Department of Anesthesiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2010 Mar 5;285(10):7281-7. doi: 10.1074/jbc.M109.050294. Epub 2009 Dec 10.
In gamma-aminobutyric acid type A (GABA(A)) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the beta2-subunit may be involved in GABA(A) receptor activation. Specifically, residues Gly(170)-Gln(185) of the beta2-subunit were mutated to alanine, co-expressed with wild-type alpha1- and gamma2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC(50) whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC(50). None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC(50). Taken together, these results indicate that beta2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that beta2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA(A) receptor.
在γ-氨基丁酸 A 型 (GABA(A)) 受体中，将配体结合与通道开放偶联的结构元件仍未得到很好的定义。在这里，定点突变被用来确定β2 亚基非 GABA 结合位点界面上的 Loop 9 是否可能参与 GABA(A) 受体的激活。具体来说，β2 亚基的残基 Gly(170)-Gln(185)突变为丙氨酸，与野生型α1 和γ2S 亚基一起在人胚肾 (HEK) 293 细胞中表达，并通过全细胞宏观记录检测它们对 GABA、静脉麻醉药异丙酚和内源性神经甾体孕烷酮的激活作用。三个突变体 G170A、V175A 和 G177A 使 GABA EC(50)分别增加了 2.5、6.7 和 5.6 倍，而一个突变体 Q185A 使 GABA EC(50)降低了 5.2 倍。这些突变都不影响异丙酚或孕烷酮增强亚最大 GABA 反应的能力，但 Q185A 突变体的异丙酚和孕烷酮直接激活的百分比分别增加了 8.3 和 3.5 倍。突变体 Q185A 受体也有增加的漏电流，对印防己毒素敏感，表明门控效率增加。进一步的 Q185E、Q185L 和 Q185W 取代表明，该位置的氨基酸的疏水性与 GABA EC(50)之间存在很强的相关性。总之，这些结果表明β2 Loop 9 参与 GABA、异丙酚和孕烷酮对受体的激活，并且β2(Q185)参与对 GABA(A)受体稳定关闭状态很重要的亲水相互作用。
