Lysophosphatidylcholine (LPC) is a bioactive lipid present at high concentrations in inflamed and injured tissues where it contributes to the initiation and maintenance of pain. One of its important molecular effectors is the transient receptor potential canonical 5 (TRPC5), but the explicit mechanism of the activation is unknown. Using electrophysiology, mutagenesis and molecular dynamics simulations, we show that LPC-induced activation of TRPC5 is modulated by xanthine ligands and depolarizing voltage, and involves conserved residues within the lateral fenestration of the pore domain. Replacement of W577 with alanine (W577A) rendered the channel insensitive to strong depolarizing voltage, but LPC still activated this mutant at highly depolarizing potentials. Substitution of G606 located directly opposite position 577 with tryptophan rescued the sensitivity of W577A to depolarization. Molecular simulations showed that depolarization widens the lower gate of the channel and this conformational change is prevented by the W577A mutation or removal of resident lipids. We propose a gating scheme in which depolarizing voltage and lipid-pore helix interactions act together to promote TRPC5 channel opening.

Department of Cellular Neurophysiology Institute of Physiology Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

Department of Physiology Faculty of Science Charles University Prague Czech Republic

Division of Biomolecular Physics Institute of Physics Faculty of Mathematics and Physics Charles University Prague Czech Republic

Leeds Institute of Cardiovascular and Metabolic Medicine and Astbury Centre for Structural Molecular Biology University of Leeds Leeds LS2 9JT UK

Zobrazit více v PubMed

Ren J, Lin J, Yu L, Yan M (2022) Lysophosphatidylcholine: potential target for the treatment of chronic pain. Int J Mol Sci. 10.3390/ijms23158274 10.3390/ijms23158274 PubMed DOI PMC

Marra S, Ferru-Clement R, Breuil V, Delaunay A, Christin M, Friend V, Sebille S, Cognard C, Ferreira T, Roux C et al (2016) Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids. EMBO J 35:414–428 10.15252/embj.201592335 PubMed DOI PMC

Maingret F, Patel AJ, Lesage F, Lazdunski M, Honore E (2000) Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK. J Biol Chem 275:10128–10133. 10.1074/jbc.275.14.10128 10.1074/jbc.275.14.10128 PubMed DOI

Andersson DA, Nash M, Bevan S (2007) Modulation of the cold-activated channel TRPM8 by lysophospholipids and polyunsaturated fatty acids. J Neurosci 27:3347–3355. 10.1523/JNEUROSCI.4846-06.2007 10.1523/JNEUROSCI.4846-06.2007 PubMed DOI PMC

Friston DA, Cuddihy J, Souza Luiz J, Truong AH, Ho L, Basra M, Santha P, Oszlacs O, de Sousa VJ, Marczylo T et al (2023) Elevated 18:0 lysophosphatidylcholine contributes to the development of pain in tissue injury. Pain 164:e103–e115. 10.1097/j.pain.0000000000002709 10.1097/j.pain.0000000000002709 PubMed DOI PMC

Benitez-Angeles M, Romero AEL, Llorente I, Hernandez-Araiza I, Vergara-Jaque A, Real FH, Gutierrez Castaneda OE, Arciniega M, Morales-Buenrostro LE, Torres-Quiroz F et al (2023) Modes of action of lysophospholipids as endogenous activators of the TRPV4 ion channel. J Physiol 601:1655–1673. 10.1113/JP284262 10.1113/JP284262 PubMed DOI

Flemming PK, Dedman AM, Xu SZ, Li J, Zeng F, Naylor J, Benham CD, Bateson AN, Muraki K, Beech DJ (2006) Sensing of lysophospholipids by TRPC5 calcium channel. J Biol Chem 281:4977–4982. 10.1074/jbc.M510301200 10.1074/jbc.M510301200 PubMed DOI

Vanden Abeele F, Zholos A, Bidaux G, Shuba Y, Thebault S, Beck B, Flourakis M, Panchin Y, Skryma R, Prevarskaya N (2006) Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids. J Biol Chem 281:40174–40182. 10.1074/jbc.M605779200 10.1074/jbc.M605779200 PubMed DOI

