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首页> 外文期刊>American Journal of Physiology >PKC-delta sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells.
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PKC-delta sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells.

机译：在HEK-293细胞中激活M3受体后，PKC-δ使Kir3.1 / 3.2通道对膜磷脂水平的变化敏感。

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摘要

G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G(q/11)-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M(3) receptor activation. In perforated patch mode, staurosporine prevented the G(q/11)-mediated, M(3) receptor, inhibition of channel activity. Recovery from M(3)-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP(2), were still irreversibly inhibited by M(3) receptor stimulation. When adenosine A(1) receptors were co-expressed, inclusion of PIP(2) rescued the A(1)-mediated response. Recordings from inside-out patches showed that catalyticallyactive PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-delta mediates channel inhibition because recombinant PKC-delta inhibited channel activity, M(3)-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-delta constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-delta to the plasma membrane on M(3) receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-delta after M(3) receptor activation in HEK-293 cells.
机译：G蛋白门控的内向整流器（Kir3）通道受G（q / 11）偶联受体的激活抑制，并且已推测其涉及信号分子蛋白激酶C（PKC）和/或磷脂酰肌醇4,5-二磷酸（ PIP（2））。它们在介导抑制该通道家族中的确切作用仍存在争议。我们在这里检查它们的相对作用，在抑制毒蕈碱M（3）受体激活后，在人类胚胎肾脏（HEK）-293细胞中稳定表达的Kir3.1 / 3.2通道的抑制作用。在穿孔的修补程序模式中，星形孢菌素阻止G（q / 11）介导的M（3）受体，抑制通道活性。从M（3）介导的抑制恢复是渥曼青霉素敏感。全细胞电流，其中贴片吸管补充了PIP（2），仍然不可逆地受到M（3）受体刺激的抑制。当腺苷A（1）受体共表达时，PIP（2）的纳入挽救了A（1）介导的反应。从里到外的贴片的记录表明，直接应用于细胞内膜表面的催化活性PKC抑制了通道：由冈田酸调节的可逆作用。突变异源通道Kir3.1S185A / Kir3.2C-S178A的产生仍然使该通道易受受体，药理学和直接激酶介导的抑制作用。生化过程中，标记的磷酸盐被并入通道中。我们建议PKC三角洲调解通道抑制，因为重组PKC三角洲抑制通道活性，M（3）介导的通道抑制，被两种类型的显性负PKC-δ构造的过表达和通过共聚焦显微镜抵消，我们已经证明了绿色荧光蛋白标记的PKC-δ易位到M（3）受体刺激的质膜上。因此，Kir3.1 / 3.2通道对膜磷脂水平的变化敏感，但这取决于HEK-293细胞中M（3）受体激活后PKC-δ的活性。

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