Pei Weimin, Ritz Mark, McCarthy Michael, Huang Zhen, Niu Li
Department of Chemistry and Center for Neuroscience Research, University at Albany, State University of New York, New York 12222, USA.
J Biol Chem. 2007 Aug 3;282(31):22731-6. doi: 10.1074/jbc.M611821200. Epub 2007 Jun 1.
AMPA glutamate ion channels are tetrameric receptors in which activation to form the open channel depends on the binding of possibly multiple glutamate molecules. However, it is unclear whether AMPA receptors bound with a different number of glutamate molecules (i.e. one being the minimal and four being the maximal number of glutamate molecules) open the channels with different kinetic constants. Using a laser pulse photolysis technique that provides microsecond time resolution, we investigated the channel-opening kinetic mechanism of a nondesensitizing AMPA receptor, i.e. GluR1Q(flip) L497Y or a leucine-to-tyrosine substitution mutant, in the entire range of glutamate concentrations to ensure receptor saturation. We found that the minimal number of glutamate molecules required to bind to the receptor and to open the channel is two (or n = 2), and that the entire channel-opening kinetics can be adequately described by just one channel-opening rate constant, k(op), which correlates to n = 2. This result suggests that higher receptor occupancy (n = 3 and 4) does not give rise to different k(op) values or, at least, not appreciably if the k(op) values are different. Furthermore, compared with the wild-type receptor (Li, G., and Niu, L. (2004) J. Biol. Chem. 279, 3990-3997), the channel-opening and channel-closing rate constants of the mutant are 1.5- and 13-fold smaller, respectively. Thus, the major effect of this mutation is to decrease the channel-closing rate constant by stabilizing the open channel conformation.
AMPA 型谷氨酸离子通道是四聚体受体，其激活形成开放通道依赖于可能多个谷氨酸分子的结合。然而，尚不清楚与不同数量谷氨酸分子结合的 AMPA 受体（即谷氨酸分子的最小数量为 1 个，最大数量为 4 个）是否以不同的动力学常数打开通道。我们使用具有微秒级时间分辨率的激光脉冲光解技术，在整个谷氨酸浓度范围内研究了一种非脱敏 AMPA 受体（即 GluR1Q(flip) L497Y 或亮氨酸到酪氨酸替代突变体）的通道开放动力学机制，以确保受体饱和。我们发现，与受体结合并打开通道所需的谷氨酸分子的最小数量为两个（即 n = 2），并且整个通道开放动力学可以仅用一个通道开放速率常数 k(op) 来充分描述，该常数与 n = 2 相关。这一结果表明，更高的受体占有率（n = 3 和 4）不会产生不同的 k(op) 值，或者至少如果 k(op) 值不同，差异也不明显。此外，与野生型受体相比（Li, G., 和 Niu, L. (2004) J. Biol. Chem. 279, 3990 - 3997），该突变体的通道开放和通道关闭速率常数分别小 1.5 倍和 13 倍。因此，该突变的主要作用是通过稳定开放通道构象来降低通道关闭速率常数。
