Molecular determinants of vanilloid sensitivity in TRPV1.
Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.
Department of Neuroscience, Amgen Inc., MS 29-2 B, One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA. ngavva@amgen.com
Narender R
NR
Gavva
Lana
L
Klionsky
Yusheng
Y
Qu
Licheng
L
Shi
Rami
R
Tamir
Steve
S
Edenson
T J
TJ
Zhang
Vellarkad N
VN
Viswanadhan
Attila
A
Toth
Larry V
LV
Pearce
Todd W
TW
Vanderah
Frank
F
Porreca
Peter M
PM
Blumberg
Jack
J
Lile
Yax
Y
Sun
Ken
K
Wild
Jean-Claude
JC
Louis
James J S
JJ
Treanor
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D002412:Cations; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005727:Ganglia, Spinal / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D020128:Inhibitory Concentration 50; D008024:Ligands; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D011817:Rabbits; D051381:Rats; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D002412:Cations; D018076:DNA, Complementary; D008024:Ligands; D010703:Phorbol Esters; D011522:Protons; D011955:Receptors, Drug; C100823:phorbol 12-phenylacetate 13-acetate 20-homovanillate; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D008715:Methionine; D002211:Capsaicin; D002118:Calcium