CELLULAR AND BIOPHYSICAL MECHANISMS OF CMTX

连接蛋白32（Cx 32）是存在于髓鞘形成的许旺细胞的结旁环和Schmidt-Lantermann切口中的间隙连接蛋白。 编码人类间隙连接蛋白的基因突变导致CMTX，这是一种X连锁形式的临床疾病，称为Charcot-Marie - Tooth病，一组遗传性周围神经病。 Cx 32中的一些突变似乎允许适当的膜靶向和功能通道的形成，一些可能导致改变的细胞加工，而另一些则完全阻止Cx 32的合成。Cx 32的突变可能在决定Cx 32离子通道的加工和生物物理行为方面很重要。 我假设CMTX患者中发现的Cx 32突变导致Cx 32丧失提供重要细胞通讯途径的能力。 各种突变可能通过改变孔的生物物理性质或通过影响蛋白质的细胞加工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）我将确定由含有突变的连接蛋白形成的通道的生物物理性质，所述突变对应于在CMTX患者中发现的那些;通道将在非洲爪蟾卵母细胞或转染细胞中表达;以及2）我将使用分子的EGFP标记形式在活细胞中观察野生型和突变形式的Cx 32在真实的时间内的细胞运输。 本提案中描述的工作将为申请人提供获得必要的知识和技术技能的机会，以便成为转化神经科学研究的独立研究者。

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.