{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T06:42:50Z","timestamp":1790404970081,"version":"4.1.0"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,1,20]],"date-time":"2021-01-20T00:00:00Z","timestamp":1611100800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31571083"],"award-info":[{"award-number":["31571083"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31970931"],"award-info":[{"award-number":["31970931"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100013105","name":"Shanghai Rising-Star Program","doi-asserted-by":"publisher","award":["14QA1400800"],"award-info":[{"award-number":["14QA1400800"]}],"id":[{"id":"10.13039\/501100013105","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Physiol."],"abstract":"<jats:p>Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2\u2013S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM<jats:sub>1234<\/jats:sub>, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM<jats:sub>3<\/jats:sub>, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM<jats:sub>1234<\/jats:sub> on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM<jats:sub>1234<\/jats:sub> or CaM<jats:sub>3<\/jats:sub> are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2\u2013S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM<jats:sub>3<\/jats:sub>, indicating that these five mutations decrease the inhibitory effect of Ca<jats:sup>2+<\/jats:sup>\/CaM. The double mutation C156A\/R159A decreases Ca<jats:sup>2+<\/jats:sup>\/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2\u2013S3 loop in CaM regulation.<\/jats:p>","DOI":"10.3389\/fphys.2020.604134","type":"journal-article","created":{"date-parts":[[2021,1,20]],"date-time":"2021-01-20T05:46:23Z","timestamp":1611121583000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["The S2\u2013S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation"],"prefix":"10.3389","volume":"11","author":[{"given":"Wenhui","family":"Zhuang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiqiang","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,1,20]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"299","DOI":"10.1080\/19336950.2018.1511512","article-title":"Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv72 potassium channel mutants","volume":"12","author":"Alaimo","year":"2018","journal-title":"Channels"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"20668","DOI":"10.1074\/jbc.M109.019539","article-title":"Calmodulin activation limits the rate of KCNQ2 K+ channel exit from the endoplasmic reticulum","volume":"284","author":"Alaimo","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"2307","DOI":"10.1113\/jphysiol.2008.152777","article-title":"Calmodulin binding to M-type K+ channels assayed by TIRF\/FRET in living cells","volume":"586","author":"Bal","year":"2008","journal-title":"J. Physiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"78","DOI":"10.1038\/384078a0","article-title":"K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current","volume":"384","author":"Barhanin","year":"1996","journal-title":"Nature"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1016\/S0169-328X(00)00204-7","article-title":"Longitudinal gradients of KCNQ4 expression in spiral ganglion and cochlear hair cells correlate with progressive hearing loss in DFNA2","volume":"82","author":"Beisel","year":"2000","journal-title":"Brain Res. Mol. Brain Res."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"9285","DOI":"10.1523\/JNEUROSCI.2110-05.2005","article-title":"Differential expression of KCNQ4 in inner hair cells and sensory neurons is the basis of progressive high-frequency hearing loss","volume":"25","author":"Beisel","year":"2005","journal-title":"J. Neurosci."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"2395","DOI":"10.1073\/pnas.1800235115","article-title":"Structural basis and energy landscape for the Ca2+ gating and calmodulation of the Kv7.2 K(+) channel","volume":"115","author":"Bernardo-Seisdedos","year":"2018","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"C669","DOI":"10.1152\/ajpcell.00191.2004","article-title":"Calmodulin interactions with IQ peptides from voltage-dependent calcium channels","volume":"288","author":"Black","year":"2005","journal-title":"Am. J. Phys. Cell Physiol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"1185","DOI":"10.1111\/j.1476-5381.2009.00111.x","article-title":"Neural KCNQ (Kv7) channels","volume":"156","author":"Brown","year":"2009","journal-title":"Br. J. Pharmacol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"836.e836","DOI":"10.1016\/j.neuron.2018.01.035","article-title":"A Calmodulin C-lobe Ca2+\/-dependent switch governs Kv7 channel function","volume":"97","author":"Chang","year":"2018","journal-title":"Neuron"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"850","DOI":"10.1038\/nrn1785","article-title":"Pathways modulating neural KCNQ\/M (Kv7) potassium channels","volume":"6","author":"Delmas","year":"2005","journal-title":"Nat. Rev. Neurosci."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"1135","DOI":"10.1096\/fj.07-9712com","article-title":"Calmodulin regulates the trafficking of KCNQ2 potassium channels","volume":"22","author":"Etxeberria","year":"2008","journal-title":"FASEB J."