Ion Channel Regulation By Signal Transduction Pathways

我们以前已经证明Rho家族GTP酶调节垂体细胞中的钾通道活性，所以我们在小胶质细胞中测试了这一想法，这些小胶质细胞被认为对大脑中对神经变性的大部分炎症反应有反应。正如我们在Glia杂志上发表的一篇论文中报道的那样，调节小胶质细胞激活和运动的趋化因子也调节小胶质细胞内的整流钾通道(Muessel等人，2013年)。我们现在正在使用一种允许在小胶质细胞中选择性表达转基因的microRNA来测试这些通道本身是否参与了激活和运动的调节，这将使它们成为治疗神经退行性疾病的重要新药靶点。 为了识别针对催产素信号转导的毒物，我们设计了一个人类细胞系(HEK293)来表达人OXTR和新一代低亲和力、遗传编码的钙指示剂GCAMP3(Tian，L.等人，2009自然方法6：875-881)。我们在96孔板荧光读取器中对细胞进行筛选。钙信号可被3 nM催产素刺激一半，并被已建立的OXTR拮抗剂60 nM tosiban阻断一半。我们确定了两种阻燃剂，三(2，3-二溴-2-丙基)磷酸(TDBPP)和三(1，3-二氯-2-丙基)磷酸(TDCPP)，它们在20-100微米的浓度范围内使用时会降低钙信号。然而，在单细胞水平上，阿托西班和阻燃剂对催产素诱导的钙信号的影响截然不同。当我们通过用thapsigargin抑制内质网(ER)中的钙泵来刺激不依赖于催产素的钙释放和进入时，这种差异的起源被揭示出来。正如催产素受体拮抗剂所预期的那样，他西班对thapsigargin产生的钙信号没有影响。相反，两种阻燃剂在内质网钙耗尽后选择性地抑制钙内流。相同浓度的阻燃剂还能抑制催产素对PD 12-17小鼠海马片CA1锥体神经元的钙依赖性突触增强作用。我们随后发现，阻燃剂阻断了TRPC5通道，该通道被认为是通过钙和PIP2及其代谢物受GQ信号调节的。然而，阻燃剂不会干扰GQ信号或钙释放。我们发现，它们完全阻断了TRPC5通道，而不是直接阻断毛孔。我们还证明了TRPC5对于催产素依赖的增强是必需的，因为它在TRPC5基因敲除的小鼠品系中缺失。我们现在正在一个异源系统的哺乳动物系统中研究GQ信号和重组通道刺激TRPC5的机制。 我们还建立了生长抑素通过一种可扩散的、cAMP依赖的机制来停止尖峰作用的机制，该机制针对涉及蛋白磷酸酶的两个孔钾通道，我们已经在一个异源系统中重组了这一信号。我们已经在啮齿动物海马神经元中证实了这一机制，无论是在急性切片中还是在分离培养中。事实上，神经元中有两种机制，其中一种的出现也受到蛋白磷酸酶的调节。

We have shown previously that Rho family GTPases regulate potassium channel activity in pituitary cells, so we tested this idea in microglia that are believed to be responsive for much of the inflammatory response in the brain to neurodegeneration. As we reported in a paper in the journal Glia, chemokines that regulate microglial activation and motility also regulate inwardly rectifying potassium channels in microglia (Muessel et al., 2013). We are now using a microRNA that allows selective expression of transgenes in microglia to test whether the channels themselves are involved in the regulation of activation and motility, which would make them important new drug targets for neurodegenerative disease. To identify toxicants targeting oxytocin signaling, we engineered a human cell line (HEK293) to express human OXTR and a new generation, low affinity, genetically encoded calcium indicator, GCAMP3 (Tian, L. et al., 2009 Nature Methods 6:875-881). We screened the cells in a 96-well plate fluorescence reader. Calcium signaling was stimulated half maximally by 3 nM oxytocin and blocked half maximally by 60 nM atosiban, an established Oxtr antagonist. We identified two flame retardants, tris (2,3-dibromo-2-propyl) phosphate (TDBPP) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) that reduced the calcium signal when they were applied at concentrations between 20-100 uM. At the single cell level, however, atosiban and the flame retardants had very different effects on the calcium signals induced by oxytocin. The origin of the difference was revealed when we stimulated calcium release and entry independently of oxytocin by inhibiting the calcium pump in the endoplasmic reticulum (ER) with thapsigargin. As expected for an oxytocin receptor antagonist, atosiban had no effect on the calcium signals produced by thapsigargin. In contrast, both flame retardants selectively suppressed calcium entry after the ER calcium was depleted. The same concentrations of the flame retardants also inhibit the calcium-dependent synaptic potentiation induced by oxytocin on CA1 pyramidal neurons in hippocampal slices from PD 12-17 mouse brains. We have subsequently shown that the flame retardants block TRPC5 channels, which were thought to be regulated by Gq signaling through calcium and PIP2 and/or its metabolites. However, the flame retardants do not interfere with Gq signaling or calcium release. We discovered that they block TRPC5 channels completely, and not by blocking the pore directly. We also showed that TRPC5 is required for oxytocin-dependent potentiation because it is missing in a TRPC5 knockout mouse strain. We are now examining the mechanism of TRPC5 stimulation by Gq signaling with recombinant channels in a heterologous system mammalian system. We have also established that somatostatin stops spiking through a diffusible, cAMP-dependent mechanism that targets two pore potassium channels involving protein phosphatases and we have reconstituted this signaling in a heterologous system. We have confirmed this mechanism in rodent hippocampal neurons, both in acute slices and in dissociated cultures. In fact there are two mechanisms in the neurons, the appearance of one of which is also regulated by protein phosphatases.