Osteopontin activates the diabetes-associated potassium channel TALK-1 in pancreatic β-cells.
Osteopontin activates the diabetes-associated potassium channel TALK-1 in pancreatic β-cells.
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DOI：
10.1371/journal.pone.0175069
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Jacobson DA
中科院分区：
文献类型：
--
作者：
Dickerson MT;Vierra NC;Milian SC;Dadi PK;Jacobson DA
葡萄糖刺激的胰岛素分泌依赖于β细胞内钙内流，这种内流受双孔结构域K+(K2P)通道Talk-1的调节。编码Talk-1的基因KCNK16的功能获得多态增加了患2型糖尿病的风险。虽然Talk-1在调节β-cell Talk-1通道中起着重要作用，但控制GSI-1通道的调控机制(S)尚不清楚。因此，我们采用了一种膜特异性酵母双杂交(MYTH)试验来鉴定人胰岛中Talk-1相互作用的蛋白，这将有助于确定调节Talk-1功能的信号方式。人胰岛c DNA文库中的21个蛋白与Talk-1相互作用。其中一些相互作用增加了Talk-1的活性，包括细胞内骨桥蛋白(IOPN)。细胞内骨桥蛋白在β细胞中高表达，并在糖尿病前期条件下上调，以帮助维持正常的β细胞功能；然而，骨桥蛋白在β细胞中的功能作用尚不清楚。我们发现iOPN与Talk-1共定位于胰腺切片，并与人胰岛Talk-1通道免疫共沉淀。由于人类β细胞表达Talk-1的两个K+通道形成变体，因此评估了iOPN对这些Talk-1变体的调节。在生理电压下，iOPN激活Talk-1转录变体3通道，而不激活Talk-1转录变体2通道。IOPN激活Talk-1通道还使HEK293细胞和原代小鼠β细胞的静息膜电位(Vm)超极化。同时在β细胞内敲除骨桥蛋白，以检测其对β-细胞Talk-1通道活动的影响。减少β细胞的iOPN可显著降低Talk-1钾电流，增加葡萄糖刺激的钙内流。重要的是，iOPN不影响其他K2P通道的功能，也不改变Talk-1缺陷的β细胞的钙内流。这些结果揭示了第一个蛋白质与Talk-1通道的相互作用，并发现与iOPN的相互作用增加了β细胞Talk-1的K+电流。Talk-1/iOPN复合体引起Vm超极化，减少β细胞葡萄糖刺激的钙内流，从而抑制GSI。
Glucose-stimulated insulin secretion (GSIS) relies on β-cell Ca2+ influx, which is modulated by the two-pore-domain K+ (K2P) channel, TALK-1. A gain-of-function polymorphism in KCNK16, the gene encoding TALK-1, increases risk for developing type-2 diabetes. While TALK-1 serves an important role in modulating GSIS, the regulatory mechanism(s) that control β-cell TALK-1 channels are unknown. Therefore, we employed a membrane-specific yeast two-hybrid (MYTH) assay to identify TALK-1-interacting proteins in human islets, which will assist in determining signaling modalities that modulate TALK-1 function. Twenty-one proteins from a human islet cDNA library interacted with TALK-1. Some of these interactions increased TALK-1 activity, including intracellular osteopontin (iOPN). Intracellular OPN is highly expressed in β-cells and is upregulated under pre-diabetic conditions to help maintain normal β-cell function; however, the functional role of iOPN in β-cells is poorly understood. We found that iOPN colocalized with TALK-1 in pancreatic sections and coimmunoprecipitated with human islet TALK-1 channels. As human β-cells express two K+ channel-forming variants of TALK-1, regulation of these TALK-1 variants by iOPN was assessed. At physiological voltages iOPN activated TALK-1 transcript variant 3 channels but not TALK-1 transcript variant 2 channels. Activation of TALK-1 channels by iOPN also hyperpolarized resting membrane potential (Vm) in HEK293 cells and in primary mouse β-cells. Intracellular OPN was also knocked down in β-cells to test its effect on β-cell TALK-1 channel activity. Reducing β-cell iOPN significantly decreased TALK-1 K+ currents and increased glucose-stimulated Ca2+ influx. Importantly, iOPN did not affect the function of other K2P channels or alter Ca2+ influx into TALK-1 deficient β-cells. These results reveal the first protein interactions with the TALK-1 channel and found that an interaction with iOPN increased β-cell TALK-1 K+ currents. The TALK-1/iOPN complex caused Vm hyperpolarization and reduced β-cell glucose-stimulated Ca2+ influx, which is predicted to inhibit GSIS.