CELLULAR AND BIOPHYSICAL MECHANISMS OF CMTX

间隙连接蛋白32(Cx32)是一种间隙连接蛋白，存在于髓鞘雪旺细胞的旁结节环和Schmidt-Lantermann切迹中。人类缝隙连接蛋白编码基因的突变导致了CMTX，这是一种X连锁的临床疾病，称为Charcot-Marie-Tooth病，是一组遗传性周围神经疾病。Cx32的一些突变似乎允许适当的膜靶向和形成功能通道，一些突变可能导致细胞加工改变，而另一些突变则完全阻止Cx32的合成。Cx32的突变在决定Cx32离子通道的加工和生物物理行为方面可能很重要。我推测，在CMTX患者中发现的Cx32突变，导致Cx32提供重要细胞通讯途径的能力丧失。各种突变可能通过改变毛孔的生物物理性质，或通过影响蛋白质的细胞处理或其组织成功能通道的能力，而导致这种功能丧失。为了评估这些可能性：1)我将确定连接蛋白形成的通道的生物物理性质，这些通道包含与在CMTX患者中发现的突变相应的突变；通道将在非洲爪哇卵母细胞或转基因细胞中表达；2)我将使用EGFP标记的分子在活细胞中实时观察野生型和突变型Cx32的细胞转运。这项建议中描述的工作将为申请者提供一个机会，以获得必要的智力和技术技能，成为翻译神经科学研究的独立研究员。

Connexin 32 (Cx32), is a gap junction protein found in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells. Mutations in the gene encoding the human gap junction protein lead to CMTX, the X-linked form of the clinical condition known as Charcot-Marie - Tooth disease, a group of inherited peripheral neuropathies. Some mutations in Cx32 appear to allow for appropriate membrane targeting and formation of functional channels, some may lead to altered cellular processing, and others prevent Cx32 synthesis entirely. Mutations in Cx32 may be important in determining the processing and biophysical behavior of the Cx32 ion-channel. I hypothesize that mutations in Cx32 found in patients with CMTX, lead to the loss of ability of Cx32 to provide a vital cellular communication pathway. Various mutations may lead to this loss-of-function by altering the biophysical properties of the pore, or by affecting cellular processing of the protein or its ability to organize into functional channels. To evaluate these possibilities: 1) I will determine the biophysical properties of the channels formed by connexins containing mutations corresponding to those found in patients with CMTX; channels will be expressed in Xenopus oocytes or transfected cells; and 2) I will observe the cellular trafficking of wild-type and mutant forms of Cx32 in real time, in living cells, using an EGFP-tagged form of the molecule. The work described in this proposal will provide the applicant with an opportunity to acquire the necessary intellectual and technical skills to be an independent investigator in translational neuroscience research.