Ion Channel Regulation By Signal Transduction Pathways

为了鉴定靶向催产素信号传导的毒物，我们工程化人细胞系（HEK 293）以表达人OXTR和新一代低亲和力遗传编码的钙指示剂GCAMP 3（Tian，L.例如，2009 Nature Methods 6：875-881）。我们在96孔板荧光读数器中筛选细胞。钙信号传导由3 nM催产素刺激一半最大值，并由60 nM阿托西班（一种已建立的Oxtr拮抗剂）阻断一半最大值。我们鉴定了两种阻燃剂，三（2，3-二溴-2-丙基）磷酸酯（TDBPP）和三（1，3-二氯-2-丙基）磷酸酯（TDCPP），当它们以20-100 μ M之间的浓度施用时，它们降低了钙信号。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有非常不同的影响。当我们通过用毒胡萝卜素抑制内质网（ER）中的钙泵而独立于催产素刺激钙的释放和进入时，发现了这种差异的起源。正如催产素受体拮抗剂所预期的那样，阿托西班对毒胡萝卜素产生的钙信号没有影响。相反，这两种阻燃剂选择性地抑制钙进入ER钙耗尽后。相同浓度的阻燃剂还抑制由催产素对来自PD 12-17小鼠脑的海马切片中的CA 1锥体神经元诱导的钙依赖性突触增强。我们随后表明，阻燃剂阻断TRPC 5通道，TRPC 5通道被认为是通过钙和PIP 2和/或其代谢产物由Gq信号转导调节的。然而，阻燃剂不干扰Gq信号传导或钙释放。我们发现它们完全阻断TRPC 5通道，而不是直接阻断孔。我们还表明，TRPC 5是催产素依赖性增强所必需的，因为它在TRPC 5敲除小鼠品系中缺失。我们现在正在研究TRPC 5刺激的机制，通过Gq信号与重组通道在异源系统哺乳动物系统。 我们还确定了生长抑素通过一种可扩散的cAMP依赖性机制停止尖峰，该机制靶向涉及蛋白磷酸酶的两孔钾通道，并且我们在异源系统中重建了这种信号传导。我们已经证实了这一机制在啮齿动物海马神经元，无论是在急性切片和解离培养。事实上，在神经元中有两种机制，其中一种机制的出现也受到蛋白磷酸酶的调节。

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.