{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T13:25:20Z","timestamp":1778678720864,"version":"3.51.4"},"reference-count":30,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2021,4,22]],"date-time":"2021-04-22T00:00:00Z","timestamp":1619049600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Society for the Promotion of Science","award":["20K16126, 15H04678, 20K07269"],"award-info":[{"award-number":["20K16126, 15H04678, 20K07269"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>A Ca2+-activated monovalent cation-selective TRPM4 channel is abundantly expressed in the heart. Recently, a single gain-of-function mutation identified in the distal N-terminus of the human TRPM4 channel (Glu5 to Lys5; E7K) was found to be arrhythmogenic because of enhanced cell membrane expression. In this study, we conducted detailed analyses of this mutant channel from more functional aspects, in comparison with its wild type (WT). In an expression system, intracellular application of a short soluble PIP2 (diC8PIP2) restored the single-channel activities of both WT and E7K, which had quickly faded after membrane excision. The potency (Kd) of diC8PIP2 for this recovery was stronger in E7K than its WT (1.44 vs. 2.40 \u03bcM). FRET-based PIP2 measurements combined with the Danio rerio voltage-sensing phosphatase (DrVSP) and patch clamping revealed that lowering the endogenous PIP2 level by DrVSP activation reduced the TRPM4 channel activity. This effect was less prominent in E7K than its WT (apparent Kd values estimated from DrVSP-mediated PIP2 depletion: 0.97 and 1.06 \u03bcM, respectively), being associated with the differential PIP2-mediated modulation of voltage dependence. Moreover, intracellular perfusion of short N-terminal polypeptides containing either the \u2018WT\u2019 or \u2018E7K\u2019 sequences respectively attenuated the TRPM4 channel activation at whole-cell and single-channel levels, but in both configurations, the E7K polypeptide exerted greater inhibitory effects. These results collectively suggest that N-terminal interaction with endogenous PIP2 is essential for the TRPM4 channel to function, the extent of which may be abnormally strengthened by the E7K mutation through modulating voltage-dependent activation. The altered PIP2 interaction may account for the arrhythmogenic potential of this mutation.<\/jats:p>","DOI":"10.3390\/cells10050983","type":"journal-article","created":{"date-parts":[[2021,4,22]],"date-time":"2021-04-22T13:59:14Z","timestamp":1619099954000},"page":"983","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["An Arrhythmic Mutation E7K Facilitates TRPM4 Channel Activation via Enhanced PIP2 Interaction"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0893-2705","authenticated-orcid":false,"given":"Yaopeng","family":"Hu","sequence":"first","affiliation":[{"name":"Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9073-5753","authenticated-orcid":false,"given":"Qin","family":"Li","sequence":"additional","affiliation":[{"name":"Biomedical Information Engineering Lab, The University of Aizu, Aizu-Wakamatsu 965-8580, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lin-Hai","family":"Kurahara","sequence":"additional","affiliation":[{"name":"Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8326-6856","authenticated-orcid":false,"given":"Narumi","family":"Shioi","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka 814-0180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Keizo","family":"Hiraishi","sequence":"additional","affiliation":[{"name":"Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6391-6126","authenticated-orcid":false,"given":"Takayuki","family":"Fujita","sequence":"additional","affiliation":[{"name":"Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4376-0806","authenticated-orcid":false,"given":"Xin","family":"Zhu","sequence":"additional","affiliation":[{"name":"Biomedical Information Engineering Lab, The University of Aizu, Aizu-Wakamatsu 965-8580, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryuji","family":"Inoue","sequence":"additional","affiliation":[{"name":"Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,4,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.hlc.2018.08.026","article-title":"Epidemiology of sudden cardiac death: Global and regional perspectives","volume":"28","author":"Wong","year":"2019","journal-title":"Heart Lung Circ."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1038\/s41569-018-0145-2","article-title":"Transient receptor potential channels in cardiac health and disease","volume":"16","author":"Hof","year":"2019","journal-title":"Nat. Rev. Cardiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1161\/01.RES.0000233356.10630.8a","article-title":"Transient receptor potential channels in cardiovascular function and disease","volume":"99","author":"Inoue","year":"2006","journal-title":"Circ. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/S0092-8674(02)00719-5","article-title":"Trpm4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization","volume":"109","author":"Launay","year":"2002","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2354","DOI":"10.1111\/j.1476-5381.2011.01715.x","article-title":"Transient receptor potential melastatin 4 inhibitor 9-phenanthrol abolishes arrhythmias induced by hypoxia and re-oxygenation in mouse ventricle","volume":"165","author":"Simard","year":"2012","journal-title":"Br. J. Pharmacol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2737","DOI":"10.1172\/JCI38292","article-title":"Impaired endocytosis of the ion channel trpm4 is associated with human progressive familial heart block type i","volume":"119","author":"Kruse","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1016\/j.cjca.2017.03.010","article-title":"Genetic determinants of hereditary bradyarrhythmias: A contemporary review of a diverse group of disorders","volume":"33","author":"Rezazadeh","year":"2017","journal-title":"Can. J. Cardiol."