MOLECULAR MECHANISMS OF NA CHANNEL SLOW INCACTIVATION

可兴奋膜的动作电位主要由电压门控钠通道的快速激活和失活决定，这种激活和失活发生在毫秒级的时间常数上。更长时间的膜去极化（数百毫秒）会导致第二种失活，从这种失活中恢复的时间常数大约是秒。对这种“缓慢失活”的分子机制的兴趣在最近的研究中得到了加强，这些研究暗示了遗传性周期性瘫痪中快速和缓慢失活的紊乱。该研究旨在确定人类骨骼肌电压门控钠通道（hSkM1）参与缓慢失活的区域。扫描点诱变先前涉及缓慢失活的区域将被执行，以确定这些区域的边界和特异性。此外，我们将利用蛋白质-蛋白质嵌合体研究中成人心脏（hH1）和骨骼肌（hSkM1） Na通道缓慢失活程度的差异来识别参与缓慢失活的新区域。嵌合蛋白的研究也将被用来检验一个假设，即孔隙形成的片段可能不同地参与了缓慢的失活。最后，通过评估在静止和缓慢失活状态下巯基反应试剂对取代半胱氨酸残基共价修饰的可用性，研究S4电压传感器在缓慢失活中的作用。我们预计，对慢失活分子机制的更详细的了解将为家族性周期性麻痹和其他膜兴奋性疾病的合理治疗策略提供框架。申请人已获得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成为一名独立研究人员。

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.