Received for publication May 18, 2006.
Revised June 30, 2006.
Accepted for publication July 13, 2006.

A polyclonal antibody to the pre-pore loop of TRPV1 blocks channel activation

Abstract

The Vanilloid receptor 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 pre-pore loop of TRPV1. Here we report on characterization of polyclonal and monoclonal antibodies to the pre-pore loop of TRPV1. A rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) acted as a full antagonist of proton activation (IC₅₀ values for pH 5 and 5.5 were 364.68 ± 29.78 nM and 28.31 ± 6.30 nM, respectively) and as a partial antagonist of capsaicin, heat, and pH 6.0 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 pre-pore region of human TRPV1, we used a recently developed rabbit immunization protocol. Whereas rabbit polyclonal antiserum blocked human TRPV1 activation, rabbit monoclonal antibodies (identified on the basis of selective binding to CHO cells expressing human TRPV1) did not block activation by either capsaicin or protons. Thus, rabbit polyclonal antibodies against rat and human TRPV1 pre-pore region appear to partially lock or stabilize the channel in the closed state, whereas rabbit anti-human TRPV1 monoclonal antibodies bind to the pre-pore region but do not lock or stabilize the channel conformation.

Key words: TRPV1, antagonist, antibody, capsaicin, monoclonal, proton

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