Li Heyi, Nowak Linda M, Gee Kyle R, Hess George P
Molecular Biology and Genetics, 216 Biotechnology Building, Cornell University, Ithaca, New York 14853-2703, USA.
Biochemistry. 2002 Apr 16;41(15):4753-9. doi: 10.1021/bi0118916.
Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).
离子型谷氨酸受体是神经系统细胞表达的一大类质膜蛋白家族的成员。一旦与谷氨酸结合，这些受体就会短暂打开跨膜通道，允许钠离子进入。细胞跨膜电位的由此产生的变化启动了一个参与向另一个细胞信号传递的过程。谷氨酸的结合在微秒时域触发通道开放，并在毫秒时域使受体发生可逆失活（脱敏）。在室温及pH 7.4条件下，将大鼠海马神经元电压钳制在-60 mV，研究了谷氨酸受体的通道开放机制。使用了两种快速化学反应技术：（1）具有4 - 10毫秒时间分辨率的细胞流动法来施加L - 谷氨酸，以及（2）激光脉冲光解技术以30微秒的时间常数从γ - O -（α - 羧基 - 2 - 硝基苄基）谷氨酸（αCNB - 笼形L - 谷氨酸）释放谷氨酸。测定了通道开放的速率和平衡常数。结果与受体在通道开放前结合两个谷氨酸分子一致，表观解离常数为600微摩尔。通道开放和关闭速率常数k(op)和k(cl)分别测定为(9.5 ± 1)×10³ s⁻¹和(1.1 ± 0.1)×10³ s⁻¹。通过激光脉冲光解测定的通道开放平衡常数Phi（= k(op)/k(cl)）的值为8.6，在细胞流动实验中为6.6。结果表明，大鼠海马神经元中至少有两种形式的谷氨酸受体，它们以不同速率脱敏。在500微摩尔谷氨酸浓度下，80%的受体以约200 s⁻¹的速率脱敏，20%以约50 s⁻¹的速率脱敏。
