PKA-mediated phosphorylation of the human KATP channel:: separate roles of Kir6.2 and SUR1 subunit phosphorylation
PKA-mediated phosphorylation of the human KATP channel:: separate roles of Kir6.2 and SUR1 subunit phosphorylation
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DOI：
10.1093/emboj/18.17.4722
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发表时间：
1999-09-01
期刊：
影响因子：
11.4
通讯作者：
Seino, S
中科院分区：
文献类型：
--
作者：
Béguin, P;Nagashima, K;Seino, S
ATP 敏感钾 (K-ATP) 通道在许多细胞功能中发挥着重要作用，例如激素分泌以及肌肉和神经元的兴奋性。经典的 ATP 敏感钾 (K-ATP) 通道是异多聚体膜蛋白，包含成孔 Kir6.2 亚基和磺酰脲受体亚基（SUR1 或 SUR2）。激素和神经递质通过蛋白激酶 A (PKA) 调节 K-ATP 通道的分子机制尚不清楚。我们突变了人类 SUR1 和 Kir6.2 的 PKA 共有序列。亚基并测试了它们在非洲爪蟾卵母细胞匀浆和完整细胞中的磷酸化能力。我们在Kir6.2（S372）和SUR1（S1571）的C端鉴定了负责PKA磷酸化的位点，在G蛋白（Gs）偶联受体或直接PKA刺激后，Kir6.2可以在完整细胞中的PKA磷酸化位点上被磷酸化。 Kir6.2 的磷酸化增加了通道活性，而 SUR1 的磷酸化通过减少突发持续时间、突发间隔和开放概率，以及增加细胞表面功能通道的数量，有助于基础通道特性。此外，可以通过在 PKA 磷酸化位点引入负电荷来模拟 PKA 的效果。这些数据证明了 K-ATP 通道的 PKA 直接磷酸化，并且可以解释 Gs 偶联受体刺激通道活性的机制。重要的是，他们还描述了异多聚体离子通道模型，其中不同亚基的磷酸化在功能上具有不同的作用。
ATP-sensitive potassium (K-ATP) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K-ATP) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2), The molecular mechanism by which hormones and neurotransmitters modulate K-ATP channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2. subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571), Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface, Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K-ATP channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.