Two calmodulin binding elements contribute distinctly to TRPA1 calcium desensitization

The "wasabi receptor," transient receptor potential ankyrin 1 (TRPA1), is a critical pain receptor that is activated by reactive environmental and endogenous irritants to initiate pain perception, local inflammation, and protective behaviors. TRPA1 is a calcium-permeable ion channel; calcium influx tightly controls channel function by first potentiating currents and then triggering rapid desensitization. Here, we provide evidence that the universal calcium sensor calmodulin (CaM) controls TRPA1 desensitization by engaging two distinct CaM-binding sites in the cytoplasmic C terminus: the distal C-terminal CaM binding element (DCTCaMBE) and the TRP CaMBD. Specifically, we found that CaM binds TRPA1 at the DCTCaMBE beginning at low calcium and the TRP CaMBD at high calcium concentrations. Pull-down experiments revealed that the DCTCaMBE and TRP CaMBD exhibit distinct CaM lobe binding specificities. Competition experiments showed that CaM can bind both sites simultaneously as isolated peptides. Molecular modeling identified residues predicted to contribute to the CaM-TRP CaMBD interaction. Mutation of these residues revealed that CaM binding to the TRP CaMBD controls a second kinetic step in TRPA1 desensitization. Finally, complete ablation of CaM binding at both the TRPA1 DCTCaMBE and TRP CaMBD showed that they contribute to a concerted desensitization mechanism. Together, these results support a model where CaM associates with TRPA1 at rest through the DCTCaMBE, which primes the channel for rapid desensitization. As intracellular calcium levels rise, CaM then binds the TRP CaMBD-through bridged or separate interactions-to promote a terminal desensitization step. Our work provides further mechanistic insight into how calcium and CaM tightly control TRPA1 channel function to promote nociceptive signaling.