Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

2nd Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague Czech Republic

Faculty of Mathematics and Physics Charles University Ke Karlovu 5 12116 Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 14220 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo namesti 2 16000 Prague Czech Republic

Zobrazit více v PubMed

Nilius B., Prenen J., Tang J., Wang C., Owsianik G., Janssens A., Voets T., Zhu M.X. Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4. J. Biol. Chem. 2005;280:6423–6433. doi: 10.1074/jbc.M411089200. PubMed DOI

Clapham D.E., Runnels L.W., Strübing C. The TRP ion channel family. Nat. Rev. Neurosci. 2001;2:387. doi: 10.1038/35077544. PubMed DOI

Ehara T., Noma A., Ono K. Calcium-activated non-selective cation channel in ventricular cells isolated from adult guinea-pig hearts. J. Physiol. 1988;403:117–133. doi: 10.1113/jphysiol.1988.sp017242. PubMed DOI PMC

Launay P., Fleig A., Perraud A.-L., Scharenberg A.M., Penner R., Kinet J.-P. TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell. 2002;109:397–407. doi: 10.1016/S0092-8674(02)00719-5. PubMed DOI

Tian J., An X., Fu M. Transient receptor potential melastatin 4 cation channel in pediatric heart block. Eur. Rev. Med. Pharmacol. Sci. 2017;21:79–84. PubMed

Nilius B., Prenen J., Droogmans G., Voets T., Vennekens R., Freichel M., Wissenbach U., Flockerzi V. Voltage dependence of the Ca2+-activated cation channel TRPM4. J. Biol. Chem. 2003;278:30813–30820. doi: 10.1074/jbc.M305127200. PubMed DOI

Mathar I., Jacobs G., Kecskes M., Menigoz A., Philippaert K., Vennekens R. Mammalian Transient Receptor Potential (TRP) Cation Channels. Springer; Berlin, Germany: 2014. Trpm4; pp. 461–487.

Duan J., Li Z., Li J., Santa-Cruz A., Sanchez-Martinez S., Zhang J., Clapham D.E. Structure of full-length human TRPM4. Proc. Natl. Acad. Sci. USA. 2018;115:2377–2382. doi: 10.1073/pnas.1722038115. PubMed DOI PMC

Autzen H.E., Myasnikov A.G., Campbell M.G., Asarnow D., Julius D., Cheng Y. Structure of the human TRPM4 ion channel in a lipid nanodisc. Science. 2018;359:228–232. doi: 10.1126/science.aar4510. PubMed DOI PMC

Winkler P.A., Huang Y., Sun W., Du J., Lü W. Electron cryo-microscopy structure of a human TRPM4 channel. Nature. 2017;552:200. doi: 10.1038/nature24674. PubMed DOI

Guo J., She J., Zeng W., Chen Q., Bai X.-c., Jiang Y. Structures of the calcium-activated, non-selective cation channel TRPM4. Nature. 2017;552:205. doi: 10.1038/nature24997. PubMed DOI PMC

Nilius B., Mahieu F., Prenen J., Janssens A., Owsianik G., Vennekens R., Voets T. The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4, 5-biphosphate. EMBO J. 2006;25:467–478. doi: 10.1038/sj.emboj.7600963. PubMed DOI PMC

Zhang Z., Okawa H., Wang Y., Liman E.R. Phosphatidylinositol 4, 5-bisphosphate rescues TRPM4 channels from desensitization. J. Biol. Chem. 2005;280:39185–39192. doi: 10.1074/jbc.M506965200. PubMed DOI

Vennekens R., Nilius B. Transient Receptor Potential (TRP) Channels. Springer; Berlin, Germany: 2007. Insights into TRPM4 function, regulation and physiological role; pp. 269–285. PubMed

Singh A.K., McGoldrick L.L., Twomey E.C., Sobolevsky A.I. Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6. Sci. Adv. 2018;4:eaau6088. doi: 10.1126/sciadv.aau6088. PubMed DOI PMC

De Groot T., Kovalevskaya N.V., Verkaart S., Schilderink N., Felici M., van der Hagen E.A., Bindels R.J., Vuister G.W., Hoenderop J.G. The molecular mechanisms of calmodulin action on TRPV5 and the modulation by parathyroid hormone. Mol. Cell. Biol. 2011;31:2845–2853. doi: 10.1128/MCB.01319-10. PubMed DOI PMC

