Abstract
The transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective monovalent cation channel belonging to the TRP channel superfamily. TRPM4 is widely expressed in various tissues and most abundantly expressed in the heart. TRPM4 plays a critical role in cardiac conduction. Patients carrying a gain-of-function or loss-of-function mutation of TRPM4 display impaired cardiac conduction. Knockout or over-expression of TRPM4 in mice recapitulates conduction defects in patients. Moreover, recent studies have indicated that TRPM4 plays a role in hypertrophy and heart failure. Whereas the role of TRPM4 mediated by cardiac myocytes has been well investigated, little is known about TRPM4 and its role in cardiac fibroblasts. Here we show that in human left ventricular fibroblasts, TRPM4 exhibits typical Ca2+-activation characteristics, linear current–voltage (I–V) relation, and monovalent permeability. TRPM4 currents recorded in fibroblasts from heart failure patients (HF) are more than 2-fold bigger than those from control individuals (CTL). The enhanced functional TRPM4 in HF is not resulted from changed channel properties, as TRPM4 currents from both HF and CTL fibroblasts demonstrate similar sensitivity to intracellular calcium activation and extracellular 9-phenanthrol (9-phen) blockade. Consistent with enhanced TRPM4 activity, the protein level of TRPM4 is about 2-fold higher in HF than that of CTL hearts. Moreover, TRPM4 current in CTL fibroblasts is increased after 24 hours of TGFβ1 treatment, implying that TRPM4 in vivo may be upregulated by fibrogenesis promotor TGFβ1. The upregulated TRPM4 in HF fibroblasts suggests that TRPM4 may play a role in cardiac fibrogenesis under various pathological conditions.
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Abbreviations
- BMI:
-
body mass index
- CAD:
-
coronary artery disease
- DM:
-
diabetes mellitus
- EF:
-
ejection fraction
- Eth:
-
ethnicity
- Gen:
-
gender
- HF:
-
heart failure
- HLD:
-
hyperlipidemia
- HTN:
-
hypertension
- Med:
-
outpatient medication
- MI:
-
myocardial infarction
- VD:
-
valve diseases
References
Autzen HE, Myasnikov AG, Campbell MG, Asarnow D, Julius D, Cheng Y (2018) Structure of the human TRPM4 ion channel in a lipid nanodisc. Science 359:228–232. https://doi.org/10.1126/science.aar4510
Barbet G, Demion M, Moura IC, Serafini N, Leger T, Vrtovsnik F, Monteiro RC, Guinamard R, Kinet JP, Launay P (2008) The calcium-activated nonselective cation channel TRPM4 is essential for the migration but not the maturation of dendritic cells. Nat Immunol 9:1148–1156. https://doi.org/10.1038/ni.1648
Barefield DY, McNamara JW, Lynch TL, Kuster DWD, Govindan S, Haar L, Wang Y, Taylor EN, Lorenz JN, Nieman ML, Zhu G, Luther PK, Varro A, Dobrev D, Ai X, Janssen PML, Kass DA, Jones WK, Gilbert RJ, Sadayappan S (2019) Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury. J Mol Cell Cardiol 129:236–246. https://doi.org/10.1016/j.yjmcc.2019.03.006
Bianchi B, Ozhathil LC, Medeiros-Domingo A, Gollob MH, Abriel H (2018) Four TRPM4 cation channel mutations found in cardiac conduction diseases lead to altered protein stability. Front Physiol 9:177. https://doi.org/10.3389/fphys.2018.00177
