Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin
Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin
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DOI：
10.1074/jbc.m806871200
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发表时间：
2009-03-27
影响因子：
4.8
通讯作者：
Balser, Jeffrey R.
中科院分区：
文献类型：
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作者：
Potet, Franck;Chagot, Benjamin;Balser, Jeffrey R.
钠通道是可兴奋细胞中的基本信号分子，并且是局部麻醉剂和细胞内游离Ca 2+（[Ca 2 +]（i））的分子靶标。Na（V）1.5的两个区域先前已被鉴定为通道失活的[Ca 2 +] i敏感性调节剂。这些包括C-末端IQ基序，其根据Ca 2+水平以不同模式结合钙调蛋白（CaM），以及直接结合Ca 2+的紧邻C-末端EF-手结构域。在这里，我们表明，IQ结构域的突变（A1924 T; Brugada综合征），减少钙调素结合稳定Na（V）1.5失活，类似地，甚至比减少[Ca 2 +]（i）更广泛。由于DIII-DIV接头是Na（V）1.5失活的重要结构，因此我们评估了该结构域的潜在CaM结合相互作用。我们确定了一个新的钙调素结合位点内的连接器，验证其与钙调素的相互作用，通过NMR光谱，并揭示了其微摩尔亲和力的等温滴定量热法。在该CaM结合结构域中，三个连续的疏水残基（Phe（1520）-Ile（1521）-Phe（1522））突变为丙氨酸，重现了C-末端IQ结构域突变时观察到的电生理表型：NaV1.5失活稳定;此外，CaM结合结构域的突变消除了充分描述的利多卡因失活稳定。钙调素与C-末端IQ结构域和DIII-DIV接头的直接物理相互作用，结合钙调素结合位点突变时表型的相似性，表明这些细胞质结构可以通过钙调素的作用在功能上偶联。这些发现与遗传改变通道中的Na+通道功能以及[Ca 2 +]（i）影响心脏传导的病理生理条件有关。
Sodium channels are fundamental signaling molecules in excitable cells, and are molecular targets for local anesthetic agents and intracellular free Ca2+ ([Ca2+](i)). Two regions of Na(V)1.5 have been identified previously as [Ca2+]i-sensitive modulators of channel inactivation. These include a C-terminal IQ motif that binds calmodulin (CaM) in different modes depending on Ca2+ levels, and an immediately adjacent C-terminal EF-hand domain that directly binds Ca2+. Here we show that a mutation of the IQ domain (A1924T; Brugada Syndrome) that reduces CaM binding stabilizes Na(V)1.5 inactivation, similarly and more extensively than even reducing [Ca2+](i). Because the DIII-DIV linker is an essential structure in Na(V)1.5 inactivation, we evaluated this domain for a potential CaM binding interaction. We identified a novel CaM binding site within the linker, validated its interaction with CaM by NMR spectroscopy, and revealed its micromolar affinity by isothermal titration calorimetry. Mutation of three consecutive hydrophobic residues (Phe(1520)-Ile(1521)-Phe(1522)) to alanines in this CaM-binding domain recapitulated the electrophysiology phenotype observed with mutation of the C-terminal IQ domain: NaV1.5 inactivation was stabilized; moreover, mutations of either CaM-binding domain abolish the well described stabilization of inactivation by lidocaine. The direct physical interaction of CaM with the C-terminal IQ domain and the DIII-DIV linker, combined with the similarity in phenotypes when CaM-binding sites in either domain are mutated, suggests these cytoplasmic structures could be functionally coupled through the action of CaM. These findings have bearing upon Na+ channel function in genetically altered channels and under pathophysiologic conditions where [Ca2+](i) impacts cardiac conduction.