Villalobo A., González-Muñoz M., Berchtold M.W. Proteins with calmodulin-like domains: Structures and functional roles. Cell. Mol. Life Sci. 2019;76:2299–2328. doi: 10.1007/s00018-019-03062-z. PubMed DOI PMC

Tabernero L., Taylor D.A., Chandross R.J., VanBerkum M.F., Means A.R., Quiocho F.A., Sack J.S. The structure of a calmodulin mutant with a deletion in the central helix: Implications for molecular recognition and protein binding. Structure. 1997;5:613–622. doi: 10.1016/S0969-2126(97)00217-7. PubMed DOI

Rhoads A.R., Friedberg F. Sequence motifs for calmodulin recognition. FASEB J. 1997;11:331–340. doi: 10.1096/fasebj.11.5.9141499. PubMed DOI

Zhu M.X. Multiple roles of calmodulin and other Ca2+-binding proteins in the functional regulation of TRP channels. Pflügers Archiv. 2005;451:105–115. doi: 10.1007/s00424-005-1427-1. PubMed DOI

Hasan R., Zhang X. Ca2+ regulation of TRP ion channels. Int. J. Mol. Sci. 2018;19:1256. doi: 10.3390/ijms19041256. PubMed DOI PMC

Rohacs T., Nilius B. Regulation of transient receptor potential (TRP) channels by phosphoinositides. Pflügers Archiv. Eur. J. Physiol. 2007;455:157–168. doi: 10.1007/s00424-007-0275-6. PubMed DOI

Lemmon M.A., Ferguson K.M., O’Brien R., Sigler P.B., Schlessinger J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc. Natl. Acad. Sci. USA. 1995;92:10472–10476. doi: 10.1073/pnas.92.23.10472. PubMed DOI PMC

Yamaguchi S., Tanimoto A., Iwasa S., Otsuguro K.-i. TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI (4, 5) P2. Int. J. Mol. Sci. 2019;20:2012. doi: 10.3390/ijms20082012. PubMed DOI PMC

Bousova K., Jirku M., Bumba L., Bednarova L., Sulc M., Franek M., Vyklicky L., Vondrasek J., Teisinger J. PIP2 and PIP3 interact with N-terminus region of TRPM4 channel. Biophys. Chem. 2015;205:24–32. doi: 10.1016/j.bpc.2015.06.004. PubMed DOI

Ufret-Vincenty C.A., Klein R.M., Hua L., Angueyra J., Gordon S.E. Localization of the PIP2 sensor of TRPV1 ion channels. J. Biol. Chem. 2011;286:9688–9698. doi: 10.1074/jbc.M110.192526. PubMed DOI PMC

Yin Y., Le S.C., Hsu A.L., Borgnia M.J., Yang H., Lee S.-Y. Structural basis of cooling agent and lipid sensing by the cold-activated TRPM8 channel. Science. 2019;363:eaav9334. doi: 10.1126/science.aav9334. PubMed DOI PMC

Hughes T.E., Pumroy R.A., Yazici A.T., Kasimova M.A., Fluck E.C., Huynh K.W., Samanta A., Molugu S.K., Zhou Z.H., Carnevale V. Structural insights on TRPV5 gating by endogenous modulators. Nat. Commun. 2018;9:4198. doi: 10.1038/s41467-018-06753-6. PubMed DOI PMC

Fine M., Schmiege P., Li X. Structural basis for PtdInsP 2-mediated human TRPML1 regulation. Nat. Commun. 2018;9:4192. doi: 10.1038/s41467-018-06493-7. PubMed DOI PMC

Stokum J.A., Kwon M.S., Woo S.K., Tsymbalyuk O., Vennekens R., Gerzanich V., Simard J.M. SUR1-TRPM4 and AQP4 form a heteromultimeric complex that amplifies ion/water osmotic coupling and drives astrocyte swelling. Glia. 2018;66:108–125. doi: 10.1002/glia.23231. PubMed DOI PMC

Woo S.K., Kwon M.S., Ivanov A., Gerzanich V., Simard J.M. The sulfonylurea receptor 1 (Sur1)-transient receptor potential melastatin 4 (Trpm4) channel. J. Biol. Chem. 2013;288:3655–3667. doi: 10.1074/jbc.M112.428219. PubMed DOI PMC

