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首页> 外文期刊>IBRO Reports >Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells
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Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells

机译：钙调蛋白调节表达的Ca 2 + 依赖性的失活和表达水平，表达的Ca V 2.2在HEK293T细胞中

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Ca 2+ influx through voltage-gated Ca 2+ channels (Ca V s) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca 2+ -dependent inactivation (CDI) of several types of Ca V s; however, little is known about how CaM modulates Ca V 2.2. Here, we expressed Ca V 2.2 with CaM or CaM mutants with a Ca 2+ -binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that Ca V 2.2 displayed a fast inactivation with Ca 2+ but not Ba 2+ as the charge carrier; when Ca V 2.2 was co-expressed with CaM mutants with a Ca 2+ -binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of Ca V 2.2 in the presence or absence of Ca 2+ . However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. Ca V 2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM 12 (Ca 2+ -binding deficiency at the N-lobe) overexpression; in addition, CaM 12 enhanced the total expression level of Ca V 2.2, but the ratio of Ca V 2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca 2+ -binding abilities, modulates not only the inactivation of Ca V 2.2 but also its expression to regulate Ca 2+ -related physiological activities. Highlights ? CaM binds to the conserved IQ motif of Ca V 2.2 and triggers inactivation. ? Mutations in the conserved IQ motif decrease the current density. ? The IQ motif is the main CaM binding site. ? CaM with Ca 2+ -binding deficiency at the N-lobe increases the Ca V 2.2 level.
机译：Ca 2+通过质膜上的电压门控Ca 2+通道（Ca V s）流入是引起细胞内Ca 2+浓度（[Ca 2+] i）升高的主要途径，从而激活了各种生理活动。 。已知钙调蛋白（CaM）参与多种Ca V s的Ca 2+依赖性失活（CDI）。然而，对于CaM如何调节Ca V 2.2的了解甚少。在这里，我们用HEM293T细胞中具有Ca 2+结合缺陷的CaM或CaM突变体表达了Ca V 2.2，并测量了电流以表征CDI。结果表明，Ca V 2.2表现出以Ca 2+为快速载体而没有以Ba 2+为电荷载体的失活。当Ca V 2.2与具有Ca 2+结合缺陷的CaM突变体共表达时，失活水平降低。使用谷胱甘肽S-转移酶标记的CaM或CaM突变体作为诱饵，我们发现CaM可以在存在或不存在Ca 2+的情况下与Ca V 2.2的胞内C端片段发生相互作用。但是，当在CaM结合位点的保守氨基酸残基中产生突变时，CaM及其突变体无法与该片段相互作用。 CaV 2.2在CaM结合位点发生突变，显示电流大大降低，可以通过CaM 12过表达（N瓣处的Ca 2+结合缺陷）来挽救。另外，CaM 12增强了Ca V 2.2的总表达水平，但膜中存在的Ca V 2.2与总级分之比保持不变。总之，我们的数据表明具有不同Ca 2+结合能力的CaM不仅调节Ca V 2.2的失活，而且还调节其表达以调节Ca 2+相关的生理活性。强调 ？ CaM与Ca V 2.2的保守IQ主题结合并触发失活。 ？保守的IQ基序中的突变降低了电流密度。 ？ IQ基序是CaM的主要结合位点。 ？在N瓣处具有Ca 2+结合缺陷的CaM增加Ca V 2.2的水平。

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