Nicotinic receptor pore mutations create a sensitive inhibitory site for ethanol
Nicotinic receptor pore mutations create a sensitive inhibitory site for ethanol
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DOI：
10.1111/j.1530-0277.2000.tb02104.x
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发表时间：
2000-09-01
影响因子：
3.2
通讯作者：
Zhou, Q
中科院分区：
文献类型：
--
作者：
Forman, SA;Zhou, Q
背景资料：乙醇（EtOH）抑制和增强配体门控离子通道功能可能是由于这些膜蛋白上的网站直接相互作用。外周烟碱受体具有长链醇类抑制但乙醇不抑制的孔位点，这是因为弱结合（低亲和力）或不能损害离子移位（低功效）。我们测试是否烟碱孔突变，增加疏水性和/或大小可以创建一个网站，EtOH抑制在生理concentration.Methods：我们研究了重组表达的小鼠肌肉受体与膜片钳电生理学测量EtOH的影响都在单通道电导和多通道电流引起的快速激动剂灌流。我们研究了在位置α 252具有相似残基大小但不同疏水性的突变体对，以确定是否大小或疏水性决定EtOH sensitivity.Results：抑制野生型电流被认为是在EtOH浓度>300 mM. Receptors，包含α S252 I（丝氨酸到异亮氨酸）突变显着抑制由100 mM EtOH。在β亚基（β T263 I）上添加第二个同源突变进一步增强了敏感性，并产生了被50 mM EtOH显著抑制的受体。单烟碱通道的开放态电导在EtOH的存在下降低，这与EtOH对多通道电流的抑制密切相关。在位置α 252的两个等排突变对，只有疏水侧链显着增加受体的敏感性EtOH.Conclusions：野生型烟碱受体有一个非常低的亲和力EtOH，但只有一个或两个突变的孔网站创建受体抑制生理EtOH浓度。乙醇的抑制作用主要是由于在开放状态下的通道相互作用。在氨基酸α 252处，侧链疏水性而不是大小决定了受体对EtOH抑制的敏感性。我们建议，类似的网站可能存在于其他乙醇敏感的离子通道，如NMDA受体和神经元烟碱受体的孔内。
Background: Ethanol (EtOH) inhibition and enhancement of ligand-gated ion channel functions may be due to direct interactions with sites on these membrane proteins. Peripheral nicotinic receptors have pore sites that long-chain alcohols inhibit but EtOH does not, either because of weak binding (low affinity) or inability to impair ion translocation (low efficacy). We tested whether nicotinic pore mutations that increase hydrophobicity and/or size can create a site where EtOH inhibits at physiological concentrations.Methods: We studied recombinant expressed mouse muscle receptors with patch-clamp electrophysiology to measure EtOH effects both on single-channel conductance and on multichannel currents elicited with rapid agonist superfusion. We studied pairs of mutants with similar residue sizes but different hydrophobicities at position alpha 252 to determine if size or hydrophobicity determines EtOH sensitivity.Results: Inhibition of wild-type currents is seen at EtOH concentrations >300 mM. Receptors that contain the alpha S252I (serine to isoleucine) mutation are significantly inhibited by 100 mM EtOH. Adding a second homologous mutation on the beta subunit (beta T263I) further enhances sensitivity and creates receptors that are inhibited significantly by 50 mM EtOH. The open-state conductance of single nicotinic channels is reduced in the presence of EtOH, which closely parallels EtOH inhibition of multichannel currents. In two isosteric mutant pairs at position alpha 252, only hydrophobic side-chains significantly increase receptor sensitivity to EtOH.Conclusions: Wild-type nicotinic receptors have a very low affinity for EtOH, but only one or two mutations in the pore site create receptors inhibited by physiological EtOH concentrations. Ethanol inhibition is due primarily to channel interactions in the open state. At amino acid alpha 252, side-chain hydrophobicity, not size, determines receptor sensitivity to EtOH inhibition. We propose that similar sites may exist within the pores of other EtOH-sensitive ion channels, such as NMDA receptors and neuronal nicotinic receptors.