Pratt E.B., Tewson P., Bruederle C.E., Skach W.R., Shyng S.-L. N-terminal transmembrane domain of SUR1 controls gating of Kir6. 2 by modulating channel sensitivity to PIP2. J. Gen. Physiol. 2011;137:299–314. doi: 10.1085/jgp.201010557. PubMed DOI PMC

Galizia L., Pizzoni A., Fernandez J., Rivarola V., Capurro C., Ford P. Functional interaction between AQP2 and TRPV4 in renal cells. J. Cell. Biochem. 2012;113:580–589. doi: 10.1002/jcb.23382. PubMed DOI

Yap K.L., Kim J., Truong K., Sherman M., Yuan T., Ikura M. Calmodulin target database. J. Struct. Funct. Genom. 2000;1:8–14. doi: 10.1023/A:1011320027914. PubMed DOI

Roche J.V., Törnroth-Horsefield S. Aquaporin protein-protein interactions. Int. J. Mol. Sci. 2017;18:2255. doi: 10.3390/ijms18112255. PubMed DOI PMC

Bily J., Grycova L., Holendova B., Jirku M., Janouskova H., Bousova K., Teisinger J. Characterization of the S100A1 protein binding site on TRPC6 C-terminus. PLoS ONE. 2013;8:e62677. doi: 10.1371/journal.pone.0062677. PubMed DOI PMC

Grycova L., Holendova B., Bumba L., Bily J., Jirku M., Lansky Z., Teisinger J. Integrative binding sites within intracellular termini of TRPV1 receptor. PLoS ONE. 2012;7:e48437. doi: 10.1371/journal.pone.0048437. PubMed DOI PMC

Holakovska B., Grycova L., Bily J., Teisinger J. Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails. Amino Acids. 2011;40:741–748. doi: 10.1007/s00726-010-0712-2. PubMed DOI

Prosser B.L., Hernández-Ochoa E.O., Schneider M.F. S100A1 and calmodulin regulation of ryanodine receptor in striated muscle. Cell Calcium. 2011;50:323–331. doi: 10.1016/j.ceca.2011.06.001. PubMed DOI PMC

Bousova K., Herman P., Vecer J., Bednarova L., Monincova L., Majer P., Vyklicky L., Vondrasek J., Teisinger J. Shared CaM-and S100A1-binding epitopes in the distal TRPM 4 N terminus. FEBS J. 2018;285:599–613. doi: 10.1111/febs.14362. PubMed DOI

Lau S.-Y., Procko E., Gaudet R. Distinct properties of Ca2+–calmodulin binding to N-and C-terminal regulatory regions of the TRPV1 channel. J. Gen. Physiol. 2012;140:541–555. doi: 10.1085/jgp.201210810. PubMed DOI PMC

Meador W.E., Means A.R., Quiocho F.A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992;257:1251–1255. doi: 10.1126/science.1519061. PubMed DOI

Maximciuc A.A., Putkey J.A., Shamoo Y., MacKenzie K.R. Complex of calmodulin with a ryanodine receptor target reveals a novel, flexible binding mode. Structure. 2006;14:1547–1556. doi: 10.1016/j.str.2006.08.011. PubMed DOI

Wright N.T., Prosser B.L., Varney K.M., Zimmer D.B., Schneider M.F., Weber D.J. S100A1 and calmodulin compete for the same binding site on ryanodine receptor. J. Biol. Chem. 2008;283:26676–26683. doi: 10.1074/jbc.M804432200. PubMed DOI PMC

Prosser B.L., Wright N.T., Hernandez-Ochoa E.O., Varney K.M., Liu Y., Olojo R.O., Zimmer D.B., Weber D.J., Schneider M.F. S100A1 binds to the calmodulin-binding site of ryanodine receptor and modulates skeletal muscle excitation-contraction coupling. J. Biol. Chem. 2008;283:5046–5057. doi: 10.1074/jbc.M709231200. PubMed DOI PMC

Grycova L., Holendova B., Lansky Z., Bumba L., Jirku M., Bousova K., Teisinger J. Ca2+ Binding Protein S100A1 Competes with Calmodulin and PIP2 for Binding Site on the C-Terminus of the TPRV1 Receptor. ACS Chem. Neurosci. 2014;6:386–392. doi: 10.1021/cn500250r. PubMed DOI

Holakovska B., Grycova L., Jirku M., Sulc M., Bumba L., Teisinger J. Calmodulin and S100A1 protein interact with N terminus of TRPM3 channel. J. Biol. Chem. 2012;287:16645–16655. doi: 10.1074/jbc.M112.350686. PubMed DOI PMC

