Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function.
Phosphorylation sites in the Hook domain of CaVβ subunits differentially modulate CaV1.2 channel function.
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DOI：
10.1016/j.yjmcc.2015.08.006
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发表时间：
2015-10
影响因子：
5
通讯作者：
Catterall WA
中科院分区：
文献类型：
--
作者：
Brunet S;Emrick MA;Sadilek M;Scheuer T;Catterall WA
L 型钙电流的调节对于许多细胞类型的发育、功能和调节至关重要。传导 L 型钙电流的 CaV1.2 通道受到许多蛋白激酶的调节，但在大多数情况下这些激酶的作用位点仍然未知。我们结合了质谱 (LC-MS/MS) 和全细胞膜片钳技术，以确定体内 CaVβ 亚基的磷酸化位点，并在体外测试这些位点的突变对 CaV1.2 通道功能的影响。以兔骨骼肌纯化的CaV1.1通道为底物进行磷酸化蛋白质组分析，发现CaVβ1a亚基HOOK结构域中的Ser193和Thr205在体内均被磷酸化。 Ser193 位于酪蛋白激酶 II 的潜在共有序列中，但它在体外未被该激酶磷酸化。相比之下，Thr205 位于 cAMP 依赖性磷酸化的共有序列中，并且在体外被 PKA 强烈磷酸化。这两个位点在多种 CaVβ 亚基异构体中是保守的，包括心脏 CaV1.2 通道的主要 CaVβ 亚基 CaVβ2b。为了分别评估这些位点磷酸化的潜在调节作用和α11.2亚基磷酸化的作用，我们在CaVβ2b中插入了磷酸化或磷酸化抑制突变，并分析了它们对转染的非肌肉细胞中CaV1.2通道功能的影响。拟磷突变 CaVβ2bS152E 降低了峰值通道电流，并将激活和失活的电压依赖性转变为更正的膜电位。磷酸抑制突变 CaVβ2bS152A 具有相反的作用。磷酸模拟突变CaVβ2bT164D和磷酸抑制突变CaVβ2bT164A之间的峰值CaV1.2电流或电压依赖性没有差异。然而，对于磷酸模拟突变CaVβ2bT164D，钙依赖性失活显着增加。这种效应是亚基特异性的，因为棕榈酰化亚型 CaVβ2a 中的相应突变没有影响。总体而言，我们的数据确定了体内 CaVβ 亚基 HOOK 结构域的两个保守磷酸化位点，并揭示了这些位点中磷酸模拟突变的差异调节作用。这些结果揭示了通过磷酸化 CaV1.2 通道 β 亚基的 HOOK 结构域来调节 CaV1.2 通道的新维度。
Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. CaV1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of CaVβ subunits in vivo and test the impact of mutations of those sites on CaV1.2 channel function in vitro. Using the CaV1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser193 and Thr205 in the HOOK domain of CaVβ1a subunits were both phosphorylated in vivo. Ser193 is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr205 is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple CaVβ subunit isoforms, including the principal CaVβ subunit of cardiac CaV1.2 channels, CaVβ2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in CaVβ2b and analyzed their effects on CaV1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation CaVβ2bS152E decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation CaVβ2bS152A had opposite effects. There were no differences in peak CaV1.2 currents or voltage dependence between the phosphomimetic mutation CaVβ2bT164D and the phosphoinhibitory mutation CaVβ2bT164A. However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation CaVβ2bT164D. This effect was subunit-specific, as the corresponding mutation in the palmitoylated isoform, CaVβ2a, had no effect. Overall, our data identify two sites of conserved phosphorylation of the HOOK domain of CaVβ subunits in vivo and reveal differential modulatory effects of phosphomimetic mutations in these sites. These results reveal a new dimension of regulation of CaV1.2 channels through phosphorylation of the HOOK domains of their β subunits.