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

描述(由申请人提供)： 钙激活的氯离子通道(CaCCs)电流在背根神经节(DRG)神经元中已被很好地描述，由于细胞内高氯离子浓度，CACC开放的性质是兴奋性的。此外，CACC在神经损伤后的DRG神经元中表达上调，可能在神经病理性疼痛或再生中发挥作用。我们的目标是进一步了解这一通道及其在神经病理性疼痛中所起的作用，以便有朝一日将其用作治疗靶点。虽然CACC电流已经被很好地描述，但该通道直到最近才被鉴定为TMEM16A，一个先前未知功能的蛋白质。我们推测TMEM16A CACC在DRG神经元生理学中起一定作用。对新生DRG神经元的初步研究表明，这些神经元中的一部分确实显示出显著的CACC电流，并且来自DRG组织的RT-PCR显示TMEM16A CACC的表达。在培养的TMEM16A-/-小鼠DRG神经元的实验中，没有观察到CACC电流，这表明TMEM16A可能是DRG CACC电流的起因。本文提出的项目的最初目标是利用生理学和分子技术来表征显示CACC电流的DRG神经元，然后检查TMEM16A通道丢失的后果。在对CACC阳性的DRG神经元进行电生理鉴定后，将进行单细胞RT-PCR以确定哪些细胞表达TMEM16A。一种识别TMEM16A蛋白的抗体将用于确定在完整的神经节中是否存在表达该通道的DRG神经元的不同亚群。然后，研究将转移到检测TMEM16A在神经损伤期间的表达。在坐骨神经损伤后的多个时间点，将通过定量RT-PCR检测TMEM16A的表达。为了确定TMEM16A在神经损伤后的分布是否发生转移，将进行免疫组织化学染色，并与先前获得的对照结果进行比较。最后，将对TMEM16A在神经损伤后的功能进行研究。将使用一种新的技术，其中TMEM16A/-神经元通过体细胞重组(MADM)在杂合背景中产生。如前所述，将使用损伤研究来研究这些单个神经元的再生能力。

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.