REGULATION OF T-TYPE CALCIUM CHANNEL TRAFFICKING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 T型钙通道是一类重要的电压门控性离子通道，调节神经元的电生理特性以及各种钙依赖的细胞过程。T型钙通道调节神经细胞的动作电位波形和重复放电的时间模式，因此在确定神经元的电生理表型中起着关键作用。T型钙通道的激活也可能起到 在伤害性感觉或感觉性疼痛传递中起关键作用。例如，消除T型钙通道会导致疼痛诱发反应的显著降低。 尽管T型钙通道在感觉知觉和伤害性感受中的作用已经确定，但我们对感觉神经元寿命中调节T型通道表达的分子和细胞机制知之甚少。我们之前已经确定 T型钙通道在感觉神经元中的表达受发育调节。在发育的早期阶段，T型钙通道的表达非常低，出生后表达增加了几倍。这一建议旨在验证神经营养因子调节发育中的感觉神经元中T型钙通道表达的假设。为了检验这一假设，我们 将以鸡背根神经节(DRG)神经元为模型。这些感觉神经元将包括痛感在内的各种感觉刺激传递到中枢神经系统。 根据其电生理和形态特征，背根神经节神经元可分为C型和A型神经元。疼痛传递神经元似乎是C型的。这些细胞是 具有细胞体小和动作电位延长的特点。传递疼痛的DRG神经元似乎也标记凝集素凝集素。这将允许我们只对C型疼痛进行染色 我们研究中的敏感背根节神经元。首先，我们将在分离的背根节神经元上建立T型钙通道的发育表达。将在胚胎第6天、第10天、第15天和第20天从鸡胚中分离出神经元。T型钙通道的表达将由凝集素标记的DRG神经元的电压钳记录来确定。不同神经元类型的DRG神经元表达T型钙通道的能力将通过比较 凝集素阳性和阴性神经元中T型钙通道的表达。其次，我们将研究DRG神经元对各种营养因子的暴露是否调节T-型钙通道的表达。背根神经节神经元的存活和分化依赖于神经营养因子神经生长因子(NGF)。其他因素如细胞因子睫状神经营养因子(CNTF)或白血病抑制因子(LIF)也可能调节生存和 背根神经节神经元的分化。因此，我们将测试在NGF、CNTF或LIF存在的情况下，DRG神经元更有可能表达T型钙通道。在E6或发育后期分离神经元，并在10 ng/mLNGF、CNTF或LIF存在下培养24至72小时。通道的表达将通过电生理记录在凝集素标记的神经元中进行评估。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. T-type Ca2+ channels are an important group of voltage-gated ion channels that regulate the electrophysiological properties of neurons as well as various Ca2+-dependent cellular processes. T-type Ca2+ channels regulate the action potential waveform and the temporal pattern of repetitive firing of nerve cells and therefore play a critical role in defining the electrophysiological phenotype of neurons. Activation of T-type Ca2+ channels may also play a critical role in nociception or sensory pain transmission. For example, elimination of T-type Ca2+ channels results in a significant decrease in pain-evoked responses. Although the role of T-type calcium channels in sensory perception and nociception is well established we have very little understanding of the molecular and cellular mechanisms that regulate T-type channel expression in a sensory neuron lifetime. We have previously determined that the expression of T-type Ca2+ channels in sensory neurons is developmentally regulated. At arly stages of development, T-type Ca2+ channel expression is very low and is increased several fold by birth. This proposal is designed to test the hypothesis that neurotrophic factors regulate the expression of T-type Ca2+ channels in developing sensory neurons. To test this hypothesis we will use the chicken dorsal root ganglia (DRG) neurons as a model. These are sensory neurons that transmit a variety of sensory stimuli including pain sensations to the central nervous system. Based on their electrophysiological and morphological properties, DRG neurons can be divided into C-type and A-type neurons. Pain transmitting neurons appear to be C-type. These cells are characterized by their small cell bodies and prolonged action potentials. Pain-transmitting DRG neurons also appear to label for lectin agglutinin. This will allow us to stain only C-type, pain sensitive DRG neurons in our studies. First, we will establish the developmental expression of Ttype Ca2+ channels in isolated DRG neurons. Neurons will be isolated from chicken embryos at embryonic day (E) 6, E10, E15 and E20. T-type Ca2+ channel expression will be determined by voltage-clamp recordings of lectin-labeled DRG neurons. The ability of different neuronal types of DRG neurons to express T-type Ca2+ channels will be investigated by comparing the level of T-type Ca2+ channel expression in lectin-positive and lectin-negative neurons. Second, we will investigate whether exposure of DRG neurons to various trophic factors regulate the expression of T-type Ca2+ channels. The survival and differentiation of DRG neurons depends on the neurotrophin nerve growth factor (or NGF). Other factors like the cytokines ciliary neurotrophic factor (or CNTF) or leukemia inhibitory factor (or LIF) also may regulate the survival and differentiation of DRG neurons. Therefore, we will test whether in the presence NGF, CNTF or LIF, DRG neurons are more likely to express T-type Ca2+ channels. Neurons will be isolated at E6 or at later stages of development and culture for 24 to 72 hr in the presence of 10 ng/mL of NGF, CNTF or LIF. Channel expression will be assessed by electrophysiological recordings in lectin-labeled neurons.