Structure of the full-length TRPV2 channel by cryo-EM.

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels

Structural insights on TRPV5 gating by endogenous modulators.

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery

Allosteric Antagonist Modulation of TRPV2 by Piperlongumine Impairs Glioblastoma Progression.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer

Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies.

Transient receptor potential vanilloid 2 (TRPV2) is a Ca(2+)-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

Effect of IGF-1 on endogenous TRPV2 trafficking in F11 cells.

<p><i>A,</i> F11 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. <i>B,</i> Biotinylation of surface proteins from F11 cells was performed following the procedure from Figure 5B. <i>C,</i> F11 cells treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.</p

Immunostaining with TRPV2 antibodies.

<p><i>A,</i> HeLa cells transiently expressing TRPV2-1D4 immunolabeled with indicated TRPV2 antibodies (green) and 1D4 antibody (red). Scale bar represents 10 µm. <i>B,</i> HeLa cells transiently expressing TRPV1-1D4 immunolabeled with TRPV2 17A11 (green) and 1D4 antibody (red). Scale bar represents 10 µm.</p

Detection of endogenous TRPV2.

<p><i>A,</i> Western blot analysis with anti-TRPV2 2D6 of F11 cells treated with control siRNA or TRPV2 siRNA (100 nM, 48 h). Quantification of the band corresponding to the molecular weight of TRPV2 was measured using LiCor Odyssey software. TRPV2 band intensity was normalized to actin. Error bars represent S.E.M. from 3 separate experiments. <i>B,</i> Immunoprecipitation of TRPV2 with 10 µg of indicated antibodies from 2.5 mg mouse brain lysate and 2.5 mg mouse heart lysate. TRPV2 was detected by western blot with IR dye-labeled 2D6 antibody. Input represents 100 µg of total protein. Membranes from yeast overexpressing recombinant rat TRPV2 were loaded as a control.</p

Detection of recombinant TRPV2 and mapping of the TRPV2 binding region.

<p><i>A,</i> Schematic of the domain arrangement for a TRPV2 monomer with the approximate epitope sites for indicated TRPV2 antibodies. ARD, ankryin repeat domain; TM, transmembrane domain. <i>B,</i> Western blots with indicated TRPV2 antibodies against purified full-length rat TRPV2, purified rat TRPV2 ankryin repeat domain and purified rat TRPV2 C-terminus. <b>C,</b> Western blots with indicated TRPV2 antibodies against extracts from HeLa cells transiently transfected with empty vector, TRPV1-1D4 and TRPV2-1D4.</p