Rimola V, Hahnefeld L, Zhao J, Jiang C, Angioni C, Schreiber Y, Osthues T, Pierre S, Geisslinger G, Ji RR et al (2020) Lysophospholipids contribute to oxaliplatin-induced acute peripheral pain. J Neurosci 40:9519–9532. 10.1523/JNEUROSCI.1223-20.2020 10.1523/JNEUROSCI.1223-20.2020 PubMed DOI PMC

Sadler KE, Moehring F, Shiers SI, Laskowski LJ, Mikesell AR, Plautz ZR, Brezinski AN, Mecca CM, Dussor G, Price TJ et al (2021) Transient receptor potential canonical 5 mediates inflammatory mechanical and spontaneous pain in mice. Sci Translat Med. 10.1126/scitranslmed.abd770210.1126/scitranslmed.abd7702 PubMed DOI PMC

Bernal L, Sotelo-Hitschfeld P, Konig C, Sinica V, Wyatt A, Winter Z, Hein A, Touska F, Reinhardt S, Tragl A et al (2021) Odontoblast TRPC5 channels signal cold pain in teeth. Sci Adv. 10.1126/sciadv.abf5567 10.1126/sciadv.abf5567 PubMed DOI PMC

Akbulut Y, Gaunt HJ, Muraki K, Ludlow MJ, Amer MS, Bruns A, Vasudev NS, Radtke L, Willot M, Hahn S et al (2015) (-)-Englerin A is a potent and selective activator of TRPC4 and TRPC5 calcium channels. Angew Chem 54:3787–3791. 10.1002/anie.201411511 10.1002/anie.201411511 PubMed DOI PMC

Carson C, Raman P, Tullai J, Xu L, Henault M, Thomas E, Yeola S, Lao J, McPate M, Verkuyl JM et al (2015) Englerin A Agonizes the TRPC4/C5 cation channels to inhibit tumor cell line proliferation. PLoS ONE. 10.1371/journal.pone.0127498 10.1371/journal.pone.0127498 PubMed DOI PMC

Wright DJ, Simmons KJ, Johnson RM, Beech DJ, Muench SP, Bon RS (2020) Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site. Communications Biology 3:704. 10.1038/s42003-020-01437-8 10.1038/s42003-020-01437-8 PubMed DOI PMC

Dryn DO, Melnyk MI, Bon RS, Beech DJ, Zholos AV (2023) Pico145 inhibits TRPC4-mediated mI(CAT) and postprandial small intestinal motility. Biomed Pharmacother. 10.1016/j.biopha.2023.115672 10.1016/j.biopha.2023.115672 PubMed DOI

Duan J, Li J, Chen GL, Ge Y, Liu J, Xie K, Peng X, Zhou W, Zhong J, Zhang Y et al (2019) Cryo-EM structure of TRPC5 at 2.8-A resolution reveals unique and conserved structural elements essential for channel function. Sci Adv 5:7935. 10.1126/sciadv.aaw793510.1126/sciadv.aaw7935 PubMed DOI PMC

Song K, Wei M, Guo W, Quan L, Kang Y, Wu JX, Chen L (2021) Structural basis for human TRPC5 channel inhibition by two distinct inhibitors. Elife. 10.7554/eLife.63429 10.7554/eLife.63429 PubMed DOI PMC

Lichtenegger M, Tiapko O, Svobodova B, Stockner T, Glasnov TN, Schreibmayer W, Platzer D, de la Cruz GG, Krenn S, Schober R et al (2018) An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel. Nat Chem Biol 14:396–404. 10.1038/s41589-018-0015-6 10.1038/s41589-018-0015-6 PubMed DOI PMC

Storch U, Mederos YSM, Gudermann T (2021) A greasy business: Identification of a diacylglycerol binding site in human TRPC5 channels by cryo-EM. Cell Calcium. 10.1016/j.ceca.2021.102414 10.1016/j.ceca.2021.102414 PubMed DOI

