Novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide - Protein kinase A-dependent endocytosis
Novel mechanism for the suppression of a voltage-gated potassium channel by glucose-dependent insulinotropic polypeptide - Protein kinase A-dependent endocytosis
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DOI：
10.1074/jbc.m504913200
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发表时间：
2005-08-05
影响因子：
4.8
通讯作者：
McIntosh, CHS
中科院分区：
文献类型：
--
作者：
Kim, SJ;Choi, WS;McIntosh, CHS
葡萄糖通过ATP敏感（K-ATP）和钙激活（K-CA）钾通道调节胰岛素分泌的机制已被广泛研究，但对胰腺β细胞中电压门控（K-V）钾通道的作用知之甚少。肠促胰岛素激素，葡萄糖依赖性促胰岛素多肽（GIP）通过增强膜去极化的基础事件和对胞吐作用产生直接影响来刺激胰岛素分泌。在本研究中，我们确定了GIP在调节K（V）1.4通道内吞作用中的新作用。在表达GIP受体的HEK 293细胞中，GIP通过激活蛋白激酶A（PKA）降低KV 1.4的A型峰离子电流幅度。使用在潜在PKA磷酸化位点具有Ala-Ser/Thr取代的KV1.4的突变形式，显示C-末端磷酸化与GIP介导的电流幅度降低相关。蛋白酶K消化和免疫细胞化学研究的突变KV1.4定位后GIP刺激表明磷酸化依赖的快速内吞作用的KV1.4。在人β细胞中也证实了KV1.4蛋白的表达; GIP处理导致A型钾电流峰值振幅与HEK 293细胞中的A型钾电流峰值振幅相似的降低。在INS-1 β细胞（克隆832/13）中，野生型（WT）KV1.4或PKA磷酸化抗性T601 A突变体形式的瞬时过表达导致葡萄糖刺激的胰岛素分泌减少; WT KV1.4过表达增强了GIP诱导的胰岛素分泌，而T601 A细胞中不存在这种反应。这些结果强烈支持一个重要的新的作用，GIP在调节KV1.4细胞表面表达和调制的A型钾电流，这可能是至关重要的促胰岛素作用。
The mechanisms involved in glucose regulation of insulin secretion by ATP-sensitive ( K-ATP) and calcium-activated ( K-CA) potassium channels have been extensively studied, but less is known about the role of voltage-gated ( K-V) potassium channels in pancreatic beta-cells. The incretin hormone, glucose-dependent insulinotropic polypeptide ( GIP) stimulates insulin secretion by potentiating events underlying membrane depolarization and exerting direct effects on exocytosis. In the present study, we identified a novel role for GIP in regulating K(V)1.4 channel endocytosis. In GIP receptor-expressing HEK293 cells, GIP reduced A-type peak ionic current amplitude of KV1.4 via activation of protein kinase A (PKA). Using mutant forms of KV1.4 with Ala-Ser/Thr substitutions in a potential PKA phosphorylation site, C-terminal phosphorylation was shown to be linked to GIP-mediated current amplitude decreases. Proteinase K digestion and immunocytochemical studies on mutant KV1.4 localization following GIP stimulation demonstrated phosphorylation-dependent rapid endocytosis of KV1.4. Expression of KV1.4 protein was also demonstrated in human beta-cells; GIP treatment resulting in similar decreases in A-type potassium current peak amplitude to those in HEK293 cells. Transient overexpression in INS-1 beta-cells ( clone 832/13) of wild-type ( WT) KV1.4, or a T601A mutant form resistant to PKA phosphorylation, resulted in reduced glucose- stimulated insulin secretion; WT KV1.4 overexpression potentiated GIP-induced insulin secretion, whereas this response was absent in T601A cells. These results strongly support an important novel role for GIP in regulating KV1.4 cell surface expression and modulation of A-type potassium currents, which is likely to be critically important for its insulinotropic action.