Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition
Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition
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DOI：
10.1113/jphysiol.1996.sp021319
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发表时间：
1996-04-15
影响因子：
5.5
通讯作者：
Smart, TG
中科院分区：
文献类型：
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作者：
Krishek, BJ;Amato, A;Smart, TG
1. 在体外培养的大鼠交感神经元、表达重组GABA(A)受体的非洲爪蟾卵母细胞和人胚胎肾（HEK）细胞中，研究了外源H+对GABA(A)受体的调节作用，这些受体由α、β 1、β 2、γ 2S和δ亚基组合组成。将外部pH值从7.4改变会减少交感神经元中gaba激活的电流。gaba感应电流的pH滴定符合pH模型，该模型预测H+与两个位点（pK(a)值分别为6.4和7.2）相互作用。对于α 1 β 1 GABA(A)受体，低外部pH（< 7.4）增强了对GABA的反应。pH滴定法预测存在两个位点，pK(a)值分别为6.6和7.5。低pH下GABA浓度-响应曲线左移，高pH下无竞争性抑制（> 7.4）。4. α 1 β 1 γ 2S受体结构不受外部pH的影响，而将β 1亚基交换为β 2则对pH敏感，预测pK(a)值为5.16和9.44.5。低pH增强了α - 1 β - 1 δ亚基对GABA的反应，而高pH引起抑制（pK(a)值分别为6.6和9.9）。GABA浓度响应曲线增强（pH值为5.4）或降低（pH值为9.4），但GABA EC没有变化（50）。亚基和表位特异性抗血清对α 1， β 1和δ亚基的免疫沉淀表明，这些亚基可以在细胞膜中共同组装。α 1 β 1 γ 2S δ构建体的表达导致了“钟形”pH滴定关系。增加或降低外部pH值可抑制GABA.8的反应。HEK细胞中表达的重组GABA(A)受体的pH敏感性与爪蟾卵母细胞的数据基本一致。然而，在高pH值（> 7.4）下，快速将GABA应用于α 1 β 1构建体，导致峰值增加，稳态电流降低，相应的脱敏率增加。GABA(A)受体功能的调节明显不受内部pH的影响。而且，pH值在5 ~ 9.5之间对两性离子GABA分子上的电荷分布没有显著影响。总之，本研究表明，外部pH可以增强、影响甚微或降低gaba激活的反应，这显然取决于受体亚基组成。讨论了GABA(A)受体对H+敏感性的潜在重要性。
1. Modulation of GABA(A) receptors by external H+ was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A), receptors composed of combinations of alpha, beta 1, beta 2, gamma 2S and delta subunits.2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H+ interact with two sites (pK(a) values of 6.4 and 7.2).3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with pK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted pK(a) values of 5.16 and 9.44.5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (pK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50).6. Immunoprecipation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes.7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA.8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of desensitization.9. Modulation of GABA(A) receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules.10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H+ sensitivity of GABA(A) receptors is discussed.