Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations.
Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations.
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DOI：
10.1371/journal.pbio.1001429
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发表时间：
2012
期刊：
影响因子：
9.8
通讯作者：
Dutzler R
中科院分区：
文献类型：
--
作者：
Zimmermann I;Marabelli A;Bertozzi C;Sivilotti LG;Dutzler R
原核五聚体配体门控离子通道 ELIC 受到二价阳离子的抑制，二价阳离子占据特定的细胞外位点并干扰通道门控。二价阳离子对五聚体配体门控离子通道（pLGIC）的调节被认为在其生理背景下的调节中发挥着重要作用。钙或锌等离子通过与 pLGIC 神经递质受体的胞外结构域结合来影响 pLGIC 神经递质受体的活性，并增强或抑制通道激活。在这里，我们通过电生理学和 X 射线晶体学研究了二价离子对 ELIC（结构已知的原核 pLGIC 同系物）的影响。我们发现二价阳离子抑制激动剂半胱胺对 ELIC 的激活，降低其效力，并在较高浓度下降低其最大反应。与钡复合的通道的晶体结构揭示了几个不同结合位点的存在。通过诱变，我们证实负责二价抑制的位点位于胞外结构域的外缘、相邻亚基之间的界面处，但距激动剂结合区有一定距离。在这里，二价阳离子通过羧酸侧链与蛋白质相互作用，并且该位点在结构上与其他蛋白质中描述的钙结合位点相似。有证据表明，其他 pLGIC 可能受到与相似区域结合的二价离子的调节，即使相互作用的残基在家族内并不保守。我们的研究提供了 ELIC 变构调节的结构和功能见解，并且对整个家庭具有潜在的相关性。五聚体配体门控离子通道 (pLGIC) 是离子型神经递质受体，可介导化学突触的电信号传导。 pLGIC 家族包括乙酰胆碱、血清素、GABA 和甘氨酸受体，它们具有相似的结构组织和激活机制：通道在没有配体的情况下关闭，而在神经递质与细胞外结构域中的保守位点结合时打开。在许多家庭成员中，神经递质的激活可能受到调节剂（包括几种治疗用途的药物）的影响，调节剂与通道上的不同位点结合。通道功能也可以通过二价阳离子来调节，二价阳离子在生理浓度下可以增强或抑制 pLGIC。在这里，我们分析了 pLGIC ELIC（结构已知的原核生物家族成员）中的这一机制。我们发现二价阳离子（例如钙或锌）通过占据远离配体结合区域的细胞外位点从而干扰门控来抑制 ELIC。尽管不同家族成员之间的相互作用位点并不保守，但我们提供的证据表明其他 pLGIC 的调节涉及同一区域。因此，我们的研究提供了对真核生物和原核生物中 pLGIC 家族普遍存在的调节过程的见解。
The prokaryotic pentameric ligand-gated ion channel ELIC is inhibited by divalent cations, which occupy a specific extracellular site and interfere with channel gating. The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family. Pentameric ligand-gated ion channels (pLGICs) are ionotropic neurotransmitter receptors that mediate electrical signaling at chemical synapses. The pLGIC family includes receptors for acetylcholine, serotonin, GABA and glycine, which share a similar structural organization and activation mechanism: the channels are closed in the absence of ligands and open when neurotransmitters bind to a conserved site in the extracellular domain. In many family members, activation by the neurotransmitter can be affected by modulators (including several drugs in therapeutic use), which bind to different sites on the channel. Channel function can be modulated also by divalent cations, which either potentiate or inhibit pLGICs at physiological concentrations. Here, we analyze this mechanism in the pLGIC ELIC, a prokaryotic family member of known structure. We show that divalent cations such as calcium or zinc inhibit ELIC by occupying an extracellular site remote from the ligand-binding region thereby interfering with gating. Although the site of interaction is not conserved between different family members, we present evidence that regulation of other pLGICs involves the same region. Our study has thus provided insights into a regulatory process that appears to be general for the pLGIC family in both eukaryotes and prokaryotes.
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