Alcohol and Brain Research Laboratories, Departments of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA.
J Pharmacol Exp Ther. 2012 May;341(2):543-51. doi: 10.1124/jpet.111.190942. Epub 2012 Feb 22.
Recent studies highlighted the importance of loop 2 of α1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the β hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC₅₀ but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC₅₀ while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC₅₀ and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the β hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs.
最近的研究强调了α1 甘氨酸受体（GlyRs）loop 2 在配体结合能量传递到通道门的过程中的重要性。在 loop 2 的β发夹中位置 52 改变极性的突变显著影响了对乙醇的敏感性。本研究将调查范围扩展到带电残基。我们发现，在位置 52 用带负电荷的谷氨酸取代丙氨酸（A52E）会显著左移甘氨酸浓度反应曲线并增加对乙醇的敏感性，而带负电荷的天冬氨酸取代（A52D）则显著右移甘氨酸 EC₅₀，但不影响乙醇敏感性。值得注意的是，位置 52 的不带电荷的谷氨酰胺（A52Q）仅导致甘氨酸 EC₅₀ 发生较小的右移，同时增加了对乙醇的敏感性，就像 A52E 一样。相比之下，较短的不带电荷的天冬酰胺（A52N）导致甘氨酸 EC₅₀ 发生最大的右移，并将乙醇敏感性降低至野生型的一半。总的来说，这些发现表明，位置 52 处氨基酸的特定几何形状决定的电荷相互作用（例如，天冬氨酸和谷氨酸之间的 1 Å 链长差异）在受体对激动剂和乙醇的敏感性中发挥着不同的作用。我们根据一个原核离子通道的同源模型来解释这些结果，并提出这些突变形成了与 β 发夹中跨残基的盐桥（A52E-R59 和 A52N-D57）。我们假设这些静电相互作用扭曲了 loop 2，从而改变了激动剂的激活和乙醇的调节。这一知识将有助于确定导致乙醇在 GlyRs 中作用的关键物理化学参数。
