Calmodulin binding is required for calcium mediated TRPA1 desensitization.

Calcium (Ca) ions affect nearly all aspects of biology. Excessive Ca entry is cytotoxic and Ca-mobilizing receptors have evolved diverse mechanisms for tight regulation that often include Calmodulin (CaM). TRPA1, an essential Ca-permeable ion channel involved in pain signaling and inflammation, exhibits complex Ca regulation with initial channel potentiation followed by rapid desensitization. The molecular mechanisms of TRPA1 Ca regulation and whether CaM plays a role remain elusive. We find that TRPA1 binds CaM best at basal Ca concentration, that they co-localize in resting cells, and that CaM suppresses TRPA1 activity. Combining biochemical, biophysical, modeling, NMR spectroscopy, and functional approaches, we identify an evolutionarily conserved, high-affinity CaM binding element in the distal TRPA1 C-terminus (DCTCaMBE). Genetic or biochemical perturbation of Ca/CaM binding to the TRPA1 DCTCaMBE yields hyperactive channels that exhibit drastic slowing of desensitization with no effect on potentiation. Ca/CaM TRPA1 regulation does not require the N-lobe, raising the possibility that CaM is not the Ca sensor, . Higher extracellular Ca can partially rescue slowed desensitization suggesting Ca/CaM binding to the TRPA1 DCTCaMBE primes an intrinsic TRPA1 Ca binding site that, upon binding Ca, triggers rapid desensitization. Collectively, our results identify a critical regulatory element in an unstructured TRPA1 region highlighting the importance of these domains, they reveal Ca/CaM is an essential TRPA1 auxiliary subunit required for rapid desensitization that establishes proper channel function with implications for all future TRPA1 work, and they uncover a mechanism for receptor regulation by Ca/CaM that expands the scope of CaM biology.