Arige Vikas, Wagner Larry E, Malik Sundeep, Baker Mariah R, Fan Guizhen, Serysheva Irina I, Yule David I
Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, USA.
Department of Biochemistry and Molecular Biology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas, USA.
J Biol Chem. 2025 Mar;301(3):108302. doi: 10.1016/j.jbc.2025.108302. Epub 2025 Feb 11.
The regulation of inositol 1,4,5-trisphosphate (IP) receptor (IPR) activity is thought to define the spatiotemporal patterns of Ca signals necessary for the appropriate activation of downstream effectors. The binding of both IP and Ca is obligatory for IPR channel opening. Ca however regulates IPR activity in a biphasic manner. Ca binding to a high-affinity pocket formed by the third armadillo repeat domain and linker domain promotes IPR channel opening without altering the Ca dependency for channel inactivation. These data suggest that a distinct low-affinity Ca-binding site is responsible for the reduction in IPR activity at higher [Ca]. We mutated a cluster of acidic residues in the second armadillo repeat domain and central linker domain of IPR type 1, reported to coordinate Ca in the cryo-EM structures of the IPR type 3. This "CD Ca-binding site" is well conserved in all IPR subtypes. CD site Ca-binding mutants where the negatively charged glutamic acid residues were mutated to alanine exhibited enhanced sensitivity to IP-generating agonists. Ca-binding mutants displayed spontaneous elemental Ca puffs, and the number of IP-induced Ca puffs was augmented in cells stably expressing Ca-binding site mutants. The inhibitory effect of high [Ca] on single-channel open probability (P) was reduced in mutant channels, and this effect was dependent on [ATP]. This indicates that Ca binding to the putative CD Ca inhibitory site facilitates the reduction in IPR channel activation at subsaturating, likely physiological cytosolic [ATP], and suggest that at higher [ATP], additional Ca-binding motifs may contribute to the biphasic regulation of IP-induced Ca release.
肌醇1,4,5 - 三磷酸（IP）受体（IPR）活性的调节被认为决定了下游效应器适当激活所需的Ca信号的时空模式。IPR通道开放需要IP和Ca的结合。然而，Ca以双相方式调节IPR活性。Ca与由第三个犰狳重复结构域和连接结构域形成的高亲和力口袋结合可促进IPR通道开放，而不会改变通道失活的Ca依赖性。这些数据表明，一个独特的低亲和力Ca结合位点负责在较高[Ca]时IPR活性的降低。我们对1型IPR的第二个犰狳重复结构域和中央连接结构域中的一组酸性残基进行了突变，据报道这些残基在3型IPR的冷冻电镜结构中参与Ca的配位。这个“CD Ca结合位点”在所有IPR亚型中都高度保守。将带负电荷的谷氨酸残基突变为丙氨酸的CD位点Ca结合突变体对产生IP的激动剂表现出增强的敏感性。Ca结合突变体显示出自发性元素Ca微泡，并且在稳定表达Ca结合位点突变体的细胞中，IP诱导的Ca微泡数量增加。在突变通道中，高[Ca]对单通道开放概率（P）的抑制作用降低，并且这种作用依赖于[ATP]。这表明Ca与假定的CD Ca抑制位点结合有助于在亚饱和、可能是生理胞质[ATP]水平下降低IPR通道激活，并表明在较高[ATP]时，额外的Ca结合基序可能有助于IP诱导的Ca释放的双相调节。
