Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

Abstract

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosettabased molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor.on channels constitute the second largest family of drug targets for therapeutics (1-3); therefore, understanding their gating mechanisms by small-molecule ligands is critical for both basic and translational research. Capsaicin activation of the pain-sensing transient receptor potential vanilloid 1 (TRPV1) ion channel represents an outstanding model system for understanding liganddependent gating process (4), because capsaicin not only potently activates the channel with a submicromolar EC 50 (5) but also effectively stabilizes the channel at high open probability (6-8). Moreover, capsaicin activation is highly selective for TRPV1 channel (5). Previous mutagenesis (9, 10) and cryo-EM studies (11,12) have shown that capsaicin binds near the third (S3) and fourth (S4) transmembrane segments of TRPV1 (Fig. 1A). Based on the high-resolution cryo-EM structures, we have used a combination of computational and functional assays to reveal that capsaicin takes a "tail-up, head-down" configuration in the ligand-binding pocket (6) (Fig. 1B). The aliphatic tail forms extensive but nonspecific van der Waals (VDW) interactions with residues lining the binding pocket, whereas the vanillyl group (head) and the amide group (neck) of capsaicin form a hydrogen bond with E571 on S4-S5 linker and T551 on S4, respectively. To activate TRPV1, VDW interactions first secure capsaicin in the pocket and the neck of capsaicin forms a hydrogen bond with T551. This is followed by the formation of another hydrogen bond between the head and E571, which stabilizes the outward conformation of S4-S5 linker to open the chann...

Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM

“…28 ). Moreover, the vanilloid-insensitive TRPV2 channel can be transformed into the vanilloid-sensitive channel by modification of four critical residues in this vanilloid-binding pocket 31,32 . One of these residues is located on the S3 helix: for mouse TRPV1, it is a polar Ser513, and for mouse TRPV2, it is a hydrophobic Phe467 (Fig.…”

“…Changing TRPV2 Phe467 to Ser467 facilitates capsaicin entering into the vanilloid-binding pocket of TRPV2 (refs. 31,32 ). Changing TRPV1 Ser513 to Tyr513, a residue that is equally as bulky as Phe467 in TRPV2, diminishes TRPV1 activation by capsaicin 31,32 .…”

Exploring Functional Roles of TRPV1 Intracellular Domains with Unstructured Peptide-Insertion Screening

“…The S4-S5 linker forms a horizontal helix connecting the peripheral S1-S4 domain to the central pore domain 22 . Recent studies confirmed that it serves a critical role in coupling capsaicin binding to channel activation [16][17][18][19] . We made a series of insertions of 2-to-12 amino acids in length into this key structure, at three different positions (Fig.…”

“…Among TRPV1 stimulations, the capsaicin-channel interaction is the best-understood case in both location and detailed atomic interactions [14][15][16][17][18][19] . None of the functional insertion mutations disrupted capsaicin activation, in agreement with the notion that capsaicin-activation machinery mostly resides within the transmembrane region.…”

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore

“…Rosetta has previously been successfully used to calculate gating charges (Khalili-Araghi et al, 2010), simulate voltagesensor movements (Decaen et al, 2008(Decaen et al, , 2009(Decaen et al, , 2011YarovYarovoy et al, 2012;Tuluc et al, 2016), construct open and closed state conformations of Kv channels (Yarov-Yarovoy et al, 2006a;Pathak et al, 2007), and model the interactions of scorpion toxins with voltage-gated Na 1 channels (Cestèle et al, 2006;Wang et al, 2011;Zhang et al, 2011Zhang et al, , 2012. More recently, Rosetta has been used to simulate the access and binding of local anesthetics and anticonvulsants to Na 1 channels (Boiteux et al, 2014), study the interaction of capsaicin with the TRPV1 channel (Yang et al, 2015), and rationally design a vanilloid-sensitive TRPV2 channel (Yang et al, 2016). Similarly, the KCa3.1 structural models we developed in our current study accurately identified key residues for binding of several small molecule probes and explain their atomistic mechanisms of action.…”