title: Role of the outer pore domain in transient receptor potential vanilloid 1 dynamic permeability to large cations.
abstract: Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.
authors.affiliation: From the Departments of Neurosurgery, Biological Chemistry, and Neuroscience, Neurosurgery Pain Research Institute, and Center for Sensory Biology and.
authors.forename: Clare H
authors.initials: CH
authors.lastname: Munns
authors.affiliation: the Department of Neural and Pain Sciences, University of Maryland School of Dentistry, Baltimore, Maryland 21201.
authors.forename: Man-Kyo
authors.initials: MK
authors.lastname: Chung
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, the Departamento de Física, Facultad de Ciencias, Universidad Nacional de Colombia, 111321 Bogotá, D.C., Colombia, and the Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, 110231 Bogotá, D.C., Colombia.
authors.forename: Yuly E
authors.initials: YE
authors.lastname: Sanchez
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
authors.forename: L Mario
authors.initials: LM
authors.lastname: Amzel
authors.affiliation: From the Departments of Neurosurgery, Biological Chemistry, and Neuroscience, Neurosurgery Pain Research Institute, and Center for Sensory Biology and caterina@jhmi.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Caterina
mesh_terms: D000818:Animals; D001583:Benzoxazoles / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002211:Capsaicin / Q000494:pharmacology; D002412:Cations / Q000493:pharmacokinetics*; D004224:Diterpenes / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D008536:Meglumine / Q000493:pharmacokinetics; D051379:Mice; D008958:Models, Molecular; D020125:Mutation, Missense; D010539:Permeability / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011806:Quinolinium Compounds / Q000493:pharmacokinetics; D051381:Rats; D050916:TRPV Cation Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
chemical_list: D001583:Benzoxazoles; D002412:Cations; D004224:Diterpenes; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; C493922:Trpv1 protein, rat; C089813:YO-PRO 1; D008536:Meglumine; C024353:resiniferatoxin; D002211:Capsaicin
keywords: Capsaicin; Imaging; N-Methyl-D-glucamine; Patch Clamp; Permeability; Pore; Resiniferatoxin; Site-directed Mutagenesis; TRPV1; Transient Receptor Potential Channels (TRP Channels)