Burt R, Graves BM, Gao M, Li C, Williams DL, Fregoso SP, Hoover DB, Li Y, Wright GL, Wondergem R (2013) 9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes. Cell Calcium 54:193–201. https://doi.org/10.1016/j.ceca.2013.06.003
Chatelier A, Mercier A, Tremblier B, Theriault O, Moubarak M, Benamer N, Corbi P, Bois P, Chahine M, Faivre JF (2012) A distinct de novo expression of Nav1.5 sodium channels in human atrial fibroblasts differentiated into myofibroblasts. J Physiol 590:4307–4319. https://doi.org/10.1113/jphysiol.2012.233593
Chen B, Gao Y, Wei S, Low SW, Ng G, Yu D, Tu TM, Soong TW, Nilius B, Liao P (2019) TRPM4-specific blocking antibody attenuates reperfusion injury in a rat model of stroke. Arch Eur J Physiol 471:1455–1466. https://doi.org/10.1007/s00424-019-02326-8
Cheng H, Beck A, Launay P, Gross SA, Stokes AJ, Kinet JP, Fleig A, Penner R (2007) TRPM4 controls insulin secretion in pancreatic beta-cells. Cell Calcium 41:51–61. https://doi.org/10.1016/j.ceca.2006.04.032
Clapham DE (2003) TRP channels as cellular sensors. Nature 426:517–524
Colquhoun D, Neher E, Reuter H, Stevens CF (1981) Inward current channels activated by intracellular Ca in cultured cardiac cells. Nature 294:752–754
Crnich R, Amberg GC, Leo MD, Gonzales AL, Tamkun MM, Jaggar JH, Earley S (2010) Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells. Am J Physiol Cell Physiol 299:C682–C694. https://doi.org/10.1152/ajpcell.00101.2010
Daumy X, Amarouch MY, Lindenbaum P, Bonnaud S, Charpentier E, Bianchi B, Nafzger S, Baron E, Fouchard S, Thollet A, Kyndt F, Barc J, Le Scouarnec S, Makita N, Le Marec H, Dina C, Gourraud JB, Probst V, Abriel H, Redon R, Schott JJ (2016) Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I. Int J Cardiol 207:349–358. https://doi.org/10.1016/j.ijcard.2016.01.052
Demion M, Bois P, Launay P, Guinamard R (2007) TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells. Cardiovasc Res 73:531–538. https://doi.org/10.1016/j.cardiores.2006.11.023
Demion M, Thireau J, Gueffier M, Finan A, Khoueiry Z, Cassan C, Serafini N, Aimond F, Granier M, Pasquie JL, Launay P, Richard S (2014) Trpm4 gene invalidation leads to cardiac hypertrophy and electrophysiological alterations. PLoS One 9:e115256. https://doi.org/10.1371/journal.pone.0115256
Dragun M, Gazova A, Kyselovic J, Hulman M, Matus M (2019) TRP channels expression profile in human end-stage heart failure. Medicina 55. https://doi.org/10.3390/medicina55070380
Du J, Xie J, Yue L (2009) Intracellular calcium activates TRPM2 and its alternative spliced isoforms. Proc Natl Acad Sci 107:7239–7244. https://doi.org/10.1073/pnas.0811725106
Du J, Xie J, Zhang Z, Tsujikawa H, Fusco D, Silverman D, Liang B, Yue L (2010) TRPM7-mediated Ca2+ signals confer fibrogenesis in human atrial fibrillation. Circ Res 106:992–1003. https://doi.org/10.1161/CIRCRESAHA.109.206771
Duan J, Li Z, Li J, Santa-Cruz A, Sanchez-Martinez S, Zhang J, Clapham DE (2018) Structure of full-length human TRPM4. Proc Natl Acad Sci U S A 115:2377–2382. https://doi.org/10.1073/pnas.1722038115
Earley S (2013) TRPM4 channels in smooth muscle function. Arch Eur J Physiol 465:1223–1231. https://doi.org/10.1007/s00424-013-1250-z
Earley S, Waldron BJ, Brayden JE (2004) Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ Res 95:922–929. https://doi.org/10.1161/01.RES.0000147311.54833.03
