MOLECULAR MECHANISMS OF NA CHANNEL SLOW INCACTIVATION

可兴奋膜的动作电位主要由电压门控钠(Na)通道的快速激活和失活决定，这种激活和失活以毫秒量级的时间常数发生。更长时间的膜去极化(数百毫秒)会导致第二种类型的失活，从失活中恢复的时间常数约为秒。最近的研究表明，遗传性周期性麻痹中存在快失活和慢失活的错乱，这提高了人们对这种“慢失活”的分子机制的兴趣。这项拟议的研究试图确定人类骨骼肌电压门控钠通道(HSkM1)参与缓慢失活的区域。对先前涉及缓慢失活的区域进行扫描点突变，以确定这些区域的边界和特异性。此外，在利用蛋白质-蛋白质嵌合体的研究中，我们将利用成人心肌(HH1)和骨骼肌(HSkM1)钠通道之间缓慢失活程度的差异，来识别参与缓慢失活的新区域。嵌合蛋白研究也将被用来检验这一假说，即形成孔的片段可能与缓慢失活有关。最后，将通过评估取代半胱氨酸残基对硫醇反应试剂在静止和缓慢失活状态下的共价修饰的可用性来检验S4电压传感器在缓慢失活中的贡献。我们预计，对缓慢失活的分子机制(S)的更详细的了解将为家族性周期性麻痹以及其他膜兴奋性障碍的合理治疗策略提供一个框架。申请人拥有医学博士和博士学位，目前正在完成神经病学住院医师的临床培训。他预计，拟议的培训将使他能够在三年资助期内确立自己的独立研究人员的地位。

The action potential of excitable membranes is determined primarily by the fast activation and inactivation of voltage gated sodium (Na) channels, which occur with time constants on the order of milliseconds. More prolonged membrane depolarizations (hundreds of milliseconds) result in a second type of inactivation, recovery from which takes place with time constants on the order of seconds. Interest in the molecular mechanism of this "slow inactivation" has been heightened by recent studies implicating derangements of both fast and slow inactivation in inherited periodic paralyses. The proposed study seeks to define regions of the human skeletal muscle voltage gated Na channel (hSkM1) which participate in slow inactivation. Scanning point mutagenesis of regions previously implicated in slow inactivation will be performed to define the boundaries and specificity of these regions. In addition, we will take advantage of the difference in the extent of slow inactivation between adult cardiac (hH1) and skeletal muscle (hSkM1) Na channels in studies utilizing protein-protein chimeras, to identify novel regions which participate in slow inactivation. Chimeric protein studies will also be used to test the hypothesis that pore-forming segments may be differentially involved in slow inactivation. Finally, the contribution of the S4 voltage sensors in slow inactivation will be examined by assessing the availability of substituted cysteine residues to covalent modification by thiol-reactive reagents in both resting and slow inactivated states. We anticipate that more detailed knowledge of the molecular mechanism(s) of slow inactivation will provide a framework for rational therapeutic strategies for familial periodic paralyses and perhaps other disorders of membrane excitability. The applicant has received both M.D. and Ph.D. degrees, and is currently completing clinical training as a resident in Neurology. He anticipates that the proposed training will enable him to establish himself as an independent researcher within the three year funding period.