Calmodulin binding is required for calcium mediated TRPA1 desensitization.

Abstract

Calcium (Ca²⁺) ions affect nearly all aspects of biology. Excessive Ca²⁺ entry is cytotoxic and Ca²⁺-mobilizing receptors have evolved diverse mechanisms for tight regulation that often include Calmodulin (CaM). TRPA1, an essential Ca²⁺-permeable ion channel involved in pain signaling and inflammation, exhibits complex Ca²⁺ regulation with initial channel potentiation followed by rapid desensitization. The molecular mechanisms of TRPA1 Ca²⁺ regulation and whether CaM plays a role remain elusive. We find that TRPA1 binds CaM best at basal Ca²⁺ 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 Ca²⁺/CaM binding to the TRPA1 DCTCaMBE yields hyperactive channels that exhibit drastic slowing of desensitization with no effect on potentiation. Ca²⁺/CaM TRPA1 regulation does not require the N-lobe, raising the possibility that CaM is not the Ca²⁺ sensor, per se. Higher extracellular Ca²⁺ can partially rescue slowed desensitization suggesting Ca²⁺/CaM binding to the TRPA1 DCTCaMBE primes an intrinsic TRPA1 Ca²⁺ binding site that, upon binding Ca²⁺, triggers rapid desensitization. Collectively, our results identify a critical regulatory element in an unstructured TRPA1 region highlighting the importance of these domains, they reveal Ca²⁺/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 Ca²⁺/CaM that expands the scope of CaM biology.