Jirku M., Lansky Z., Bednarova L., Sulc M., Monincova L., Majer P., Vyklicky L., Vondrasek J., Teisinger J., Bousova K. The characterization of a novel S100A1 binding site in the N-terminus of TRPM1. Int. J. Biochem. Cell Biol. 2016;78:186–193. doi: 10.1016/j.biocel.2016.07.014. PubMed DOI

Uhlén M., Fagerberg L., Hallström B.M., Lindskog C., Oksvold P., Mardinoglu A., Sivertsson Å., Kampf C., Sjöstedt E., Asplund A. Tissue-based map of the human proteome. Science. 2015;347:1260419. doi: 10.1126/science.1260419. PubMed DOI

Sierra-Valdez F., Azumaya C.M., Romero L.O., Nakagawa T., Cordero-Morales J.F. Structure–function analyses of the ion channel TRPC3 reveal that its cytoplasmic domain allosterically modulates channel gating. J. Biol. Chem. 2018;293:16102–16114. doi: 10.1074/jbc.RA118.005066. PubMed DOI PMC

Huynh K.W., Cohen M.R., Jiang J., Samanta A., Lodowski D.T., Zhou Z.H., Moiseenkova-Bell V.Y. Structure of the full-length TRPV2 channel by cryo-EM. Nat. Commun. 2016;7:1–8. doi: 10.1038/ncomms11130. PubMed DOI PMC

Zubcevic L., Le S., Yang H., Lee S.-Y. Conformational plasticity in the selectivity filter of the TRPV2 ion channel. Nat. Struct. Mol. Biol. 2018;25:405–415. doi: 10.1038/s41594-018-0059-z. PubMed DOI PMC

Zhu M.X., Tang J. TRPC channel interactions with calmodulin and IP3 receptors. Novartis Found. Symp. 2004;258:44–58. doi: 10.1002/0470862580.ch4. PubMed DOI

Martin G.M., Yoshioka C., Rex E.A., Fay J.F., Xie Q., Whorton M.R., Chen J.Z., Shyng S.-L. Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating. Elife. 2017;6:e24149. doi: 10.7554/eLife.24149. PubMed DOI PMC

Subbotina E., Williams N., Sampson B.A., Tang Y., Coetzee W.A. Functional characterization of TRPM4 variants identified in sudden unexpected natural death. Forensic Sci. Int. 2018;293:37–46. doi: 10.1016/j.forsciint.2018.10.006. PubMed DOI

Lindsay C., Sitsapesan M., Chan W.M., Venturi E., Welch W., Musgaard M., Sitsapesan R. Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour. Sci. Rep. 2018;8:1–13. doi: 10.1038/s41598-018-33328-8. PubMed DOI PMC

Kohda D. “Multiple partial recognitions in dynamic equilibrium” in the binding sites of proteins form the molecular basis of promiscuous recognition of structurally diverse ligands. Biophys. Rev. 2018;10:421–433. doi: 10.1007/s12551-017-0365-4. PubMed DOI PMC

Brix J., Dietmeier K., Pfanner N. Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J. Biol. Chem. 1997;272:20730–20735. doi: 10.1074/jbc.272.33.20730. PubMed DOI

Hainzl T., Huang S., Meriläinen G., Brännström K., Sauer-Eriksson A.E. Structural basis of signal-sequence recognition by the signal recognition particle. Nat. Struct. Mol. Biol. 2011;18:389. doi: 10.1038/nsmb.1994. PubMed DOI

Hansen S.B., Tao X., MacKinnon R. Structural basis of PIP 2 activation of the classical inward rectifier K+ channel Kir2. 2. Nature. 2011;477:495. doi: 10.1038/nature10370. PubMed DOI PMC

Suh B.-C., Hille B. Regulation of ion channels by phosphatidylinositol 4, 5-bisphosphate. Curr. Opin. Neurobiol. 2005;15:370–378. doi: 10.1016/j.conb.2005.05.005. PubMed DOI

Lakowicz J.R., Ray K., Chowdhury M., Szmacinski H., Fu Y., Zhang J., Nowaczyk K. Plasmon-controlled fluorescence: A new paradigm in fluorescence spectroscopy. Analyst. 2008;133:1308–1346. doi: 10.1039/b802918k. PubMed DOI PMC

