Ion Channel Regulation By Signal Transduction Pathways

我们重点研究了三种特定的离子通道：CaV1.2，BKCa，GIRK。 CaV1.2通道显示受二氢吡啶、蛋白质磷酸化和钙调节的模式门控。我们从单通道记录中获得了新的证据，表明二氢吡啶通过改变其对磷酸化的敏感性来调节通道活性。此外，钙通过使通道蛋白去磷酸化来抑制通道活性。 BKCa钾通道由KCNMA1基因(SLO)编码，调节内分泌细胞和神经末梢的动作电位时程。在无脊椎动物模式生物中，关于乙醇对运动协调作用的遗传学研究已经发现BKCa通道是乙醇作用的一个重要目标。其他研究已经确定Gs-cAMP-PKA信号系统是乙醇作用的目标。因此，我们研究了乙醇对模式遗传生物秀丽线虫BKca的调节机制。我们鉴定了ceBKca上的PKA位点；当它发生突变时，蠕虫对高达500毫米的乙醇不具抵抗力！通过在CE神经元中靶向表达哺乳动物抑制蛋白来抑制Ser/Thr蛋白磷酸酶PP1，也可以阻止乙醇干扰运动。我们已经证实了乙醇对大鼠海马片PP1活性的这种依赖性，其中乙醇抑制了CA1锥体树突上的LTP。在与Birnbaumer博士的团队合作中，TRPC通道参与了通过GQ偶联受体的信号反应而进入钙的过程。 GIRK通道调节神经元、内分泌细胞和心肌细胞的兴奋性。我们利用串联的重组通道在单通道水平上研究了G蛋白、磷脂和蛋白磷酸化对GIRK通道的调节，以避免表达变化引起的混乱。特别是，我们使用生长抑素刺激GIRK通道的活动来研究GO的效应。

We have focused on three specific ion channels: CaV1.2, BKca, GIRK. CaV1.2 channels show modal gating that is regulated by dihydropyridine, protein phosphorylatiion, and calcium. We have obtained new evidence from single channel recordings that dihydropyridines modulate channel activity by altering their susceptibility to phosphorylation. Furthermore, calcium inhibits channel activity through dephosphorylation of the channel protein. The BKca potassium channels, which are encoded by the KCNMA1 gene (slo) and regulate action potential duration in endocrine cells and nerve terminals. Genetic studies of ethanol action on locomotor coordination in invertebrate model organisms have identified BKca channels as a prominent target of ethanol action. Other studies have identified the Gs-cAMP-PKA signaling system as a target of ethanol action. Therefore, we have studied the mechanism of BKca regulation by ethanol in a model genetic organism, C. elegans. We identified the PKA site on ceBKca; when it was mutated the worms were impervious to ethanol up t o 500 mM ! Inhibition of the Ser/Thr protein phosphatase PP1 by targeted expression of a mammalian inhibitor protein in ce neurons also prevented ethanol from disrupting locomotion. We have confirmed this dependence of ethanol action on PP1 activity in rat hippocampal slices, in which ethanol inhibits LTP on CA1 pyramidal dendrites. In collaboration with Dr. Birnbaumer's group, TRPC channels have been implicated in calcium entry in response to signaling through Gq coupled receptors. GIRK channels regulate excitability of neurons, endocrine cells and cardiac myocytes. We have investigated GIRK channel regulation by G proteins, phospholipids, and protein phosphorylation using concatenated recombinant channels at the single channel level to avoid confusion by changes in expression. In particular we have used somatostatin stimulation of GIRK channel activity to investigate the effector for Go.