钙调蛋白及其突变体与电压门控钠离子通道1.2异亮氨酸-谷氨酰胺基序的结合作用

目的: 探讨在不同钙离子浓度下电压门控钠离子通道（Na_V）1.2与钙调蛋白（CaM）的结合，并分析CaM的钙离子结合位点突变后与Na_V1.2结合能力的变化。方法: 应用PCR技术构建Na_V1.2蛋白片段异亮氨酸-谷胺酰胺（IQ）基序的cDNA，采用QIAGEN点突变技术构建CaM突变体（CaM₁₂、CaM₃₄、CaM₁₂₃₄），应用牵出（pull-down）试验技术检测有钙（100 nmol/L钙离子浓度）和无钙条件下Na_V1.2 IQ与CaM及其突变体（CaM₁₂、CaM₃₄、CaM₁₂₃₄）的结合情况。结果: CaM与Na_V1.2 IQ在无钙和有钙情况下均可互相结合，而单独重组谷胱甘肽-S-转移酶（GST）不具有与CaM结合的能力。无钙条件下CaM与Na_V1.2 IQ的结合量大于有钙条件下两者的结合量（P < 0.05）；无钙条件下，CaM野生型与Na_V1.2 IQ结合量大于CaM突变体与Na_V1.2 IQ的结合量，其中CaM₁₂₃₄的结合能力在三个突变体中最弱（P < 0.05）。结论: CaM及其突变体对Na_V1.2 IQ的结合具有钙离子依赖性，这一CaM调控Na_V1.2新机制为离子通道疾病研究提供了一定依据。

Effect of calmodulin and its mutants on binding to NaV1.2 IQ

Objective: To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (Na_V1.2 IQ). Methods: The cDNA of Na_V1.2 IQ was constructed by PCR technique, CaM mutants CaM₁₂, CaM₃₄ and CaM₁₂₃₄ were constructed with Quickchange^TM site-directed mutagenesis kit (QIAGEN). The binding of Na_V1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. Results: Na_V1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and Na_V1.2 IQ at Ca²⁺]-free was greater than that at 100 nmol/L Ca²⁺] (P < 0.05). In the absence of calcium, the binding amount of CaM wild-type to Na_V1.2 IQ was greater than that of its mutant, and the binding affinity of CaM₁₂₃₄ to Na_V1.2 IQ was the weakest among the three mutants (P < 0.05). Conclusions: The binding ability of CaM and CaM mutants to Na_V1.2 IQ is Ca²⁺-dependent. This study has revealed a new mechanism of Na_V1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.