Interactions of Calmodulin and α-Actinin with the NR1 Subunit Modulate Ca2+-Dependent Inactivation of NMDA Receptors
Interactions of Calmodulin and α-Actinin with the NR1 Subunit Modulate Ca2+-Dependent Inactivation of NMDA Receptors
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DOI：
10.1523/jneurosci.19-04-01165.1999
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发表时间：
1999-02
期刊：
影响因子：
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通讯作者：
J. Krupp;B. Vissel;C. G. Thomas;S. Heinemann;G. Westbrook
中科院分区：
文献类型：
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作者：
J. Krupp;B. Vissel;C. G. Thomas;S. Heinemann;G. Westbrook
谷氨酸受体与各种调节蛋白和细胞骨架蛋白相关。然而，对这些相互作用的功能意义的理解仍然是初步的。海马神经元的研究表明，这种相互作用可能涉及钙诱导的NMDA受体开放概率（失活）的降低。因此，我们使用人胚肾（HEK）293细胞中表达的NR 1/NR 2A异聚体，研究了NR 1亚基及其两种结合伴侣（钙调素和α-辅肌动蛋白）的细胞内结构域在此过程中的作用。NR 1（C 0结构域）胞内C末端的前30个残基的存在是失活所必需的。C 0最后5个残基的突变减少了失活，并在α-辅肌动蛋白和Ca 2 +/钙调蛋白与各自C 0衍生肽的结合中产生平行变化。虽然钙调蛋白减少通道活动在切除的补丁，钙调蛋白抑制剂并没有阻止失活在全细胞记录，表明在完整的细胞失活是更复杂的钙调蛋白结合到C 0。过表达假定的Ca 2+不敏感，但不是Ca 2+敏感，形式的α-辅肌动蛋白减少失活，这种作用是克服了列入钙调蛋白在全细胞吸管。C 0结构域也直接影响通道门控，因为缺少钙调蛋白或α-辅肌动蛋白结合位点的截短C 0结构域的NR 1亚基具有低的开放概率。我们认为，C 0从α-辅肌动蛋白解离后，可通过两个不同但会聚的钙依赖性过程发生失活：钙调蛋白竞争性置换α-辅肌动蛋白和α-辅肌动蛋白结合钙后降低α-辅肌动蛋白对C 0的亲和力。
Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and α-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of α-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of α-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or α-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from α-actinin by two distinct but converging calcium-dependent processes: competitive displacement of α-actinin by calmodulin and reduction in the affinity of α-actinin for C0 after binding of calcium to α-actinin.