Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation

Eldo V. Kuzhikandathil, Haibin Wang, Tamas Szabo, Natasha Morozova, Peter M. Blumberg, Gerry S. Oxford

Research output: Contribution to journalArticle

103 Citations (Scopus)

Abstract

The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [3H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [3H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

Original languageEnglish (US)
Pages (from-to)8697-8706
Number of pages10
JournalJournal of Neuroscience
Volume21
Issue number22
StatePublished - Nov 15 2001
Externally publishedYes

Fingerprint

TRPV Cation Channels
Mutation
Protons
Ligand-Gated Ion Channels
Amino Acids
Capsaicin
Cricetulus
vanilloid receptor subtype 1
Ovary
Permeability
Protein Isoforms
Hot Temperature
Binding Sites
resiniferatoxin

Keywords

  • Capsaicin
  • CHO cell
  • Dominant negative
  • Mutation
  • Pain
  • Resiniferatoxin
  • VR1

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Kuzhikandathil, E. V., Wang, H., Szabo, T., Morozova, N., Blumberg, P. M., & Oxford, G. S. (2001). Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation. Journal of Neuroscience, 21(22), 8697-8706.

Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation. / Kuzhikandathil, Eldo V.; Wang, Haibin; Szabo, Tamas; Morozova, Natasha; Blumberg, Peter M.; Oxford, Gerry S.

In: Journal of Neuroscience, Vol. 21, No. 22, 15.11.2001, p. 8697-8706.

Research output: Contribution to journalArticle

Kuzhikandathil, EV, Wang, H, Szabo, T, Morozova, N, Blumberg, PM & Oxford, GS 2001, 'Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation', Journal of Neuroscience, vol. 21, no. 22, pp. 8697-8706.
Kuzhikandathil, Eldo V. ; Wang, Haibin ; Szabo, Tamas ; Morozova, Natasha ; Blumberg, Peter M. ; Oxford, Gerry S. / Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation. In: Journal of Neuroscience. 2001 ; Vol. 21, No. 22. pp. 8697-8706.
@article{9caa60a150084417b72acd6dc24a1e6c,
title = "Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation",
abstract = "The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [3H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [3H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.",
keywords = "Capsaicin, CHO cell, Dominant negative, Mutation, Pain, Resiniferatoxin, VR1",
author = "Kuzhikandathil, {Eldo V.} and Haibin Wang and Tamas Szabo and Natasha Morozova and Blumberg, {Peter M.} and Oxford, {Gerry S.}",
year = "2001",
month = "11",
day = "15",
language = "English (US)",
volume = "21",
pages = "8697--8706",
journal = "Journal of Neuroscience",
issn = "0270-6474",
publisher = "Society for Neuroscience",
number = "22",

}

TY - JOUR

T1 - Functional analysis of capsaicin receptor (vanilloid receptor subtype 1) multimerization and agonist responsiveness using a dominant negative mutation

AU - Kuzhikandathil, Eldo V.

AU - Wang, Haibin

AU - Szabo, Tamas

AU - Morozova, Natasha

AU - Blumberg, Peter M.

AU - Oxford, Gerry S.

PY - 2001/11/15

Y1 - 2001/11/15

N2 - The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [3H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [3H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

AB - The recently cloned vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that is activated by capsaicin, protons, and heat. We have attempted to develop a dominant negative isoform by targeting several mutations of VR1 at highly conserved amino acids or at residues of potential functional importance and expressing the mutants in Chinese hamster ovary cells. Mutation of three highly conserved amino acid residues in the putative sixth transmembrane domain disrupts activation of the VR1 receptor by both capsaicin and resiniferatoxin. The vanilloid binding site in this mutant is intact, although the affinity for [3H]resiniferatoxin (RTX) is diminished by nearly 40-fold. Interestingly, this mutant retains a significant but diminished response to protons, supporting the existence of multiple gating mechanisms for different stimuli. The mutant appears to function by interfering with the gating induced by vanilloids rather than the expression level or permeability of the receptor. In addition, this mutant was found to function as a strong dominant negative mutation when coexpressed with wild-type VR1, providing functional evidence that the VR1 receptor forms a multimeric complex. Analysis of both current density and [3H]RTX affinity in cells cotransfected with different ratios of wild-type and mutant VR1 is consistent with tetrameric stoichiometry for the native capsaicin receptor.

KW - Capsaicin

KW - CHO cell

KW - Dominant negative

KW - Mutation

KW - Pain

KW - Resiniferatoxin

KW - VR1

UR - http://www.scopus.com/inward/record.url?scp=0035889970&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035889970&partnerID=8YFLogxK

M3 - Article

C2 - 11698581

AN - SCOPUS:0035889970

VL - 21

SP - 8697

EP - 8706

JO - Journal of Neuroscience

JF - Journal of Neuroscience

SN - 0270-6474

IS - 22

ER -