Abstract
Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function.
Highlights
The transient receptor potential (TRP) family of nonselective cation channels contains 28 recently identified mammalian homologs grouped into six subfamilies based on sequence homology: vanilloid (TRPV), canonical (TRPC), melastatin (TRPM), ankyrin (TRPA), mucolipin (TRPML), and polycystin (TRPP) [1]
Divergence in the sequence between Transient receptor potential vanilloid 2 (TRPV2) and other TRPV subfamily members resides in the distal C-terminus; most available polyclonal TRPV2 antibodies were generated against synthetic or recombinant peptides derived from the C-terminus
Progress in understanding the cellular function of TRPV2 has been slowed by the lack of tools available to study the channel [12]
Summary
The transient receptor potential (TRP) family of nonselective cation channels contains 28 recently identified mammalian homologs grouped into six subfamilies based on sequence homology: vanilloid (TRPV), canonical (TRPC), melastatin (TRPM), ankyrin (TRPA), mucolipin (TRPML), and polycystin (TRPP) [1]. Considerable challenges in elucidating the function of TRP channels include the absence of the specific activators, inhibitors and antibodies for each individual family member [2]. The controversial function of TRPV subfamily members provides a good example of this current problem in TRP field. The TRPV subfamily consists of six members (TRPV1–6) [1]. TRPV1 has been the most comprehensively studied TRP channel due to its role in noxious pain sensation [3]. The active ingredient in chili peppers, is a specific activator of TRPV1 and was used for identification and characterization of the channel properties [4]. In addition to TRPV1 knockout mice, have consistently indicated that TRPV1 acts as a heat and pain sensor in vivo [5].
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