Harper C.C., Berg J.M., Gould S.J. PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12. J. Biol. Chem. 2003;278:7897–7901. doi: 10.1074/jbc.M206651200. PubMed DOI

Kohler J.J., Schepartz A. Kinetic studies of fos- jun- DNA complex formation: DNA binding prior to dimerization. Biochemistry. 2001;40:130–142. doi: 10.1021/bi001881p. PubMed DOI

Humphrey W., Dalke A., Schulten K. VMD: Visual molecular dynamics. J. Mol. Graph. 1996;14:33–38. doi: 10.1016/0263-7855(96)00018-5. PubMed DOI

Kozakov D., Hall D.R., Xia B., Porter K.A., Padhorny D., Yueh C., Beglov D., Vajda S. The ClusPro web server for protein–protein docking. Nat. Protoc. 2017;12:255. doi: 10.1038/nprot.2016.169. PubMed DOI PMC

Kozakov D., Beglov D., Bohnuud T., Mottarella S.E., Xia B., Hall D.R., Vajda S. How good is automated protein docking? Proteins Struct. Funct. Bioinform. 2013;81:2159–2166. doi: 10.1002/prot.24403. PubMed DOI PMC

Kozakov D., Brenke R., Comeau S.R., Vajda S. PIPER: An FFT-based protein docking program with pairwise potentials. Proteins Struct. Funct. Bioinform. 2006;65:392–406. doi: 10.1002/prot.21117. PubMed DOI

Lindorff-Larsen K., Piana S., Palmo K., Maragakis P., Klepeis J.L., Dror R.O., Shaw D.E. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins Struct. Funct. Bioinform. 2010;78:1950–1958. doi: 10.1002/prot.22711. PubMed DOI PMC

Jorgensen W.L., Chandrasekhar J., Madura J.D., Impey R.W., Klein M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983;79:926–935. doi: 10.1063/1.445869. DOI

Salomon-Ferrer R., Götz A.W., Poole D., Le Grand S., Walker R.C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald. J. Chem. Theory Comput. 2013;9:3878–3888. doi: 10.1021/ct400314y. PubMed DOI

Le Grand S., Götz A.W., Walker R.C. SPFP: Speed without compromise—A mixed precision model for GPU accelerated molecular dynamics simulations. Comput. Phys. Commun. 2013;184:374–380. doi: 10.1016/j.cpc.2012.09.022. DOI

Cheatham T.I., Miller J., Fox T., Darden T., Kollman P. Molecular dynamics simulations on solvated biomolecular systems: The particle mesh Ewald method leads to stable trajectories of DNA, RNA, and proteins. J. Am. Chem. Soc. 1995;117:4193–4194. doi: 10.1021/ja00119a045. DOI

Miyamoto S., Kollman P.A. Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comput. Chem. 1992;13:952–962. doi: 10.1002/jcc.540130805. DOI

Feenstra K.A., Hess B., Berendsen H.J. Improving efficiency of large time-scale molecular dynamics simulations of hydrogen-rich systems. J. Comput. Chem. 1999;20:786–798. doi: 10.1002/(SICI)1096-987X(199906)20:8<786::AID-JCC5>3.0.CO;2-B. PubMed DOI

Vanommeslaeghe K., MacKerell A., Jr. CHARMM additive and polarizable force fields for biophysics and computer-aided drug design. Biochim. Biophys. Acta (BBA)-Gen. Subj. 2015;1850:861–871. doi: 10.1016/j.bbagen.2014.08.004. PubMed DOI PMC

Phillips J.C., Braun R., Wang W., Gumbart J., Tajkhorshid E., Villa E., Chipot C., Skeel R.D., Kale L., Schulten K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005;26:1781–1802. doi: 10.1002/jcc.20289. PubMed DOI PMC

Ryckaert J.-P., Ciccotti G., Berendsen H.J. Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 1977;23:327–341. doi: 10.1016/0021-9991(77)90098-5. DOI

Roe D.R., Cheatham T.E., III PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 2013;9:3084–3095. doi: 10.1021/ct400341p. PubMed DOI

Biovia D.S. Discovery Studio Modeling Environment. Dassault Systèmes; San Diego, CA, USA: 2016. Release 2017.

Interaction of Calmodulin with TRPM: An Initiator of Channel Modulation

TRPM7 N-terminal region forms complexes with calcium binding proteins CaM and S100A1