title: Distinct properties of Ca2+-calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel.
abstract: Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca(2+)-CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca(2+)-CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca(2+)-CaM, respectively. This structure is similar to canonical Ca(2+)-CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca(2+)-CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca(2+)-CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca(2+)-CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Sze-Yi
authors.initials: SY
authors.lastname: Lau
authors.forename: Erik
authors.initials: E
authors.lastname: Procko
authors.forename: Rachelle
authors.initials: R
authors.lastname: Gaudet
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D017089:Ankyrin Repeat; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002211:Capsaicin / Q000494:pharmacology; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D050916:TRPV Cation Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D002147:Calmodulin; D050916:TRPV Cation Channels; C494484:TRPV1 receptor; D002211:Capsaicin; D002118:Calcium