A Polyclonal Antibody to the Prepore Loop of Transient Receptor Potential Vanilloid Type 1 Blocks Channel Activation

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 Ϯ 29.78 and 28.31 Ϯ 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 (capsaicinsensitive) 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.Transient receptor potential vanilloid type 1 (VR1 or TRPV1), a nonselective cation channel, is activated by chemical ligands (capsaicin, anandamide, and protons, pH Յ 5.7) and heat (Ͼ42°C), acting as an integrator of multiple noxious stimuli (Caterina et al

Mouse Monoclonal Antibodies to Transient Receptor Potential Ankyrin 1 Act as Antagonists of Multiple Modes of Channel Activation

“…We and several others have shown that polyclonal (Zhou et al, 1998;Klionsky et al, 2006;Naylor et al, 2008) and monoclonal (Gómez-Varela et al, 2007;this study) antibodies to the third extracellular loop (loop 3) that forms the pore in different ion channels can act as antagonists (see reviews by Naylor and Beech, 2009;Sun and Li, 2013). Interestingly, most of the antibodies blocked their respective channel's activation by multiple ligands, which bind to different locations in the channel (e.g., capsaicin at transmembrane regions 2-4 and protons that act through loop 3 in TRPV1; Klionsky et al, 2006), suggesting that high-affinity binding of antibodies to the pore loop holds the channel in a closed confirmation or blocks the pore.…”

“…Interestingly, most of the antibodies blocked their respective channel's activation by multiple ligands, which bind to different locations in the channel (e.g., capsaicin at transmembrane regions 2-4 and protons that act through loop 3 in TRPV1; Klionsky et al, 2006), suggesting that high-affinity binding of antibodies to the pore loop holds the channel in a closed confirmation or blocks the pore. Although we do not know exactly where 2B10 and 2D1 bind to the TRPA1 channels, we propose that these mAbs perhaps act through high-affinity binding to the pore loop region, based on their ability to block diverse modes of activation.…”

“…In all, these studies suggest that TRPA1 is a potential target for novel therapeutics (McMahon and Wood, 2006;Viana and FerrerMontiel, 2009;Lapointe and Altier, 2011;Strassmaier and Bakthavatchalam, 2011;Meseguer et al, 2014). Based on the published feasibility (Xu et al, 2005;Klionsky et al, 2006;Gómez-Varela et al, 2007;Naylor and Beech, 2009;Sun and Li, 2013), we attempted to generate monoclonal antagonist antibodies to TRPA1. Here, we report the generation and pharmacological characterization of monoclonal antibodies (mAbs) that antagonize cold, exogenous, and endogenous ligand activation of human TRPA1.…”

Uncoupling Proton Activation of Vanilloid Receptor TRPV1

“…A glutamic residue (E600) on the extracellular side of the fifth transmembrane segment is crucial for the low pH potentiation (Jordt et al, 2000). An antibody to the region inhibits the low pH activity of the channel (Klionsky et al, 2006). Another acidic residue, E648, located in the linker between the selectivity filter and the sixth transmembrane domain, was proposed to mediate the direct response to low pH (Jordt et al, 2000).…”

Signaling mechanisms involved in the intestinal pro-secretory actions of hydrogen sulfide

“…However, antagonist binding to sites other than the capsaicin site may not modulate polymodal receptor activation induced by stimuli other than capsaicin. 42 Ruthenium red is an unspecific TRP channel blocker also antagonizing capsaicin activation of TRPV1 channels. Unlike capsazepine, AMG 9810, SB 705498 or BCTC, ruthenium red blocks the pore of the TRPV1 channel.…”