Channel-Opening Kinetic Mechanism of Wild-Type GluK1 Kainate Receptors and a C-Terminal Mutant
Channel-Opening Kinetic Mechanism of Wild-Type GluK1 Kainate Receptors and a C-Terminal Mutant
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DOI：
10.1021/bi201446z
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发表时间：
2012-01-24
期刊：
影响因子：
2.9
通讯作者：
Niu, Li
中科院分区：
文献类型：
--
作者：
Han, Yan;Wang, Congzhou;Niu, Li
GluK1是嗜离子性谷氨酸受体家族中的一个盐酸盐受体亚基，表达时可形成功能通道，例如在HEK-293细胞中。然而，对GluK1的通道打开机制了解甚少。研究GluK1通道的一个主要挑战是其明显的低水平表面表达，这导致即使对饱和浓度的激动剂，全细胞电流反应也很低。低水平的表面表达被认为是由内质网（ER）保留信号序列引起的。当这个序列基序存在于野生型GluK1-2b的c端时，受体在内质网中被显著保留。相反，当该序列缺失时，如野生型GluK1-2a（即在c端有不同的可选剪接异构体），或中断时，如GluK1-2b突变体（即R896A， R897A， R900A和K901A），表面表达水平更高，全细胞电流响应更大。本文采用激光脉冲光解技术表征了这三种GluK1受体在HEK-293细胞中表达的通道打开动力学机制。我们的研究结果表明，野生型GluK1-2a、野生型GluK1-2b和GluK1-2b突变体具有相同的通道打开和通道关闭速率常数。这些结果表明，c端ER保留信号序列附近或内部的氨基酸序列影响受体转运和/或表达，但不影响通道门控特性。此外，与GluK2盐酸盐受体相比，GluK1通道的打开、关闭和脱敏速度至少快2倍，但其EC50值与GluK2相似。
GluK1 is a kainate receptor subunit in the ionotropic glutamate receptor family and can form functional channels when expressed, for instance, in HEK-293 cells. However, the channel-opening mechanism of GluK1 is poorly understood. One major challenge to studying the GluK1 channel is its apparent low level of surface expression, which results in a low whole-cell current response even to a saturating concentration of agonist. A low level of surface expression is thought to be contributed by an endoplasmic reticulum (ER) retention signal sequence. When this sequence motif is present as in the C-terminus of wild-type GluK1-2b, the receptor is significantly retained in the ER. Conversely, when this sequence is either lacking, as in wild-type GluK1-2a (i.e., a different alternatively spliced isoform at the C-terminus), or disrupted, as in a GluK1-2b mutant (i.e., R896A, R897A, R900A, and K901A), there is a higher level of surface expression and a greater whole-cell current response. Here we characterize the channel-opening kinetic mechanism for these three GluK1 receptors expressed in HEK-293 cells by using a laser-pulse photolysis technique. Our results show that wild-type GluK1-2a, wild-type GluK1-2b, and the GluK1-2b mutant have identical channel opening and channel closing rate constants. These results indicate that the amino acid sequence near or within the C-terminal ER retention signal sequence, which affects receptor trafficking and/or expression, does not affect channel gating properties. Furthermore, as compared with the GluK2 kainate receptor, the GluK1 channel is faster to open, close, and desensitize by at least 2-fold, yet the EC50 value of GluK1 is similar to that of GluK2.