MOLECULAR PHYSIOLOGY OF HYPERKALEMIC PERIODIC PARALYSIS

高钾血症周期性麻痹的分子生理学

基本信息

批准号：
2735510
负责人：
LAWRENCE J HAYWARD
金额：
$ 10.92万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2002-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2735510
关键词：
animal genetic material tag chloride channels genetically modified animals hyperkalemias laboratory mouse mathematical model model design /development muscle cells muscle function muscle metabolism paralysis point mutation potassium channel potassium ion sodium channel tissue /cell culture voltage /patch clamp voltage gated channel

项目摘要

Hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita (PMC), and sodium channel myotonia (SCM), are dominantly inherited myotonic disorders caused by missense mutations in the skeletal muscle sodium (Na) channel. Na channels generate the muscle action potential and influence excitability of the muscle membrane through voltage-dependent mechanisms that regulate channel availability. The proposed study will investigate the pathophysiology of these diseases by (1) analyzing electrophysiological properties of mutant Na channel expressed in a myogenic cell line and (2) evaluating the physiological consequences of over-expressed wild type or mutant Na channels upon muscle function in a transgenic mouse model. Whole-cell Na currents will be measured from MM14 muscle cells transfected with tetrodotoxin-resistant versions of wild-type or mutant Na channel cDNAs to assess muscle-specific influences upon Na channel properties. In eight transgenic mouse lines, wild type or mutant Na channel gene propagation will be verified by Southern blotting, and specificity of expression will be quantitated by an RNAse protection assay. Electromyographic screening of mice for myotonia will be performed in the absence and presence of provokation by potassium loading, exercise or cooling. Muscle force and relaxation properties of intact muscle fibers in vitro will be measured under conditions of elevated extracellular potassium. Patch clamp analysis of Na currents from sarcolemmal membrane blebs will compare native channel properties with those measured previously in heterologous cells. Histological studies will look for evidence of progressive vacuolar myopathy. These studies will test whether introduction of mutant but not wild-type Na channels in mice is sufficient to induce HyperPP through a "gain of function" mechanism. Sodium channel blockers or other drugs will be compared for effectiveness in preventing abnormal muscle excitation or ameliorating muscle cell degeneration. Kinetic models will then integrate the behavior of muscle Na, K, and Cl channels to provide quantitative predictions for understanding Na channel function and the biophysical pathology of skeletal muscle in HyperPP and related myotonic disorders. The applicant is a board-certified neurologist who has obtained experience in molecular biology and electrophysiology during three years of a postdoctoral fellowship in the laboratories of Dr. Robert H. Brown, Jr. , and Dr. Stephen C. Cannon. The proposed research will establish the critical tolls needed for the applicant to become an independent investigator focusing on an expanding family of ion channel diseases.

高钾性周期性麻痹 (HyperPP)、副肌强直先天性肌强直 (PMC) 和钠通道性肌强直 (SCM) 是错义引起的显性遗传性肌强直性疾病骨骼肌钠 (Na) 通道发生突变。钠通道产生肌肉动作电位和影响肌肉膜的兴奋性通过电压依赖性调节渠道可用性的机制。拟议的研究将通过以下方式研究这些疾病的病理生理学：(1) 分析突变型Na通道的电生理特性在生肌细胞系中表达，并且（2）评估过度表达野生型或突变体的生理后果 Na 在转基因小鼠模型中影响肌肉功能。将从 MM14 肌肉细胞测量全细胞 Na 电流用野生型或河豚毒素抗性版本转染突变 Na 通道 cDNA 用于评估肌肉特异性影响取决于Na通道特性。在八个转基因小鼠品系中，野生类型或突变型Na通道基因传播将通过以下方式验证 Southern blotting，并对表达的特异性进行定量通过 RNAse 保护测定。肌电图筛查小鼠肌强直将在缺席和存在的情况下进行钾负荷、运动或冷却引起的刺激。肌肉体外完整肌纤维的力和松弛特性在细胞外钾升高的条件下进行测量。肌膜 Na 电流的膜片钳分析 blobs 会将本机通道属性与测量的通道属性进行比较之前在异源细胞中。组织学研究将寻找进行性空泡肌病的证据。这些研究将测试是否引入突变型而非野生型Na通道小鼠体内足以通过“获得功能”机制。钠通道阻滞剂或其他药物将比较预防异常肌肉的有效性兴奋或改善肌肉细胞退化。动力学然后模型将整合肌肉 Na、K 和 Cl 的行为为了解 Na 提供定量预测的渠道骨骼肌的通道功能和生物物理病理学 HyperPP 和相关的肌强直性疾病。申请人是获得委员会认证的神经科医生，拥有以下方面的经验：三年的分子生物学和电生理学 Robert H. 博士实验室的博士后奖学金小布朗和斯蒂芬·C·坎农博士。拟议的研究将确定申请人成为所需的关键通行费一位独立调查员，专注于一个不断扩大的家庭离子通道疾病。