The transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific nonselective cation channel that is gated in response to various noxious stimuli: pungent vanilloids, low pH, noxious heat, and depolarizing voltages. By its analogy to K+ channels, the S6 inner helix domain of TRPV1 (Y666-G683) is a prime candidate to form the most constricted region of the permeation pathway and might therefore encompass an as-yet-unmapped gate of the channel. Using alanine-scanning mutagenesis, we identified 16 of 17 residues, that when mutated affected the functionality of the TRPV1 channel with respect to at least one stimulus modality. T670A was the only substitution producing the wild-type channel phenotype, whereas Y666A and N676A were nonfunctional but present at the plasma membrane. The periodicity of the functional effects of mutations within the TRPV1 inner pore region is consistent with an alpha-helical structure in which T670 and A680 might play the roles of two bending "hinges."

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

Zobrazit více v PubMed

Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. Molecular-dynamics with coupling to an external bath. J Chem Phys. 1984;81:3684–3690.

Brauchi S, Orio P, Latorre R. Clues to understanding cold sensation: thermodynamics and electrophysiological analysis of the cold receptor TRPM8. Proc Natl Acad Sci USA. 2004;101:15494–15499. PubMed PMC

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997;389:816–824. PubMed

Dittert I, Benedikt J, Vyklicky L, Zimmermann K, Reeh PW, Vlachova V. Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions. J Neurosci Methods. 2006;151:178–185. PubMed

Dodier Y, Banderali U, Klein H, Topalak O, Dafi O, Simoes M, Bernatchez G, Sauve R, Parent L. Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis. J Biol Chem. 2004;279:6853–6862. PubMed

Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998;280:69–77. PubMed

Ferrer-Montiel A, Garcia-Martinez C, Morenilla-Palao C, Garcia-Sanz N, Fernandez-Carvajal A, Fernandez-Ballester G, Planells-Cases R. Molecular architecture of the vanilloid receptor. Insights for drug design. Eur J Biochem. 2004;271:1820–1826. PubMed

Flynn GE, Zagotta WN. A cysteine scan of the inner vestibule of cyclic nucleotide-gated channels reveals architecture and rearrangement of the pore. J Gen Physiol. 2003;121:563–582. PubMed PMC

Gunthorpe MJ, Smith GD, Davis JB, Randall AD. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells. Pflügers Arch. 2001;442:668–674. PubMed

Hackos DH, Chang TH, Swartz KJ. Scanning the intracellular S6 activation gate in the shaker K+ channel. J Gen Physiol. 2002;119:521–532. PubMed PMC

Hui K, Liu B, Qin F. Capsaicin activation of the pain receptor, VR1: multiple open states from both partial and full binding. Biophys J. 2003;84:2957–2968. PubMed PMC

Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R. Crystal structure and mechanism of a calcium-gated potassium channel. Nature. 2002;417:515–522. PubMed

Kuzhikandathil EV, Wang H, Szabo T, Morozova N, Blumberg PM, Oxford GS. Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation. J Neurosci. 2001;21:8697–8706. PubMed PMC

Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr. 1993;26:283–291.

Liu B, Hui K, Qin F. Thermodynamics of heat activation of single capsaicin ion channels VR1. Biophys J. 2003;85:2988–3006. PubMed PMC

McPhee JC, Ragsdale DS, Scheuer T, Catterall WA. A critical role for transmembrane segment IVS6 of the sodium channel alpha subunit in fast inactivation. J Biol Chem. 1995;270:12025–12034. PubMed

Mitcheson JS, Chen J, Lin M, Culberson C, Sanguinetti MC. A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci USA. 2000;97:12329–12333. PubMed PMC

Mohapatra DP, Wang SY, Wang GK, Nau C. A tyrosine residue in TM6 of the vanilloid receptor TRPV1 involved in desensitization and calcium permeability of capsaicin-activated currents. Mol Cell Neurosci. 2003;23:314–324. PubMed

