Li Gang, Oswald Robert E, Niu Li
Department of Chemistry, Center for Neuroscience Research, University at Albany, State University of New York, Albany, New York 12222, USA.
Biochemistry. 2003 Oct 28;42(42):12367-75. doi: 10.1021/bi034797t.
GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.
GluR6是红藻氨酸亚型的离子型谷氨酸受体亚基。它在突触可塑性和癫痫中起着至关重要的作用。我们在HEK - 293细胞中表达了这种重组受体，并使用笼形谷氨酸（γ - O -（α - 羧基 - 2 - 硝基苄基）谷氨酸）的激光脉冲光解技术来表征谷氨酸诱导的通道开放反应。该技术允许笼形谷氨酸以约30微秒的时间常数通过光解释放出谷氨酸。在激光光解之前，笼形谷氨酸既不激活GluR6通道，也不抑制或增强谷氨酸反应。在跨膜电压为 - 60 mV、pH 7.4和22摄氏度的条件下，通道开放和关闭速率常数分别测定为(1.1 ± 0.4)×10⁴和(4.2 ± 0.2)×10² s⁻¹。发现谷氨酸的内在解离常数和通道开放概率分别为450 ± 200 μM和0.96。这些常数源自通道激活的最小动力学机制，该机制涉及两个谷氨酸分子的结合。该机制将受体的开放通道形式的时间进程描述为谷氨酸浓度的函数。根据获得的通道开放速率常数，预测GluR6Q通道最短的上升时间（受体电流响应的20 - 80%）或最快的开放时间为120微秒。受体的开放通道形式决定跨膜电压变化，进而控制两个神经元之间的突触信号传递。在配套论文中报道的GluR6Q和GluR2Q(flip)之间通道开放动力学速率常数的比较表明，例如，在谷氨酸浓度为100 μM时，与GluR2Q(flip)成分相比，整合的神经元信号将主要由较慢的GluR6Q受体反应主导。