Strubing C, Krapivinsky G, Krapivinsky L, Clapham DE (2003) Formation of novel TRPC channels by complex subunit interactions in embryonic brain. J Biol Chem 278:39014–39019. 10.1074/jbc.M306705200 10.1074/jbc.M306705200 PubMed DOI

Hofmann T, Schaefer M, Schultz G, Gudermann T (2002) Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci U S A 99:7461–7466. 10.1073/pnas.102596199 10.1073/pnas.102596199 PubMed DOI PMC

Erkan-Candag H, Clarke A, Tiapko O, Gsell MA, Stockner T, Groschner K (2022) Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state. EMBO Rep 23:e54276 10.15252/embr.202154276 PubMed DOI PMC

Bai Y, Yu X, Chen H, Horne D, White R, Wu X, Lee P, Gu Y, Ghimire-Rijal S, Lin DC et al (2020) Structural basis for pharmacological modulation of the TRPC6 channel. Elife. 10.7554/eLife.53311 10.7554/eLife.53311 PubMed DOI PMC

Rubaiy HN, Ludlow MJ, Henrot M, Gaunt HJ, Miteva K, Cheung SY, Tanahashi Y, Hamzah N, Musialowski KE, Blythe NM et al (2017) Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels. J Biol Chem 292:8158–8173. 10.1074/jbc.M116.773556 10.1074/jbc.M116.773556 PubMed DOI PMC

Jung S, Muhle A, Schaefer M, Strotmann R, Schultz G, Plant TD (2003) Lanthanides potentiate TRPC5 currents by an action at extracellular sites close to the pore mouth. J Biol Chem 278:3562–3571. 10.1074/jbc.M211484200 10.1074/jbc.M211484200 PubMed DOI

Semtner M, Schaefer M, Pinkenburg O, Plant TD (2007) Potentiation of TRPC5 by protons. J Biol Chem 282:33868–33878. 10.1074/jbc.M702577200 10.1074/jbc.M702577200 PubMed DOI

Obukhov AG, Nowycky MC (2008) TRPC5 channels undergo changes in gating properties during the activation-deactivation cycle. J Cell Physiol 216:162–171. 10.1002/jcp.21388 10.1002/jcp.21388 PubMed DOI PMC

Zholos AV (2014) Trpc5. Handb Exp Pharmacol 222:129–156. 10.1007/978-3-642-54215-2_6 10.1007/978-3-642-54215-2_6 PubMed DOI

Beech DJ (2013) Characteristics of transient receptor potential canonical calcium-permeable channels and their relevance to vascular physiology and disease. Circ J 77:570–579. 10.1253/circj.cj-13-0154 10.1253/circj.cj-13-0154 PubMed DOI

Zimmermann K, Lennerz JK, Hein A, Link AS, Kaczmarek JS, Delling M, Uysal S, Pfeifer JD, Riccio A, Clapham DE (2011) Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system. Proc Natl Acad Sci USA 108:18114–18119. 10.1073/pnas.1115387108 10.1073/pnas.1115387108 PubMed DOI PMC

Gomis A, Soriano S, Belmonte C, Viana F (2008) Hypoosmotic- and pressure-induced membrane stretch activate TRPC5 channels. J Physiol 586:5633–5649 10.1113/jphysiol.2008.161257 PubMed DOI PMC

Beech DJ, Bahnasi YM, Dedman AM, Al-Shawaf E (2009) TRPC channel lipid specificity and mechanisms of lipid regulation. Cell Calcium 45:583–588. 10.1016/j.ceca.2009.02.006 10.1016/j.ceca.2009.02.006 PubMed DOI PMC

Yoo J, Cui Q (2009) Curvature generation and pressure profile modulation in membrane by lysolipids: insights from coarse-grained simulations. Biophys J 97:2267–2276. 10.1016/j.bpj.2009.07.051 10.1016/j.bpj.2009.07.051 PubMed DOI PMC

