IONIC CHANNELS IN DISSOCIATED SMOOTH MUSCLE CELLS

这一建议是对花生四烯酸的影响进行研究的结果， 脂肪酸和其他长链脂肪酸对平滑肌离子通道的影响 细胞这些研究沿着目前收集的初步数据 这些建议使得这些脂肪酸不太可能以非- 特异性的方式对脂质双层，并强烈表明，他们的作用， 离子通道本身或密切相关的蛋白质上。本 一项提案针对两个基本问题，并采用NMDA受体 大电导Ca~（++）激活K~（++）通道应答。一是做 长链脂肪酸如花生四烯酸的作用涉及脂肪酸 通道蛋白上的酸结合位点具体来说，在这种情况下， NMDA受体是NMDAR 1亚基上结合位点A区 我们已经发现它与胞浆蛋白超家族同源 脂肪酸结合蛋白？嵌合通道和具有单- 残基突变将在几个电生理研究 表达系统来回答这个问题。此外，2D NMR将 用于研究推定的NMDAR 1脂肪酸结合的结构 将被过度表达的区域。二、生理作用是什么 长链脂肪酸对离子通道的作用脂肪酸 介导神经递质对离子通道的作用或 拉伸，后者通过机械敏感磷脂酶？ADIFAB，a 将使用长链脂肪酸的荧光比率指示剂 来测量响应拉伸的脂肪酸产生， 神经递质作用。作为脂肪酸汇或阻断剂的试剂 它们的生产也将用于研究的一个子集， ADIFAB。 在中风和心肌缺血中， 酸是基因分级的，它们可能会导致细胞损伤， 如在中风中对NMDA受体的作用，或者可能发挥 通过对心脏K+通道的作用而具有保护作用。新 基于对长链脂肪酸对离子的作用的理解的疗法 通道对于这两种情况都是可能的，但似乎特别可能在 在中风的情况下，NMDA受体活化似乎是主要的 病理生理学中的因素。

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.