Ehara T, Noma A, Ono K (1988) Calcium-activated non-selective cation channel in ventricular cells isolated from adult guinea-pig hearts. J Physiol 403:117–133. https://doi.org/10.1113/jphysiol.1988.sp017242
Feng J, Armillei MK, Yu AS, Liang BT, Runnels LW, Yue L (2019) Ca(2+) signaling in cardiac fibroblasts and fibrosis-associated heart diseases. Journal of cardiovascular development and disease 6. https://doi.org/10.3390/jcdd6040034
Fonfria E, Murdock PR, Cusdin FS, Benham CD, Kelsell RE, McNulty S (2006) Tissue distribution profiles of the human TRPM cation channel family. J Recept Signal Transduct Res 26:159–178. https://doi.org/10.1080/10799890600637506
Frede W, Medert R, Poth T, Gorenflo M, Vennekens R, Freichel M, Uhl S (2020) TRPM4 modulates right ventricular remodeling under pressure load accompanied with decreased expression level. J Card Fail 26:599–609. https://doi.org/10.1016/j.cardfail.2020.02.006
Grand T, Demion M, Norez C, Mettey Y, Launay P, Becq F, Bois P, Guinamard R (2008) 9-phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels. Br J Pharmacol 153:1697–1705. https://doi.org/10.1038/bjp.2008.38
Gualandi F, Zaraket F, Malagu M, Parmeggiani G, Trabanelli C, Fini S, Dang X, Wei X, Fang M, Bertini M, Ferrari R, Ferlini A (2017) Mutation load of multiple ion channel gene mutations in Brugada syndrome. Cardiology 137:256–260. https://doi.org/10.1159/000471792
Guinamard R, Bouvagnet P, Hof T, Liu H, Simard C, Salle L (2015) TRPM4 in cardiac electrical activity. Cardiovasc Res 108:21–30. https://doi.org/10.1093/cvr/cvv213
Guinamard R, Chatelier A, Demion M, Potreau D, Patri S, Rahmati M, Bois P (2004) Functional characterization of a Ca(2+)-activated non-selective cation channel in human atrial cardiomyocytes. J Physiol 558:75–83. https://doi.org/10.1113/jphysiol.2004.063974
Guinamard R, Demion M, Magaud C, Potreau D, Bois P (2006) Functional expression of the TRPM4 cationic current in ventricular cardiomyocytes from spontaneously hypertensive rats. Hypertension 48:587–594. https://doi.org/10.1161/01.HYP.0000237864.65019.a5
Guo J, She J, Zeng W, Chen Q, Bai XC, Jiang Y (2017) Structures of the calcium-activated, non-selective cation channel TRPM4. Nature 552:205–209. https://doi.org/10.1038/nature24997
Harada M, Luo X, Qi XY, Tadevosyan A, Maguy A, Ordog B, Ledoux J, Kato T, Naud P, Voigt N, Shi Y, Kamiya K, Murohara T, Kodama I, Tardif JC, Schotten U, Van Wagoner DR, Dobrev D, Nattel S (2012) Transient receptor potential canonical-3 channel-dependent fibroblast regulation in atrial fibrillation. Circulation 126:2051–2064. https://doi.org/10.1161/CIRCULATIONAHA.112.121830
Hedon C, Lambert K, Chakouri N, Thireau J, Aimond F, Cassan C, Bideaux P, Richard S, Faucherre A, Le Guennec JY, Demion M (2020) New role of TRPM4 channel in the cardiac excitation-contraction coupling in response to physiological and pathological hypertrophy in mouse. Prog Biophys Mol Biol. https://doi.org/10.1016/j.pbiomolbio.2020.09.006
Hof T, Chaigne S, Recalde A, Salle L, Brette F, Guinamard R (2019) Transient receptor potential channels in cardiac health and disease. Nat Rev Cardiol 16:344–360. https://doi.org/10.1038/s41569-018-0145-2
Hof T, Salle L, Coulbault L, Richer R, Alexandre J, Rouet R, Manrique A, Guinamard R (2016) TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres. J Physiol 594:295–306. https://doi.org/10.1113/JP271347