Anishkin A, Loukin SH, Teng J, Kung C (2014) Feeling the hidden mechanical forces in lipid bilayer is an original sense. Proc Nat Acad Sci 111:7898–7905. 10.1073/pnas.1313364111 10.1073/pnas.1313364111 PubMed DOI PMC

Zhang K, Julius D, Cheng Y (2021) Structural snapshots of TRPV1 reveal mechanism of polymodal functionality. Cell. 10.1016/j.cell.2021.08.012 10.1016/j.cell.2021.08.012 PubMed DOI PMC

Diver MM, King JVL, Julius D, Cheng Y (2022) Sensory TRP channels in three dimensions. Ann Rev Biochem. 10.1146/annurev-biochem-032620-105738 10.1146/annurev-biochem-032620-105738 PubMed DOI PMC

Levental I, Lyman E (2023) Regulation of membrane protein structure and function by their lipid nano-environment. Nat Rev Mol Cell Biol 24:107–122. 10.1038/s41580-022-00524-4 10.1038/s41580-022-00524-4 PubMed DOI PMC

Khoury S, Colas J, Breuil V, Kosek E, Ahmed AS, Svensson CI, Marchand F, Deval E, Ferreira T (2023) Identification of lipid biomarkers for chronic joint pain associated with different joint diseases. Biomolecules 13:342. 10.3390/biom13020342 10.3390/biom13020342 PubMed DOI PMC

Obukhov AG, Nowycky MC (2005) A cytosolic residue mediates Mg2+ block and regulates inward current amplitude of a transient receptor potential channel. J Neurosci 25:1234–1239. 10.1523/JNEUROSCI.4451-04.2005 10.1523/JNEUROSCI.4451-04.2005 PubMed DOI PMC

Yau WM, Wimley WC, Gawrisch K, White SH (1998) The preference of tryptophan for membrane interfaces. Biochemistry 37:14713–14718. 10.1021/bi980809c 10.1021/bi980809c PubMed DOI

Duan J, Li J, Zeng B, Chen GL, Peng X, Zhang Y, Wang J, Clapham DE, Li Z, Zhang J (2018) Structure of the mouse TRPC4 ion channel. Nat Commun 9:3102. 10.1038/s41467-018-05247-9 10.1038/s41467-018-05247-9 PubMed DOI PMC

Won J, Kim J, Jeong H, Kim J, Feng S, Jeong B, Kwak M, Ko J, Im W, So I et al (2023) Molecular architecture of the Galpha(i)-bound TRPC5 ion channel. Nat Commun 14:2550. 10.1038/s41467-023-38281-3 10.1038/s41467-023-38281-3 PubMed DOI PMC

Yang Y, Wei M, Chen L (2022) Structural identification of riluzole-binding site on human TRPC5. Cell Discov 8:67. 10.1038/s41421-022-00410-5 10.1038/s41421-022-00410-5 PubMed DOI PMC

Fan C, Choi W, Sun W, Du J, Lu W (2018) Structure of the human lipid-gated cation channel TRPC3. Elife. 10.7554/eLife.36852 10.7554/eLife.36852 PubMed DOI PMC

Ningoo M, Plant LD, Greka A, Logothetis DE (2021) PIP2 regulation of TRPC5 channel activation and desensitization. J Biol Chem. 10.1016/j.jbc.2021.100726 10.1016/j.jbc.2021.100726 PubMed DOI PMC

Yang F, Xu L, Lee BH, Xiao X, Yarov-Yarovoy V, Zheng J (2020) An unorthodox mechanism underlying voltage sensitivity of TRPV1 ion channel. Adv Sci (Weinh) 7:2000575. 10.1002/advs.202000575 10.1002/advs.202000575 PubMed DOI PMC

Babinski K, Catarsi S, Biagini G, Seguela P (2000) Mammalian ASIC2a and ASIC3 subunits co-assemble into heteromeric proton-gated channels sensitive to Gd3+. J Biol Chem 275:28519–28525 10.1074/jbc.M004114200 PubMed DOI

