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A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation.
J Pharmacol Exp Ther. 2006 Oct; 319(1):192-8.JP

Abstract

Transient receptor potential vanilloid type 1 (TRPV1) can be activated by multiple chemical and physical stimuli such as capsaicin, anandamide, protons, and heat. Capsaicin interacts with the binding pocket constituted by transmembrane regions 3 and 4, whereas protons act through residues in the prepore loop of TRPV1. Here, we report on characterization of polyclonal and monoclonal antibodies to the prepore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC(50) values for pH 5 and 5.5 were 364.68 +/- 29.78 and 28.31 +/- 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6 potentiated chemical ligand (anandamide and capsaicin) activation (50-79% inhibition). Ab-156H antagonism of TRPV1 is not affected by the conformation of the capsaicin-binding pocket because it is equally potent at wild-type (capsaicin-sensitive) rat TRPV1 and its T550I mutant (capsaicin-insensitive). With the goal of generating monoclonal antagonist antibodies to the prepore region of human TRPV1, we used a recently developed rabbit immunization protocol. Although rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to Chinese hamster ovary cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 prepore region seem to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the prepore region but do not lock or stabilize the channel conformation.

Authors+Show Affiliations

Department of Neuroscience, Amgen Inc., MS-29-2-B, Thousand Oaks, CA 91320-1799, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

16844842

Citation

Klionsky, Lana, et al. "A Polyclonal Antibody to the Prepore Loop of Transient Receptor Potential Vanilloid Type 1 Blocks Channel Activation." The Journal of Pharmacology and Experimental Therapeutics, vol. 319, no. 1, 2006, pp. 192-8.
Klionsky L, Tamir R, Holzinger B, et al. A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation. J Pharmacol Exp Ther. 2006;319(1):192-8.
Klionsky, L., Tamir, R., Holzinger, B., Bi, X., Talvenheimo, J., Kim, H., Martin, F., Louis, J. C., Treanor, J. J., & Gavva, N. R. (2006). A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation. The Journal of Pharmacology and Experimental Therapeutics, 319(1), 192-8.
Klionsky L, et al. A Polyclonal Antibody to the Prepore Loop of Transient Receptor Potential Vanilloid Type 1 Blocks Channel Activation. J Pharmacol Exp Ther. 2006;319(1):192-8. PubMed PMID: 16844842.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation. AU - Klionsky,Lana, AU - Tamir,Rami, AU - Holzinger,Bob, AU - Bi,Xiaojuan, AU - Talvenheimo,Jane, AU - Kim,Helen, AU - Martin,Frank, AU - Louis,Jean-Claude, AU - Treanor,James J S, AU - Gavva,Narender R, Y1 - 2006/07/14/ PY - 2006/7/18/pubmed PY - 2006/11/3/medline PY - 2006/7/18/entrez SP - 192 EP - 8 JF - The Journal of pharmacology and experimental therapeutics JO - J. Pharmacol. Exp. Ther. VL - 319 IS - 1 N2 - Transient receptor potential vanilloid type 1 (TRPV1) can be activated by multiple chemical and physical stimuli such as capsaicin, anandamide, protons, and heat. Capsaicin interacts with the binding pocket constituted by transmembrane regions 3 and 4, whereas protons act through residues in the prepore loop of TRPV1. Here, we report on characterization of polyclonal and monoclonal antibodies to the prepore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC(50) values for pH 5 and 5.5 were 364.68 +/- 29.78 and 28.31 +/- 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6 potentiated chemical ligand (anandamide and capsaicin) activation (50-79% inhibition). Ab-156H antagonism of TRPV1 is not affected by the conformation of the capsaicin-binding pocket because it is equally potent at wild-type (capsaicin-sensitive) rat TRPV1 and its T550I mutant (capsaicin-insensitive). With the goal of generating monoclonal antagonist antibodies to the prepore region of human TRPV1, we used a recently developed rabbit immunization protocol. Although rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to Chinese hamster ovary cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 prepore region seem to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the prepore region but do not lock or stabilize the channel conformation. SN - 0022-3565 UR - https://www.unboundmedicine.com/medline/citation/16844842/A_polyclonal_antibody_to_the_prepore_loop_of_transient_receptor_potential_vanilloid_type_1_blocks_channel_activation_ L2 - http://jpet.aspetjournals.org/cgi/pmidlookup?view=long&pmid=16844842 DB - PRIME DP - Unbound Medicine ER -