Nilius B, Prenen J, Janssens A, Owsianik G, Wang C, Zhu MX, Voets T. The selectivity filter of the cation channel TRPM4. J Biol Chem. 2005a;280:22899–22906. PubMed

Nilius B, Talavera K, Owsianik G, Prenen J, Droogmans G, Voets T. Gating of TRP channels: a voltage connection? J Physiol (Lond) 2005b;567:35–44. PubMed PMC

Owsianik G, Talavera K, Voets T, Nilius B. Permeation and selectivity of TRP channels. Annu Rev Physiol. 2006;68:685–717. PubMed

Planells-Cases R, Garcia-Sanz N, Morenilla-Palao C, Ferrer-Montiel A. Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia. Pflügers Arch. 2005;451:151–159. PubMed

Ragsdale DS, McPhee JC, Scheuer T, Catterall WA. Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science. 1994;265:1724–1728. PubMed

Raybaud A, Dodier Y, Bissonnette P, Simoes M, Bichet DG, Sauve R, Parent L. The role of the G(X)9G(X)3G motif in the gating of high-voltage activated Ca2+ channel. J Biol Chem. 2006;281:39424–39436. PubMed

Ryu S, Liu B, Qin F. Low pH potentiates both capsaicin binding and channel gating of VR1 receptors. J Gen Physiol. 2003;122:45–61. PubMed PMC

Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234:779–815. PubMed

Seebohm G, Strutz-Seebohm N, Ureche ON, Baltaev R, Lampert A, Kornichuk G, Kamiya K, Wuttke TV, Lerche H, Sanguinetti MC, Lang F. Differential roles of S6 domain hinges in the gating of KCNQ potassium channels. Biophys J. 2006;90:2235–2244. PubMed PMC

Szallasi A, Cruz F, Geppetti P. TRPV1: a therapeutic target for novel analgesic drugs? Trends Mol Med. 2006;12:545–554. PubMed

Tominaga M, Tominaga T. Structure and function of TRPV1. Pflügers Arch. 2005;451:143–150. PubMed

Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron. 1998;21:531–543. PubMed

Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem. 2005;26:1701–1718. PubMed

Vlachova V, Lyfenko A, Orkand RK, Vyklicky L. The effects of capsaicin and acidity on currents generated by noxious heat in cultured neo-natal rat DRG neurones. J Physiol (Lond) 533. 2001;3:717–728. PubMed PMC

Vlachova V, Teisinger J, Susankova K, Lyfenko A, Ettrich R, Vyklicky L. Functional role of C-terminal cytoplasmic tail of rat vanilloid receptor 1. J Neurosci. 2003;23:1340–1350. PubMed PMC

Voets T, Janssens A, Droogmans G, Nilius B. Outer pore architecture of a Ca2+-selective TRP channel. J Biol Chem. 2004a;279:15223–15230. PubMed

Voets T, Droogmans G, Wissenbach U, Janssens A, Flockerzi V, Nilius B. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature. 2004b;430:748–754. PubMed

Yarov-Yarovoy V, McPhee JC, Idsvoog D, Pate C, Scheuer T, Catterall WA. Role of amino acid residues in transmembrane segments IS6 and IIS6 of the Na+ channel alpha subunit in voltage-dependent gating and drug block. J Biol Chem. 2002;277:35393–35401. PubMed

Yeh BI, Kim YK, Jabbar W, Huang CL. Conformational changes of pore helix coupled to gating of TRPV5 by protons. EMBO J. 2005;24:3224–3234. PubMed PMC

Cytoplasmic Inter-Subunit Interface Controls Use-Dependence of Thermal Activation of TRPV3 Channel

C-terminal acidic cluster is involved in Ca2+-induced regulation of human transient receptor potential ankyrin 1 channel

The C-terminal basic residues contribute to the chemical- and voltage-dependent activation of TRPA1

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels