Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor.
Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor.
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DOI：
10.1111/jnc.12083
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发表时间：
2013-01
影响因子：
4.7
通讯作者：
Tran PN
中科院分区：
文献类型：
--
作者：
Laha KT;Tran PN
与其他 cys 环配体门控离子通道一样，GABAA 受体配体结合位点中芳香族残基的普遍存在，无疑对于神经递质结合和触发通道开放的能力非常重要。在这里，我们检查了 GABA 结合袋上的三个保守酪氨酸残基（β2Tyr97、β2Tyr157 和 β2Tyr205），这些残基产生了丙氨酸和苯丙氨酸的突变。我们使用 HEK-293 细胞中的异源表达来充分表征每种突变对受体功能的影响，其中包括检查表面表达、宏观电流动力学、拮抗剂的微观结合和解离率以及激动剂的微观结合率。当酪氨酸突变体表达为 αβ 受体时，GABAA 受体的组装或运输会被破坏，但有趣的是，当表达为 αβγ 受体时，GABAA 受体的组装或运输不会被破坏。每个酪氨酸的突变都会加速失活并减缓 GABA 结合。这提供了强有力的证据证明这些残基影响 GABA 的结合。定性地讲，每个酪氨酸的突变对受体功能的影响非常相似。然而，β2Tyr157 和 β2Tyr205 突变比 β2Tyr97 突变更有害，特别是对 GABA 结合率。总体而言，结果表明在结合过程中可能涉及多个酪氨酸残基的相互作用。
The prevalence of aromatic residues in the ligand binding site of the GABAA receptor, as with other cys-loop ligand-gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here we have examined three conserved tyrosine residues at the GABA binding pocket (β2Tyr97, β2Tyr157, and β2Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK-293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABAA receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β2Tyr157 and β2Tyr205 are more detrimental than β2Tyr97 mutations, particularly to the GABA binding rate. Overall the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.
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