Detailed characterization of the calmodulin binding domain on the C terminus of TRPV1 receptor

Lenka Gryčová, Zdeněk Lánský, Blanka Holakovská and Jan Teisinger

Institute of Physiology, Academy of Sciences of Czech Republic

Transient receptor potential vanilloid 1 (TRPV1) is a transmembrane ion channel that participates in physical and chemical pain evoked signal transduction. This nonselective cation channel allows monovalent and some divalent cations (Ca²⁺, Mg²⁺) to move into the cell. Many TRP channels possess multiple CaM-binding domains (CaM-BDs) located at both termini and which could show varying behaviors towards calcium. Despite the fact that CaM-BDs have no sequence homology, they share key structural features such as interspersed basic and bulky hydrophobic amino acid residues. The C-terminal region of TRPV1 (TRPV1-CT) does not possess any classical previously defined CaM-binding motif. Several possible binding motifs for CaM have been recently identified on the TRPV1-CT by sequence homology and structural analyses.

In this work we explored this unusual TRPV1 CaM-binding motif in detail. Based on our three dimensional computer homology model, we generated nine point mutant proteins of TRPV1-CT containing the putative binding site for CaM (V769A, R771A, L777A, R778A, R781A, V782A, R785A, K788A, and R797A) and evaluated their effects on TRPV1-CT interaction with CaM. The part of the rat TRPV1-CT DNA subcloned into the expression vector pET32b was used as a template for the introduction of point mutations. Recombinant fusion proteins were used for steady state anisotropy measurements and the dissociation constants for CaM on all constructions were estimated. We have found that among the basic residues the arginine R785 is the most essential residue for TRPV1-CT binding to CaM. In addition, the analysis of hydrophobic valines located at the first and the last position of a tentative ‘‘1-8-14” conserved motif revealed that the residue at the first position plays a more important role in TRPV1-CT’s binding to CaM.

This work was supported by Grant IAA600110701 of the GAAV, by grants 303/07/0915 and GACRGA CR 305/08/H037, of the GACR, by Research Project MSM0021620857 and the Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project No. AVOZ 50110509 of the Academy of Sciences of the Czech Republic.