Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.
Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels.
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DOI：
10.1085/jgp.200208783
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发表时间：
2003-07
影响因子：
3.8
通讯作者：
Shapiro, Mark S
中科院分区：
文献类型：
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作者：
Gamper, Nikita;Shapiro, Mark S
为了量化 [Ca2+]i 对 KCNQ2/3 电流的调节并测试钙调蛋白 (CaM) 是否介导此作用，对表达 KCNQ2/3 通道的 CHO 细胞进行同步全细胞记录和 Ca2+ 成像，单独或与野生型 (wt) CaM 或显性失活 (DN) CaM 一起。我们将离子霉素 (5 μM) 添加到 2 mM Ca2+ 或 EGTA 缓冲的无 Ca2+ 溶液中，将 [Ca2+]i 从 <10 nM 更改为 >400 nM。 wt CaM 的共表达使得 KCNQ2/3 电流对 [Ca2+]i 高度敏感（IC50 70 ± 20 nM，最大抑制 73%，n = 10）。然而，DN CaM 的共表达使得 KCNQ2/3 电流很大程度上对 [Ca2+]i 不敏感（最大抑制 8 ± 3%，n = 10）。在没有共转染 CaM 的细胞中，Ca2+ 敏感性有所不同，但通常较弱。 [Ca2+]i 对颈上神经节 (SCG) 神经元中 M 电流的调节遵循与用 KCNQ2/3 和 wt CaM 表达的 CHO 细胞相同的模式，表明内源性 M 电流对 [Ca2+]i 也高度敏感。免疫共沉淀显示 CaM 与 KCNQ2-5 的结合在 5 mM Ca2+ 或 5 mM EGTA 存在下相似。凝胶位移分析表明 Ca2+ 依赖性 CaM 与 KCNQ2-5 羧基末端存在的“IQ 样”基序结合。我们测试了 SCG 神经元中 M 电流的缓激肽调节是否使用 CaM。使用假病毒粒子或基因枪“基因枪”在 SCG 细胞中外源表达 Wt 或 DN CaM。使用这两种方法，wt CaM 和 DN CaM 的表达均强烈降低缓激肽对 M 电流的抑制，但对于所有组而言，毒蕈碱抑制均不受影响。用wt CaM表达的细胞也强烈降低了强直电流幅度。我们在所有细胞组中观察到缓激肽引起类似的[Ca2+]i 升高，表明CaM 不影响Ca2+ 从储备中的释放。我们得出结论，M 型电流对 [Ca2+]i 高度敏感，并且钙调蛋白充当其 Ca2+ 传感器。
To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 μM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 ± 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 ± 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2–5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an “IQ-like” motif present in the carboxy terminus of KCNQ2–5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic “gene gun.” Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.