Sanders Justin H, Taiwo Kehinde M, Adekanye Glory A, Bali Avnika, Zhang Yuekang, Paulsen Candice E
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
bioRxiv. 2024 Dec 12:2024.12.11.627969. doi: 10.1101/2024.12.11.627969.
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.
钙离子（Ca）几乎影响生物学的各个方面。过量的钙离子内流具有细胞毒性，而钙离子动员受体已经进化出多种严格调控机制，其中常常涉及钙调蛋白（CaM）。TRPA1是一种参与疼痛信号传导和炎症反应的必需的钙离子通透离子通道，它表现出复杂的钙离子调控，最初通道增强，随后迅速脱敏。TRPA1钙离子调控的分子机制以及CaM是否发挥作用仍然不清楚。我们发现，TRPA1在基础钙离子浓度下与CaM结合最佳，它们在静息细胞中共定位，并且CaM抑制TRPA1活性。结合生化、生物物理、建模、核磁共振光谱和功能方法，我们在TRPA1 C末端远端（DCTCaMBE）鉴定出一个进化上保守的高亲和力CaM结合元件。Ca/CaM与TRPA1 DCTCaMBE结合的遗传或生化扰动产生超活性通道，这些通道脱敏明显减慢，而对增强无影响。Ca/CaM对TRPA1的调控不需要N叶，这增加了CaM不是钙离子传感器的可能性。较高的细胞外钙离子可以部分挽救减慢的脱敏，这表明Ca/CaM与TRPA1 DCTCaMBE的结合引发了一个内在的TRPA1钙离子结合位点，该位点在结合钙离子后触发快速脱敏。总体而言，我们的结果在TRPA1一个无结构区域中鉴定出一个关键调控元件，突出了这些结构域的重要性；揭示了Ca/CaM是TRPA1快速脱敏所必需的辅助亚基，它建立了适当的通道功能，对所有未来的TRPA1研究都有影响；并且揭示了Ca/CaM对受体调控的一种机制，扩展了CaM生物学的范围。