Hofmann T, Chubanov V, Gudermann T, Montell C (2003) TRPM5 is a voltage-modulated and Ca(2+)-activated monovalent selective cation channel. Current biology : CB 13:1153–1158
Holzmann C, Kappel S, Kilch T, Jochum MM, Urban SK, Jung V, Stockle M, Rother K, Greiner M, Peinelt C (2015) Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells. Oncotarget 6:41783-41793. doi:10.18632/oncotarget.6157
Inoue R, Kurahara LH, Hiraishi K (2018) TRP channels in cardiac and intestinal fibrosis. Semin Cell Dev Biol. https://doi.org/10.1016/j.semcdb.2018.11.002
Jacobs G, Oosterlinck W, Dresselaers T, Geenens R, Kerselaers S, Himmelreich U, Herijgers P, Vennekens R (2015) Enhanced beta-adrenergic cardiac reserve in Trpm4(-)/(-) mice with ischaemic heart failure. Cardiovasc Res 105:330–339. https://doi.org/10.1093/cvr/cvv009
Jiang J, Li M, Yue L (2005) Potentiation of TRPM7 inward currents by protons. J Gen Physiol 126:137–150
Kecskes M, Jacobs G, Kerselaers S, Syam N, Menigoz A, Vangheluwe P, Freichel M, Flockerzi V, Voets T, Vennekens R (2015) The Ca(2+)-activated cation channel TRPM4 is a negative regulator of angiotensin II-induced cardiac hypertrophy. Basic Res Cardiol 110:43. https://doi.org/10.1007/s00395-015-0501-x
Kruse M, Pongs O (2014) TRPM4 channels in the cardiovascular system. Curr Opin Pharmacol 15:68–73. https://doi.org/10.1016/j.coph.2013.12.003
Kruse M, Schulze-Bahr E, Corfield V, Beckmann A, Stallmeyer B, Kurtbay G, Ohmert I, Brink P, Pongs O (2009) Impaired endocytosis of the ion channel TRPM4 is associated with human progressive familial heart block type I. J Clin Invest 119:2737–2744. https://doi.org/10.1172/JCI38292
Launay P, Cheng H, Srivatsan S, Penner R, Fleig A, Kinet J-P (2004) TRPM4 Regulates calcium oscillations after T cell activation. Science 306:1374–1377. https://doi.org/10.1126/science.1098845
Launay P, Fleig A, Perraud AL, Scharenberg AM, Penner R, Kinet JP (2002) TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 109:397–407
Liu H, Chatel S, Simard C, Syam N, Salle L, Probst V, Morel J, Millat G, Lopez M, Abriel H, Schott JJ, Guinamard R, Bouvagnet P (2013) Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel. PLoS One 8:e54131. https://doi.org/10.1371/journal.pone.0054131
Liu H, El Zein L, Kruse M, Guinamard R, Beckmann A, Bozio A, Kurtbay G, Megarbane A, Ohmert I, Blaysat G, Villain E, Pongs O, Bouvagnet P (2010) Gain-of-function mutations in TRPM4 cause autosomal dominant isolated cardiac conduction disease. Circ Cardiovasc Genet 3:374–385. https://doi.org/10.1161/CIRCGENETICS.109.930867
Mathar I, Kecskes M, Van der Mieren G, Jacobs G, Camacho Londono JE, Uhl S, Flockerzi V, Voets T, Freichel M, Nilius B, Herijgers P, Vennekens R (2014) Increased beta-adrenergic inotropy in ventricular myocardium from Trpm4-/- mice. Circ Res 114:283–294. https://doi.org/10.1161/CIRCRESAHA.114.302835
Mathar I, Vennekens R, Meissner M, Kees F, Van der Mieren G, Camacho Londono JE, Uhl S, Voets T, Hummel B, van den Bergh A, Herijgers P, Nilius B, Flockerzi V, Schweda F, Freichel M (2010) Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice. J Clin Invest 120:3267–3279. https://doi.org/10.1172/JCI41348
Montell C (2005) The TRP superfamily of cation channels. Sci STKE 2005:1–24
Nilius B (2007) TRP channels in disease. Biochim Biophys Acta 1772:805–812
