Calmodulin and ATP support activity of the Cav1.2 channel through dynamic interactions with the channel.
Calmodulin and ATP support activity of the Cav1.2 channel through dynamic interactions with the channel.
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DOI：
10.1113/jp273736
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发表时间：
2017-04-15
期刊：
影响因子：
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通讯作者：
Kameyama M
中科院分区：
文献类型：
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作者：
Minobe E;Mori MX;Kameyama M
Cav1.2通道通过与钙调蛋白（CaM）的相互作用维持活性。在这项研究中，我们以内外模式比较了Cav1.2通道（α1C）和突变体衍生物C末端缺失（α1CΔ）和与CaM连接的α1CΔ （α1CΔCaM）的活性。α1CΔ有CaM，但没有CaM， α1CΔCaM是活跃的，这表明即使没有远端C尾，CaM也通过与通道的动态相互作用诱导通道活性。ATP通过CaM诱导α1C活性，增强突变体通道活性。冈田酸模拟了ATP对野生型而非突变型通道的影响。这些结果支持了CaM和ATP通过动态相互作用维持Cav1.2通道活性的假设。ATP的作用包括与通道磷酸化相关和不相关的机制。CaM链接通道是在inside - out模式下研究Cav1.2通道的有用工具；只有ATP能阻止快速的下降，而缓慢的下降几乎不存在。钙调蛋白（Calmodulin, CaM）在调节Cav1.2 Ca2+通道中起关键作用。CaM直接与通道结合，维持通道活性并以Ca2+依赖性的方式调节通道。为了探索其中的分子机制，我们比较了野生型通道（α1C）和突变衍生物，C末端缺失（α1C∆）和α1C∆连接到CaM （α1C∆CaM）的活性。这些亚基在HEK293细胞中与β2a和α2δ共表达。在inside - out模式下，α1C和α1C∆在基本的内部溶液中显示出最小的打开概率（run - down），而α1C∆与CaM和α1C∆CaM保持可检测的通道活性，证实了CaM对于通道活性是必要的，但不是充分的。先前，我们报道ATP是维持α1C通道活性所必需的。与α1C不同，突变通道在早期（3-5分钟）不需要ATP激活。然而，α1C∆CaM + ATP和α1C∆CaM + ATP即使在后期（7-9 min后）仍保持活性。这些结果表明，CaM和ATP与通道的近端C末端尾部动态相互作用，从而产生通道活性。此外，蛋白磷酸酶抑制剂冈田酸可以替代ATP对α1C的影响，但不能替代突变体通道。这些结果支持了CaM和ATP维持Cav1.2通道活性的假设，进一步说明ATP具有双重作用。一个维持通道的磷酸化，另一个在远端羧基末端尾部被移除时变得明显。Cav1.2通道通过与钙调蛋白（CaM）的相互作用维持活性。在这项研究中，我们以内外模式比较了Cav1.2通道（α1C）和突变体衍生物C末端缺失（α1CΔ）和与CaM连接的α1CΔ （α1CΔCaM）的活性。α1CΔ有CaM，但没有CaM， α1CΔCaM是活跃的，这表明即使没有远端C尾，CaM也通过与通道的动态相互作用诱导通道活性。ATP通过CaM诱导α1C活性，增强突变体通道活性。冈田酸模拟了ATP对野生型而非突变型通道的影响。这些结果支持了CaM和ATP通过动态相互作用维持Cav1.2通道活性的假设。ATP的作用包括与通道磷酸化相关和不相关的机制。CaM链接通道是在inside - out模式下研究Cav1.2通道的有用工具；只有ATP能阻止快速的下降，而缓慢的下降几乎不存在。
Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent. Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+‐dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C‐terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co‐expressed with β2a and α2δ subunits in HEK293 cells. In the inside‐out mode, α1C and α1C∆ showed minimal open‐probabilities in a basic internal solution (run‐down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3–5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7–9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C‐terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl‐terminal tail is removed. Cav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant‐derivatives, C‐terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside‐out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C‐tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM‐linked channels are useful tools for investigating Cav1.2 channels in the inside‐out mode; the fast run‐down is prevented by only ATP and the slow run‐down is nearly absent.