Xu SZ, Zeng F, Boulay G, Grimm C, Harteneck C, Beech DJ (2005) Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect. Br J Pharmacol 145:405–414. 10.1038/sj.bjp.0706197 10.1038/sj.bjp.0706197 PubMed DOI PMC

Dattilo M, Penington NJ, Williams K (2020) Regulation of TRPC5 currents by intracellular ATP: single channel studies. J Cell Physiol 235:7056–7066. 10.1002/jcp.29602 10.1002/jcp.29602 PubMed DOI

Kasimova MA, Yazici AT, Yudin Y, Granata D, Klein ML, Rohacs T, Carnevale V (2018) A hypothetical molecular mechanism for TRPV1 activation that invokes rotation of an S6 asparagine. J Gen Physiol 150:1554–1566. 10.1085/jgp.201812124 10.1085/jgp.201812124 PubMed DOI PMC

Trofimov YA, Krylov NA, Efremov RG (2019) Confined dynamics of water in transmembrane pore of TRPV1 ion channel. Int J Mol Sci. 10.3390/ijms20174285 10.3390/ijms20174285 PubMed DOI PMC

Trofimov YA, Krylov NA, Minakov AS, Nadezhdin KD, Neuberger A, Sobolevsky AI, Efremov RG (2024) Dynamic molecular portraits of ion-conducting pores characterize functional states of TRPV channels. Commun Chem 7:119. 10.1038/s42004-024-01198-z 10.1038/s42004-024-01198-z PubMed DOI PMC

Trofimov YA, Minakov AS, Krylov NA, Efremov RG (2023) Structural mechanism of ionic conductivity of the TRPV1 channel. Dokl Biochem Biophys 508:1–5. 10.1134/S1607672922600245 10.1134/S1607672922600245 PubMed DOI PMC

Kwon DH, Zhang F, Suo Y, Bouvette J, Borgnia MJ, Lee SY (2021) Heat-dependent opening of TRPV1 in the presence of capsaicin. Nat Struct Mol Biol 28:554–563. 10.1038/s41594-021-00616-3 10.1038/s41594-021-00616-3 PubMed DOI PMC

Hughes TET, Pumroy RA, Yazici AT, Kasimova MA, Fluck EC, Huynh KW, Samanta A, Molugu SK, Zhou ZH, Carnevale V et al (2018) Structural insights on TRPV5 gating by endogenous modulators. Nat Commun 9:4198. 10.1038/s41467-018-06753-6 10.1038/s41467-018-06753-6 PubMed DOI PMC

Ruan Z, Haley E, Orozco IJ, Sabat M, Myers R, Roth R, Du J, Lu W (2021) Structures of the TRPM5 channel elucidate mechanisms of activation and inhibition. Nat Struct Mol Biol 28:604–613. 10.1038/s41594-021-00607-4 10.1038/s41594-021-00607-4 PubMed DOI PMC

Koldso H, Jensen MO, Jogini V, Shaw DE (2023) Functional dynamics and allosteric modulation of TRPA1. Structure 31(1556–1566):e1553. 10.1016/j.str.2023.08.01810.1016/j.str.2023.08.018 PubMed DOI

Freedy AM, Liau BB (2021) Discovering new biology with drug-resistance alleles. Nat Chem Biol 17:1219–1229. 10.1038/s41589-021-00865-9 10.1038/s41589-021-00865-9 PubMed DOI PMC

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

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. 10.1002/jcc.20084 10.1002/jcc.20084 PubMed DOI

Chovancova E, Pavelka A, Benes P, Strnad O, Brezovsky J, Kozlikova B, Gora A, Sustr V, Klvana M, Medek P et al (2012) CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol 8:e1002708. 10.1371/journal.pcbi.1002708 10.1371/journal.pcbi.1002708 PubMed DOI PMC

Thermosensing ability of TRPC5: current knowledge and unsettled questions