Nilius B, Prenen J, Droogmans G, Voets T, Vennekens R, Freichel M, Wissenbach U, Flockerzi V (2003) Voltage dependence of the Ca2+-activated cation channel TRPM4. J Biol Chem 278:30813–30820
Nilius B, Prenen J, Janssens A, Voets T, Droogmans G (2004) Decavanadate modulates gating of TRPM4 cation channels. J Physiol 560:753–765. https://doi.org/10.1113/jphysiol.2004.070839
Nilius B, Prenen J, Voets T, Droogmans G (2004) Intracellular nucleotides and polyamines inhibit the Ca2+-activated cation channel TRPM4b. Arch Eur J Physiol 448:70–75. https://doi.org/10.1007/s00424-003-1221-x
Nilius B, Vennekens R (2006) From cardiac cation channels to the molecular dissection of the transient receptor potential channel TRPM4. Arch Eur J Physiol 453:313–321. https://doi.org/10.1007/s00424-006-0088-z
Numaga-Tomita T, Oda S, Shimauchi T, Nishimura A, Mangmool S, Nishida M (2017) TRPC3 channels in cardiac fibrosis. Frontiers in cardiovascular medicine 4:56. https://doi.org/10.3389/fcvm.2017.00056
Perez CA, Huang L, Rong M, Kozak JA, Preuss AK, Zhang H, Max M, Margolskee RF (2002) A transient receptor potential channel expressed in taste receptor cells. Nat Neurosci 5:1169–1176
Piao H, Takahashi K, Yamaguchi Y, Wang C, Liu K, Naruse K (2015) Transient receptor potential melastatin-4 is involved in hypoxia-reoxygenation injury in the cardiomyocytes. PLoS One 10:e0121703. https://doi.org/10.1371/journal.pone.0121703
Pironet A, Syam N, Vandewiele F, Van den Haute C, Kerselaers S, Pinto S, Vande Velde G, Gijsbers R, Vennekens R (2019) AAV9-mediated overexpression of TRPM4 increases the incidence of stress-induced ventricular arrhythmias in mice. Front Physiol 10:802. https://doi.org/10.3389/fphys.2019.00802
Rahaman SO, Grove LM, Paruchuri S, Southern BD, Abraham S, Niese KA, Scheraga RG, Ghosh S, Thodeti CK, Zhang DX, Moran MM, Schilling WP, Tschumperlin DJ, Olman MA (2014) TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice. J Clin Invest 124:5225–5238. https://doi.org/10.1172/JCI75331
Schattling B, Steinbach K, Thies E, Kruse M, Menigoz A, Ufer F, Flockerzi V, Bruck W, Pongs O, Vennekens R, Kneussel M, Freichel M, Merkler D, Friese MA (2012) TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat Med 18:1805–1811. https://doi.org/10.1038/nm.3015
Serafini N, Dahdah A, Barbet G, Demion M, Attout T, Gautier G, Arcos-Fajardo M, Souchet H, Jouvin MH, Vrtovsnik F, Kinet JP, Benhamou M, Monteiro RC, Launay P (2012) The TRPM4 channel controls monocyte and macrophage, but not neutrophil, function for survival in sepsis. J Immunol 189:3689–3699. https://doi.org/10.4049/jimmunol.1102969
Shigeto M, Ramracheya R, Tarasov AI, Cha CY, Chibalina MV, Hastoy B, Philippaert K, Reinbothe T, Rorsman N, Salehi A, Sones WR, Vergari E, Weston C, Gorelik J, Katsura M, Nikolaev VO, Vennekens R, Zaccolo M, Galione A, Johnson PR, Kaku K, Ladds G, Rorsman P (2015) GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation. J Clin Invest 125:4714–4728. https://doi.org/10.1172/JCI81975
Simard C, Hof T, Keddache Z, Launay P, Guinamard R (2013) The TRPM4 non-selective cation channel contributes to the mammalian atrial action potential. J Mol Cell Cardiol 59:11–19. https://doi.org/10.1016/j.yjmcc.2013.01.019