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1002\/prot.22317","article-title":"Energetics of calmodulin domain interactions with the calmodulin binding domain of CaMKII","volume":"76","author":"Evans","year":"2009","journal-title":"Proteins"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"3217","DOI":"10.1016\/j.jmb.2013.06.024","article-title":"Apo states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain","volume":"425","author":"Findeisen","year":"2013","journal-title":"J. Mol. Biol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"3538","DOI":"10.1091\/mbc.e04-09-0849","article-title":"Structural requirements for differential sensitivity of KCNQ K+ channels to modulation by Ca2+\/calmodulin","volume":"16","author":"Gamper","year":"2005","journal-title":"Mol. Biol. Cell"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1085\/jgp.200208783","article-title":"Calmodulin mediates Ca2+\u2212dependent modulation of M-type K+ channels","volume":"122","author":"Gamper","year":"2003","journal-title":"J. Gen. Physiol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"4996","DOI":"10.1038\/sj.emboj.7601374","article-title":"Oxidative modification of M-type K(+) channels as a mechanism of cytoprotective neuronal silencing","volume":"25","author":"Gamper","year":"2006","journal-title":"EMBO J."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"949","DOI":"10.1016\/0092-8674(91)90547-C","article-title":"Can calmodulin function without binding calcium?","volume":"65","author":"Geiser","year":"1991","journal-title":"Cell"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"1048","DOI":"10.1161\/01.RES.0000218863.44140.f2","article-title":"KCNQ1 assembly and function is blocked by long-QT syndrome mutations that disrupt interaction with calmodulin","volume":"98","author":"Ghosh","year":"2006","journal-title":"Circ. Res."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"117","DOI":"10.3389\/fnmol.2017.00117","article-title":"Differential regulation of PI(4,5)P2 sensitivity of Kv7.2 and Kv7.3 channels by calmodulin","volume":"10","author":"Gomis-Perez","year":"2017","journal-title":"Front. Mol. Neurosci."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1124\/pr.55.4.9","article-title":"International union of pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels","volume":"55","author":"Gutman","year":"2003","journal-title":"Pharmacol. Rev."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"1811","DOI":"10.1113\/jphysiol.2007.148304","article-title":"Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications","volume":"586","author":"Hernandez","year":"2008","journal-title":"J. Physiol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1038\/35036198","article-title":"Neuronal KCNQ potassium channels: physiology and role in disease","volume":"1","author":"Jentsch","year":"2000","journal-title":"Nat. Rev. Neurosci."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"4333","DOI":"10.1073\/pnas.97.8.4333","article-title":"KCNQ4, a K+ channel mutated in a form of dominant deafness, is expressed in the inner ear and the central auditory pathway","volume":"97","author":"Kharkovets","year":"2000","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"1517","DOI":"10.1074\/jbc.M110.179010","article-title":"Cellular and molecular mechanisms of autosomal dominant form of progressive hearing loss, DFNA2","volume":"286","author":"Kim","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"e82290","DOI":"10.1371\/journal.pone.0082290","article-title":"A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+\u2212dependent modulation of KCNQ channel activity","volume":"8","author":"Kosenko","year":"2013","journal-title":"PLoS One"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"437","DOI":"10.1016\/s0092-8674(00)80556-5","article-title":"KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness","volume":"96","author":"Kubisch","year":"1999","journal-title":"Cell"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"508","DOI":"10.1126\/science.aas9466","article-title":"Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures","volume":"360","author":"Lee","year":"2018","journal-title":"Science"},{"key":"ref29","doi-asserted-by":"crossref","first-page":"8050","DOI":"10.1016\/S0021-9258(18)92938-8","article-title":"Calcium binding to calmodulin and its globular domains","volume":"266","author":"Linse","year":"1991","journal-title":"J. Biol. Chem."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1016\/j.mcn.2013.12.005","article-title":"Calmodulin orchestrates the heteromeric assembly and the trafficking of KCNQ2\/3 (Kv7.2\/3) channels in neurons","volume":"58","author":"Liu","year":"2014","journal-title":"Mol. Cell. Neurosci."},{"key":"ref31","doi-asserted-by":"crossref","first-page":"10949","DOI":"10.1016\/S0021-9258(18)90605-8","article-title":"Quantitation of energy coupling between Ca2+, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates","volume":"259","author":"Olwin","year":"1984","journal-title":"J. Biol. Chem."