2021-01-01" @default.
• W3134750987 modified "2023-10-17" @default.
• W3134750987 title "A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels" @default.
• W3134750987 cites W1970195676 @default.
• W3134750987 cites W1971017555 @default.
• W3134750987 cites W1983399409 @default.
• W3134750987 cites W1986414724 @default.
• W3134750987 cites W1987475842 @default.
• W3134750987 cites W1989608518 @default.
• W3134750987 cites W1991042645 @default.
• W3134750987 cites W2014580405 @default.
• W3134750987 cites W2031897378 @default.
• W3134750987 cites W2042810199 @default.
• W3134750987 cites W2050065095 @default.
• W3134750987 cites W2065283382 @default.
• W3134750987 cites W2065626196 @default.
• W3134750987 cites W2067174909 @default.
• W3134750987 cites W2068551052 @default.
• W3134750987 cites W2072132290 @default.
• W3134750987 cites W2072311779 @default.
• W3134750987 cites W2081355665 @default.
• W3134750987 cites W2081912151 @default.
• W3134750987 cites W2085621401 @default.
• W3134750987 cites W2091877802 @default.
• W3134750987 cites W2097694848 @default.
• W3134750987 cites W2109807846 @default.
• W3134750987 cites W2124168315 @default.
• W3134750987 cites W2131950845 @default.
• W3134750987 cites W2140994963 @default.
• W3134750987 cites W2143518969 @default.
• W3134750987 cites W2151334055 @default.
• W3134750987 cites W2161227647 @default.
• W3134750987 cites W2314139393 @default.
• W3134750987 cites W2325842765 @default.
• W3134750987 cites W2413411895 @default.
• W3134750987 cites W2811424783 @default.
• W3134750987 cites W2982396461 @default.
• W3134750987 cites W2998890960 @default.
• W3134750987 cites W3189906030 @default.
• W3134750987 doi "https://doi.org/10.1016/j.jbc.2021.100535" @default.
• W3134750987 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8086025" @default.
• W3134750987 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33713702" @default.
• W3134750987 hasPublicationYear "2021" @default.
• W3134750987 type Work @default.
• W3134750987 sameAs 3134750987 @default.
• W3134750987 citedByCount "5" @default.
• W3134750987 countsByYear W31347509872021 @default.
• W3134750987 countsByYear W31347509872023 @default.
• W3134750987 crossrefType "journal-article" @default.
• W3134750987 hasAuthorship W3134750987A5005028004 @default.
• W3134750987 hasAuthorship W3134750987A5007736608 @default.
• W3134750987 hasAuthorship W3134750987A5013997590 @default.
• W3134750987 hasAuthorship W3134750987A5021819560 @default.
• W3134750987 hasAuthorship W3134750987A5033419461 @default.
• W3134750987 hasAuthorship W3134750987A5034740703 @default.
• W3134750987 hasAuthorship W3134750987A5049876521 @default.
• W3134750987 hasAuthorship W3134750987A5053106007 @default.
• W3134750987 hasAuthorship W3134750987A5065023289 @default.
• W3134750987 hasAuthorship W3134750987A5070927098 @default.
• W3134750987 hasAuthorship W3134750987A5074434206 @default.
• W3134750987 hasAuthorship W3134750987A5082964334 @default.
• W3134750987 hasAuthorship W3134750987A5084438338 @default.
• W3134750987 hasAuthorship W3134750987A5085443086 @default.
• W3134750987 hasAuthorship W3134750987A5087424945 @default.
• W3134750987 hasBestOaLocation W31347509871 @default.
• W3134750987 hasConcept C126322002 @default.
• W3134750987 hasConcept C178790620 @default.
• W3134750987 hasConcept C185592680 @default.
• W3134750987 hasConcept C2776286485 @default.
• W3134750987 hasConcept C2778457506 @default.
• W3134750987 hasConcept C517785266 @default.
• W3134750987 hasConcept C71240020 @default.
• W3134750987 hasConcept C71924100 @default.
• W3134750987 hasConcept C83743174 @default.
• W3134750987 hasConcept C98274493 @default.
• W3134750987 hasConceptScore W3134750987C126322002 @default.
• W3134750987 hasConceptScore W3134750987C178790620 @default.
• W3134750987 hasConceptScore W3134750987C185592680 @default.
• W3134750987 hasConceptScore W3134750987C2776286485 @default.
• W3134750987 hasConceptScore W3134750987C2778457506 @default.
• W3134750987 hasConceptScore W3134750987C517785266 @default.

• next