Department of Anesthesiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2009 Nov 27;284(48):33265-74. doi: 10.1074/jbc.M109.052910. Epub 2009 Oct 5.
Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.
电压门控钠离子通道通过选择性控制其同名离子的跨膜传递，维持神经元和肌肉细胞的电节律和稳定性。这些通道对细胞兴奋性的贡献程度可以通过内源性配体进行治疗或微调。例如，细胞内钙调节钠通道失活，即钠电导负调节的过程。我们通过研究普遍存在的钙结合蛋白钙调蛋白（CaM）与同源通道域 III 和 IV（DIII-IV）之间胞质连接组成的假定钠通道失活门之间的相互作用，探索了这种效应的分子基础。使用等温滴定量热法的实验表明，CaM 以钙依赖性方式结合到 DIII-IV 连接子中心的新型双酪氨酸基序中，该基序位于先前报道的 CaM 结合位点的 N 端。重组 DIII-DIV 连接肽中芳香族残基的丙氨酸扫描表明，尽管多个侧链有助于 CaM 结合，但两个酪氨酸（Tyr(1494)和 Tyr(1495)）在结合 CaM C 结构域中起着关键作用。然后通过在存在和不存在钙的情况下测量钠通道失活动力学来确定这些观察结果的功能相关性。在膜片钳钳制转染的 tsA201 细胞上进行的实验表明，在所测试的五个位点中，只有 Y1494A 突变使钠通道稳态失活对细胞溶质钙不敏感。结果表明，钙依赖性钙调蛋白与钠通道失活门双酪氨酸基序的结合对于心脏钠通道的钙调节是必需的。