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"8081","DOI":"10.1021\/bi00348a037","article-title":"Calcium binding to complexes of calmodulin and calmodulin binding proteins","volume":"24","author":"Olwin","year":"1985","journal-title":"Biochemistry"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"6041","DOI":"10.1523\/JNEUROSCI.4275-12.2013","article-title":"Triple cysteine module within M-type K+ channels mediates reciprocal channel modulation by nitric oxide and reactive oxygen species","volume":"33","author":"Ooi","year":"2013","journal-title":"J. Neurosci."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"794","DOI":"10.1002\/pro.5560060406","article-title":"Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation","volume":"6","author":"Peersen","year":"1997","journal-title":"Protein Sci."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"1582","DOI":"10.1016\/j.str.2014.07.016","article-title":"Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin","volume":"22","author":"Sachyani","year":"2014","journal-title":"Structure"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"80","DOI":"10.1038\/384080a0","article-title":"Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel","volume":"384","author":"Sanguinetti","year":"1996","journal-title":"Nature"},{"key":"ref37","doi-asserted-by":"publisher","first-page":"1055","DOI":"10.1161\/01.res.0000218979.40770.69","article-title":"Calmodulin is essential for cardiac IKS channel gating and assembly: impaired function in long-QT mutations","volume":"98","author":"Shamgar","year":"2006","journal-title":"Circ. Res."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"2499","DOI":"10.1074\/jbc.M115.668236","article-title":"Mechanisms of calmodulin regulation of different isoforms of Kv7.4 K+ channels","volume":"291","author":"Sihn","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"5353","DOI":"10.1021\/acs.biochem.6b00477","article-title":"Structural insights into the M-channel proximal C-terminus\/calmodulin complex","volume":"55","author":"Strulovich","year":"2016","journal-title":"Biochemistry"},{"key":"ref40","doi-asserted-by":"publisher","first-page":"1042.e1049","DOI":"10.1016\/j.cell.2017.05.019","article-title":"Cryo-EM structure of a KCNQ1\/CaM complex reveals insights into congenital long QT syndrome","volume":"169","author":"Sun","year":"2017","journal-title":"Cell"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"E869","DOI":"10.1073\/pnas.1612622114","article-title":"Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv71 channel","volume":"114","author":"Tobelaim","year":"2017","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1038\/ng0196-17","article-title":"Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias","volume":"12","author":"Wang","year":"1996","journal-title":"Nat. Genet."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"1890","DOI":"10.1126\/science.282.5395.1890","article-title":"KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel","volume":"282","author":"Wang","year":"1998","journal-title":"Science"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"7991","DOI":"10.1523\/JNEUROSCI.22-18-07991.2002","article-title":"Calmodulin is an auxiliary subunit of KCNQ2\/3 potassium channels","volume":"22","author":"Wen","year":"2002","journal-title":"J. Neurosci."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"503","DOI":"10.1038\/26758","article-title":"Mechanism of calcium gating in small-conductance calcium-activated potassium channels","volume":"395","author":"Xia","year":"1998","journal-title":"Nature"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"378","DOI":"10.1016\/j.jmb.2012.11.023","article-title":"Structure of a Ca2+\/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M current modulation","volume":"425","author":"Xu","year":"2013","journal-title":"J. Mol. Biol."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"23899","DOI":"10.1074\/jbc.M702108200","article-title":"Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels","volume":"282","author":"Xu","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"28545","DOI":"10.1074\/jbc.M204130200","article-title":"The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels","volume":"277","author":"Yus-Najera","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"3243","DOI":"10.1242\/jcs.026856","article-title":"The voltage-gated Na+ channel Nav1.8 contains an ER-retention\/retrieval signal antagonized by the beta3 subunit","volume":"121","author":"Zhang","year":"2008","journal-title":"J. Cell Sci."}],"container-title":["Frontiers in Physiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphys.2020.604134\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,1,20]],"date-time":"2021-01-20T05:46:28Z","timestamp":1611121588000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fphys.2020.604134\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,20]]},"references-count":49,"alternative-id":["10.3389\/fphys.2020.604134"],"URL":"https:\/\/doi.org\/10.3389\/fphys.2020.604134","relation":{},"ISSN":["1664-042X"],"issn-type":[{"value":"1664-042X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,20]]},"article-number":"604134"}}