},{"key":"ref_8","first-page":"31","article-title":"Regulation of ion channels by membrane lipids","volume":"2","author":"Mehta","year":"2011","journal-title":"Compr. Physiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"re19","DOI":"10.1126\/stke.2001.111.re19","article-title":"The complex and intriguing lives of PIP2 with ion channels and transporters","volume":"2001","author":"Hilgemann","year":"2001","journal-title":"Sci. STKE"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1611","DOI":"10.1161\/01.CIR.0000092166.30360.78","article-title":"Dual inhibition of \u03b2-adrenergic and angiotensin ii receptors by a single antagonist: A functional role for receptor\u2013receptor interaction in vivo","volume":"108","author":"Luttrell","year":"2003","journal-title":"Circulation"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"H40","DOI":"10.1152\/ajpheart.1999.277.1.H40","article-title":"Changes in sarcolemmal plc isoenzymes in postinfarct congestive heart failure: Partial correction by imidapril","volume":"277","author":"Tappia","year":"1999","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1038\/sj.emboj.7600963","article-title":"The Ca2+-activated cation channel trpm4 is regulated by phosphatidylinositol 4, 5-biphosphate","volume":"25","author":"Nilius","year":"2006","journal-title":"EMBO J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"39185","DOI":"10.1074\/jbc.M506965200","article-title":"Phosphatidylinositol 4, 5-bisphosphate rescues trpm4 channels from desensitization","volume":"280","author":"Zhang","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1239","DOI":"10.1038\/nature03650","article-title":"Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor","volume":"435","author":"Murata","year":"2005","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"15337","DOI":"10.1074\/jbc.M007194200","article-title":"Monitoring agonist-induced phospholipase c activation in live cells by fluorescence resonance energy transfer","volume":"276","author":"Habets","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1085\/jgp.201311033","article-title":"PLC-mediated PI(4, 5) P2 hydrolysis regulates activation and inactivation of trpc6\/7 channels","volume":"143","author":"Itsuki","year":"2014","journal-title":"J. Gen. Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1161\/01.RES.88.3.325","article-title":"The transient receptor potential protein homologue trp6 is the essential component of vascular \u03b11-adrenoceptor\u2013activated Ca2+-permeable cation channel","volume":"88","author":"Inoue","year":"2001","journal-title":"Circ. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/S0896-6273(03)00395-7","article-title":"Fret two-hybrid mapping reveals function and location of L-type Ca2+ channel cam preassociation","volume":"39","author":"Erickson","year":"2003","journal-title":"Neuron"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1093\/cvr\/cvx117","article-title":"Uncovering the arrhythmogenic potential of trpm4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches","volume":"113","author":"Hu","year":"2017","journal-title":"Cardiovasc. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1085\/jgp.200910345","article-title":"Kinetics of PIP2 metabolism and kcnq2\/3 channel regulation studied with a voltage-sensitive phosphatase in living cells","volume":"135","author":"Falkenburger","year":"2010","journal-title":"J. Gen. Physiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1113\/jphysiol.2011.221358","article-title":"A self-limiting regulation of vasoconstrictor-activated trpc3\/c6\/c7 channels coupled to PI(4, 5) P2-diacylglycerol signalling","volume":"590","author":"Imai","year":"2012","journal-title":"J. Physiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s12576-018-0610-z","article-title":"The 95th annual meeting of the physiological society of Japan","volume":"68","author":"Ishikawa","year":"2018","journal-title":"J. Physiol. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.bpc.2015.06.004","article-title":"PIP2 and PIP3 interact with n-terminus region of trpm4 channel","volume":"205","author":"Bousova","year":"2015","journal-title":"Biophys. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"479","DOI":"10.4161\/chan.22177","article-title":"Ptdins (4, 5) P2 interacts with cam binding domains on trpm3 n-terminus","volume":"6","author":"Holendova","year":"2012","journal-title":"Channels"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1126\/science.aar4510","article-title":"Structure of the human trpm4 ion channel in a lipid nanodisc","volume":"359","author":"Autzen","year":"2018","journal-title":"Science"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1016\/j.celrep.2018.01.042","article-title":"Direct binding between pre-s1 and trp-like domains in trpp channels mediates gating and functional regulation by PIP2","volume":"22","author":"Zheng","year":"2018","journal-title":"Cell Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1016\/S0006-3495(78)85489-7","article-title":"Ionic channels in excitable membranes. Current problems and biophysical approaches","volume":"22","author":"Hille","year":"1978","journal-title":"Biophys. J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1038\/s41589-020-00693-3","article-title":"Ion channels as lipid sensors: From structures to mechanisms","volume":"16","author":"Thompson","year":"2020","journal-title":"Nat. Chem. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2377","DOI":"10.1073\/pnas.1722038115","article-title":"Structure of full-length human trpm4","volume":"115","author":"Duan","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1161\/01.HYP.0000237864.65019.a5","article-title":"Functional expression of the trpm4 cationic current in ventricular cardiomyocytes from spontaneously hypertensive rats","volume":"48","author":"Guinamard","year":"2006","journal-title":"Hypertension"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/10\/5\/983\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:51:37Z","timestamp":1760161897000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/10\/5\/983"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,22]]},"references-count":30,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["cells10050983"],"URL":"https:\/\/doi.org\/10.3390\/cells10050983","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,22]]}}}