Single Channel Properties and Structure of Glutamate Receptors

描述（申请人提供）：谷氨酸受体离子通道（iGluRs）介导哺乳动物中枢神经系统中绝大多数兴奋性突触传递。大量研究表明，iGluRs的生物物理和动力学特性在决定突触后电流的振幅和时间过程以及对重复的突触前刺激的突触后反应中起着关键作用。iGluRs的生物物理和动力学特性在单受体分子的研究中得到了最好的揭示，这是单通道膜片钳记录的可能。除了形成功能性受体所需的核心亚基外，在过去十年中，iGluRs还包含辅助亚基已经变得很清楚。对于iGluRs的AMPA亚型，一个这样的辅助亚基家族是跨膜AMPA受体调节蛋白（TARPs）。除了在AMPA受体运输中发挥关键作用外，我们的实验室和其他人已经证明，TARPs调节AMPA受体门控动力学的几个特征。我们所做的单通道工作强烈表明，tarp促进了模态门控，在那里，受体在低和高Popen行为之间切换（在数百毫秒的时间尺度上）。在Aim 1中，我们建议进一步探索这些初步发现，并确定不同的tarp是否有不同的效果。如果数据支持模态门控的证据，我们将测试这种行为是否影响突触对重复刺激的反应特征。我们还将通过比较共表达研究和串联结构实验的结果，研究激动剂诱导的tarp受体相互作用解偶联对单通道电流的影响，其中单个tarp直接与受体融合。在之前的资助期内，我们证明了iGluRs的盐酸盐亚型也具有调节受体动力学的辅助亚基（NETO1, NETO2）。同样，我们的初步单通道研究表明，NETO2强烈促进海碱盐受体的模态门控，这种效应对受体反应的形状和幅度有巨大影响。我们将在本建议的第2项目标中扩展这些研究。我们也有初步的数据显示，在谷氨酸饱和浓度的短暂脉冲后，GluK1受体会在数百毫秒内打开。这些结果表明，盐酸盐受体在脱敏后恢复时仍与谷氨酸结合，恢复的速率决定步骤是受体重新敏化的速率，而不是谷氨酸与脱敏受体解离的速率。在目标2中，我们将扩展和量化这些初步研究。

DESCRIPTION (provided by applicant): Glutamate-receptor ion channels (iGluRs) mediate the vast majority of excitatory synaptic transmission in the mammalian central nervous system. A host of studies have shown that the biophysical and kinetic properties of iGluRs play a key role in determining the amplitude and time-course of postsynaptic currents, as well as postsynaptic responses to repetitive presynaptic stimulation. The biophysical and kinetic properties of iGluRs are best revealed in studies of single receptor molecules, something that is possible with single-channel patch-clamp recording. In addition to the core subunits required to form functional receptors, in the last ten years it has become clear that iGluRs also contain auxiliary subunits. For the AMPA subtype of iGluRs, one family of such auxiliary subunits is the Transmembrane AMPA-receptor Regulatory Proteins (TARPs). In addition to playing a key role in AMPA receptor trafficking, our lab and others have demonstrated that TARPs modulate several features of AMPA receptor gating kinetics. Single-channel work that we have done strongly suggests that TARPs promote modal gating, where the receptors switch (on a time-scale of hundreds of milliseconds) between low and high Popen behavior. In Aim 1, we propose to further explore these initial findings and determine whether the effects differ for different TARPs. If the data support evidence of modal gating, we will test whether this behavior influences the character of synaptic responses to repetitive stimuli. We will also investigate the effect of agonist-induced uncoupling of TARP-receptor interactions on single-channel currents by comparing results from co-expression studies with those from experiments with tandem constructs where individual TARPs are fused directly to the receptors. In the prior funding period, we demonstrated that the kainate subtype of iGluRs also have auxiliary subunits (NETO1, NETO2) that modulate receptor kinetics. Again, our preliminary single-channel studies suggest that NETO2 strongly promotes modal gating of kainate receptors, an effect that has a dramatic impact on the shape and amplitude of receptor responses. We will extend these studies in Aim 2 of this proposal. We also have preliminary data showing that GluK1 receptors open hundreds of milliseconds after brief pulses of saturating concentrations of glutamate. These results suggest that kainate receptors recover from desensitization with glutamate still bound and that the rate-determining step in recovery is the rate at which the receptor re-sensitizes, rather than the rate at which glutamate dissociates from the desensitized receptor. In Aim 2 we will extend and quantify these preliminary studies.