Calmodulin binding is required for calcium mediated TRPA1 desensitization.

Justin H Sanders, Kehinde M Taiwo, Glory A Adekanye, Avnika Bali, Yuekang Zhang, Candice E Paulsen
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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.

钙调素结合是钙介导的TRPA1脱敏所必需的。
钙(ca2 +)离子几乎影响生物的所有方面。过量的ca2 +进入是细胞毒性的,ca2 +动员受体已经进化出多种严格调控机制,通常包括钙调蛋白(CaM)。TRPA1是一个重要的ca2 +渗透离子通道,参与疼痛信号传导和炎症反应,表现出复杂的ca2 +调节,初始通道增强,随后快速脱敏。TRPA1 ca2 +调控的分子机制以及CaM是否在其中发挥作用尚不清楚。我们发现TRPA1在基础ca2 +浓度下与CaM结合最好,它们在静息细胞中共定位,CaM抑制TRPA1的活性。结合生物化学、生物物理、建模、核磁共振光谱和功能方法,我们在TRPA1远端c端(DCTCaMBE)发现了一个进化上保守的、高亲和力的CaM结合元件。ca2 + /CaM与TRPA1 DCTCaMBE结合的遗传或生化扰动会产生过度活跃的通道,其脱敏速度急剧减慢,但对增强没有影响。ca2 + /CaM TRPA1调节不需要n瓣,这就提高了CaM本身不是ca2 +传感器的可能性。较高的细胞外ca2 +可以部分地挽救缓慢的脱敏,这表明ca2 + /CaM与TRPA1 DCTCaMBE的结合启动了TRPA1内在的ca2 +结合位点,在结合ca2 +后,触发快速脱敏。总的来说,我们的研究结果确定了非结构化TRPA1区域的一个关键调控元件,突出了这些结构域的重要性,他们揭示了ca2 + /CaM是TRPA1快速脱敏所需的重要辅助亚基,建立了适当的通道功能,对所有未来的TRPA1工作都有影响,他们揭示了ca2 + /CaM受体调控的机制,扩大了CaM生物学的范围。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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