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.
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