Basiry S S, Mendoza P, Lee P D, Raymond L A
Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, Vancouver, British Columbia V6T 1Z3 Canada.
J Neurosci. 1999 Jan 15;19(2):644-52. doi: 10.1523/JNEUROSCI.19-02-00644.1999.
Recent evidence suggests that the transmembrane topology of ionotropic glutamate receptors differs from other members of the ligand-gated ion channel superfamily. However, the structure of the segment linking membrane domains M3 and M4 (the M3-M4 loop) remains controversial. Although various data indicate that this loop is extracellular, other results suggest that serine residues in this segment are sites of phosphorylation and channel modulation by intracellular protein kinases. To reconcile these data, we hypothesized that the M3-M4 loop structure is dynamic and, more specifically, that the portion containing putative phosphorylation sites may be translocated across the membrane to the cytoplasmic side during agonist binding. To test this hypothesis, we mutated Ser 684, a putative cAMP-dependent protein kinase site in the kainate-type glutamate receptor GluR6, to Cys. Results of biochemical and electrophysiological experiments are consistent with Cys 684 being accessible, in the unliganded state, from the extracellular side to modification by a Cys-specific biotinylating reagent followed by streptavidin (SA). Interestingly, our data suggest that this residue becomes inaccessible to the extracellular biotinylating reagent during agonist binding. However, we find it unlikely that Cys 684 undergoes membrane translocation, because the addition of SA to Cys-biotinylated GluR6(S684C) has no effect on peak glutamate-evoked current and only a small effect on macroscopic desensitization. We conclude that residue 684 in GluR6 is extracellular in the receptor-channel's closed, unliganded state and does not cross the membrane after agonist binding. However, an agonist-induced conformational change in the receptor substantially alters accessibility of position 684 to the extracellular environment.
最近的证据表明，离子型谷氨酸受体的跨膜拓扑结构不同于配体门控离子通道超家族的其他成员。然而，连接膜结构域M3和M4的片段（M3-M4环）的结构仍存在争议。尽管各种数据表明该环位于细胞外，但其他结果表明该片段中的丝氨酸残基是细胞内蛋白激酶进行磷酸化和通道调节的位点。为了协调这些数据，我们推测M3-M4环结构是动态的，更具体地说，包含假定磷酸化位点的部分在激动剂结合期间可能会穿过膜转移到细胞质一侧。为了验证这一假设，我们将红藻氨酸型谷氨酸受体GluR6中假定的环磷酸腺苷依赖性蛋白激酶位点Ser 684突变为Cys。生化和电生理实验结果表明，在未结合配体的状态下，Cys 684可从细胞外侧被半胱氨酸特异性生物素化试剂修饰，随后与链霉亲和素（SA）结合。有趣的是，我们的数据表明，在激动剂结合期间该残基无法被细胞外生物素化试剂接近。然而，我们发现Cys 684不太可能发生膜转移，因为向半胱氨酸生物素化的GluR6(S684C)中添加SA对谷氨酸诱发的峰值电流没有影响，对宏观脱敏只有很小的影响。我们得出结论，在受体通道关闭、未结合配体的状态下，GluR6中的684位残基位于细胞外，激动剂结合后不会穿过膜。然而，激动剂诱导的受体构象变化会显著改变684位对细胞外环境的可及性。