Simard C, Magaud C, Adjlane R, Dupas Q, Salle L, Manrique A, Bois P, Faivre JF, Guinamard R (2020) TRPM4 non-selective cation channel in human atrial fibroblast growth. Arch Eur J Physiol. https://doi.org/10.1007/s00424-020-02476-0
Stallmeyer B, Zumhagen S, Denjoy I, Duthoit G, Hebert JL, Ferrer X, Maugenre S, Schmitz W, Kirchhefer U, Schulze-Bahr E, Guicheney P, Schulze-Bahr E (2012) Mutational spectrum in the Ca(2+)--activated cation channel gene TRPM4 in patients with cardiac conductance disturbances. Hum Mutat 33:109–117. https://doi.org/10.1002/humu.21599
Syam N, Chatel S, Ozhathil LC, Sottas V, Rougier JS, Baruteau A, Baron E, Amarouch MY, Daumy X, Probst V, Schott JJ, Abriel H (2016) Variants of transient receptor potential melastatin member 4 in childhood atrioventricular block. J Am Heart Assoc 5. https://doi.org/10.1161/JAHA.114.001625
Syam N, Rougier JS, Abriel H (2014) Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects. Front Cell Neurosci 8:52. https://doi.org/10.3389/fncel.2014.00052
Vennekens R, Olausson J, Meissner M, Bloch W, Mathar I, Philipp SE, Schmitz F, Weissgerber P, Nilius B, Flockerzi V, Freichel M (2007) Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4. Nat Immunol 8:312–320. https://doi.org/10.1038/ni1441
Wang C, Naruse K, Takahashi K (2018) Role of the TRPM4 channel in cardiovascular physiology and pathophysiology. Cells 7. https://doi.org/10.3390/cells7060062
Winkler PA, Huang Y, Sun W, Du J, Lu W (2017) Electron cryo-microscopy structure of a human TRPM4 channel. Nature 552:200–204. https://doi.org/10.1038/nature24674
Yan J, Bengtson CP, Buchthal B, Hagenston AM, Bading H (2020) Coupling of NMDA receptors and TRPM4 guides discovery of unconventional neuroprotectants. Science 370. https://doi.org/10.1126/science.aay3302
Yue L, Feng J, Wang Z, Nattel S (2000) Effects of ambasilide, quinidine, flecainide and verapamil on ultra-rapid delayed rectifier potassium currents in canine atrial myocytes. Cardiovasc Res 46:151–161
Yue Z, Xie J, Yu AS, Stock J, Du J, Yue L (2015) Role of TRP channels in the cardiovascular system. Am J Phys Heart Circ Phys 308:H157–H182. https://doi.org/10.1152/ajpheart.00457.2014
Yue Z, Zhang Y, Xie J, Jiang J, Yue L (2013) Transient receptor potential (TRP) channels and cardiac fibrosis. Curr Top Med Chem 13:270–282
Zhang Z, Okawa H, Wang Y, Liman ER (2005) Phosphatidylinositol 4,5-bisphosphate rescues TRPM4 channels from desensitization. J Biol Chem 280:39185–39192. https://doi.org/10.1074/jbc.M506965200
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This work was partially supported by the National Institute of Health (P01-HL06426, R01-AA024769, and R01-HL146744 to XA; and R01-HL143750 to LY) and American Heart Association (19TPA34890022 to LY).
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This article is part of the special issue on Calcium Signal Dynamics in Cardiac Myocytes and Fibroblasts: Mechanisms in Pflügers Archiv—European Journal of Physiology
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Feng, J., Zong, P., Yan, J. et al. Upregulation of transient receptor potential melastatin 4 (TRPM4) in ventricular fibroblasts from heart failure patients. Pflugers Arch - Eur J Physiol 473, 521–531 (2021). https://doi.org/10.1007/s00424-021-02525-2
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DOI: https://doi.org/10.1007/s00424-021-02525-2