Investigating the neuronal physiology of TMEM16A Ca++ activated Cl- channels

描述（由申请人提供）： 来自钙激活氯离子通道（CaCC）的电流已经在背根神经节（DRG）神经元中得到充分描述，其中（由于高细胞内氯离子浓度）CaCC开放的性质是兴奋性的。此外，神经损伤后，CaCC表达在DRG神经元中上调，导致在神经性疼痛或再生中可能的作用。我们的目标是进一步了解这一通道及其在神经性疼痛中的作用，以便有朝一日可以将其用作治疗靶点。 虽然CaCC电流已被很好地描述，但该通道最近才被鉴定为TMEM 16 A，一种先前未知功能的蛋白质。我们假设TMEM 16 A CaCC在DRG神经元生理学中起作用。对新生DRG神经元的初步研究显示，这些神经元的子集确实显示出显著的CaCC电流，并且来自DRG组织的RT-PCR显示出TMEM 16 a CaCC的表达。在来自TMEM 16 a-/-小鼠的培养的DRG神经元的实验中，未观察到CaCC电流，使得TMEM 16 A可能负责DRG CaCC电流。 本文提出的项目的最初目标是使用生理学和分子技术表征显示CaCC电流的DRG神经元，然后检查TMEM 16 A通道丢失的后果。在电生理学鉴定CaCC阳性DRG神经元后，将进行单细胞RT-PCR以确定哪些细胞表达TMEM 16 A。开发用于识别TMEM 16 A蛋白的抗体将用于确定是否存在在完整神经节中表达通道的DRG神经元的不同亚群。 然后，研究将转移到检查神经损伤期间TMEM 16 A的表达。在坐骨神经损伤后的多个时间点，通过定量RT-PCR检测TMEM 16 a的表达。为了确定神经损伤后TMEM 16 A的分布是否发生变化，将进行免疫组化并与之前获得的对照结果进行比较。 最后，将研究TMEM 16 A在神经损伤后的功能。将使用一种新技术，其中通过体细胞重组（MADM）在杂合背景中产生TMEM 16 a-/-神经元。将使用如前所述的损伤研究来研究这些个体神经元再生的能力。 公共卫生相关性： 本项目的目的是更好地了解初级感觉神经元中的钙激活氯通道。这种离子通道蛋白可能在神经损伤后上调，并可能在神经再生或神经性疼痛中发挥作用。利用遗传工具和生理学技术，我们希望阐明这种重要蛋白质的功能。

DESCRIPTION (provided by applicant): Current from calcium activated chloride channels (CaCCs) has been well described in dorsal root ganglion (DRG) neurons where (due to high intracellular chloride concentration), the nature of CaCC opening is excitatory. In addition, CaCC expression is upregulated in DRG neurons following nerve injury, leading to a possible role in neuropathic pain or regeneration. Our goal is to further understand this channel and the role it plays in neuropathic pain, so that it may someday be used as a therapeutic target. Although CaCC current has been well described, the channel has only recently been identified as TMEM16A, a protein of previously unknown function. We hypothesize that the TMEM16A CaCC plays a role in DRG neuronal physiology. Preliminary studies on neonatal DRG neurons show that a subset of these neurons does indeed display a prominent CaCC current and RT-PCR from DRG tissue shows expression of the TMEM16a CaCC. In experiments on cultured DRG neurons from TMEM16a-/- mice, CaCC current is not observed, making it likely that TMEM16A is responsible for the DRG CaCC current. The initial goal of the projects presented here are to characterize DRG neurons displaying CaCC current using physiology and molecular techniques, then to examine the consequences of the loss of the TMEM16A channel. Following electrophysiological identification of CaCC-positive DRG neurons, single-cell RT-PCR will be performed to determine which cells express TMEM16A. An antibody developed to recognize the TMEM16A protein will be used to determine if there is a distinct subpopulation of DRG neurons that express the channel in the intact ganglia. Studies will then shift to examine the expression of TMEM16A during nerve injury. At a number of time points following sciatic nerve lesion, the expression of TMEM16a will be examined via quantitative RT-PCR. To determine if the distribution of TMEM16A shifts following nerve injury, immunohistochemistry will be performed and compared to the control results obtained previously. Finally, the function of TMEM16A following nerve injury will be investigated. A novel technique will be used wherein TMEM16a-/- neurons are generated in a heterozygous background via somatic recombination (MADM). The ability of these individual neurons to regenerate will be studied using lesion studies as previously described. PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand the calcium activated chloride channel in primary sensory neurons. This ion channel protein is likely upregulated following nerve injury and may play a role in nerve regeneration or neuropathic pain. Using genetic tools and physiology techniques, we hope to elucidate the function of this important protein.