Distinct Properties of \(Ca^{2+}\)-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 \(1.95-\mathring{A}\) 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.