IONIC CHANNELS IN DISSOCIATED SMOOTH MUSCLE CELLS

这一建议是花生四烯酸疗效研究的结果。 酸等长链脂肪酸对血管平滑肌离子通道的影响 细胞。这些研究以及目前收集的初步数据 一项提议使得这些脂肪酸不太可能在非 对脂质双层的具体作用方式，强烈提示它们作用于 离子通道本身或在密切相关的蛋白质上。现在 该提案针对两个基本问题，使用了NMDA受体 大电导Ca~(++)激活K~+通道应答。首先，做 像花生四烯酸这样的长链脂肪酸的作用涉及一种脂肪 通道蛋白上的酸结合部位？具体地说，在这种情况下 NMDA受体是NMDAR1亚基上结合部位 我们已经发现它与胞浆超家族同源 脂肪酸结合蛋白？嵌合通道和单通道- 残基突变将在几个方面进行电生理研究 表达系统来回答这个问题。此外，2D核磁共振将被 用于研究推测的NMDAR1脂肪酸结合的结构 将被过度表达的区域。第二，生理作用是什么？ 长链脂肪酸对离子通道的作用？脂肪酸是不是 介导神经递质在离子通道上的作用或 拉伸，后者通过机械敏感的磷脂酶？ADIFAB，a 将使用长链脂肪酸的荧光比率指示剂 为了测量对拉伸和拉伸的反应产生的脂肪酸 神经递质作用。充当脂肪酸汇或阻滞剂的试剂 他们的成果也将用于研究的一个子集，使用 ADIFAB. 在中风和心肌缺血中，脂肪水平异常高 酸是基因分级的，它们可能会导致细胞损伤， 就像它们对中风中的NMDA受体的作用一样，或者可能施加了一种 通过对心脏K+通道的作用而发挥保护作用。新的 基于了解长链脂肪酸对离子作用的治疗 渠道在这两种情况下都是可能的，但在 NMDA受体激活似乎是中风的主要原因 在病理生理学方面的因素。

This proposal is an outgrowth of studies of the effects of arachidonic acid and other long chain fatty acids on ion channels in smooth muscle cells. These studies along with preliminary data gathered for the present proposal make it quite unlikely that these fatty acids act in a non- specific way on the lipid bilayer and suggest strongly that they act on the ion channels themselves or on a closely related protein. The present proposal is directed at two basic questions and employs the NMDA receptor and large conductance Ca++activated K+ channels to answer them. First, do the effects of long chain fatty acids like arachidonate involve a fatty acid binding site on the channel protein? And specifically, in the case of the NMDA receptor, is that binding site a region on the NMDAR1 subunit which we have discovered is homologous to the superfamily of cytosolic fatty acid binding proteins? Chimeric channels and channels with single- residue mutations will be studied electrophysiologically in several expression systems to answer this question. In addition 2D NMR will be used to study the structure of the putative NMDAR1 fatty acid binding region which will be overexpressed. Second, what is the physiological role of long-chain fatty acid action on ion channels? Do the fatty acids mediate the action of neurotransmitters on ion channels or the effects of stretch, the latter through mechanosensitive phospholipases? ADIFAB, a fluorescent ratiometric indicator of long-chain fatty acids, will be used to measure fatty acid production in response to stretch and neurotransmitter action. Agents that act as sinks for fatty acids or block their production will also be employed in a subset of the studies using ADIFAB. In both stroke and myocardial ischemia abnormally high levels of fatty acids are gene rated, and they may either contribute to cellular injury, as in their action on NMDA receptors in stroke, or perhaps exert a protective effect as by their action on K+ channels in heart. New therapies based on an understanding of long chain fatty acid action on ion channels are possible for both conditions but seem especially likely in the case of stroke where NMDA receptor activation appears to be a major factor in the pathophysiology.