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首页> 美国卫生研究院文献>other >Free-energy Landscapes of Ion-channel Gating Are Malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels
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Free-energy Landscapes of Ion-channel Gating Are Malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels

机译：离子通道门控的自由能态具有可塑性：结合配体数量的变化伴随着乙酰胆碱受体通道中过渡态位置的变化

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摘要

Acetylcholine-receptor channels (AChRs) are allosteric membrane proteins that mediate synaptic transmission by alternatively opening and closing (‘gating’) a cation-selective transmembrane pore. Although ligand binding is not required for the channel to open, the binding of agonists (for example, acetylcholine) increases the closed ⇌ open equilibrium constant because the ion-impermeable → ion-permeable transition of the ion pathway is accompanied by a low → high affinity change at the agonist-binding sites. The fact that the gating conformational change of muscle AChRs can be kinetically modeled as a two-state reaction has paved the way to the experimental characterization of the corresponding transition state, which represents a snapshot of the continuous sequence of molecular events separating the closed and open states. Previous studies of fully (di-) liganded AChRs, combining single-channel kinetic measurements, site-directed mutagenesis, and data analysis in the framework of the linear free-energy relationships of physical organic chemistry, have suggested a transition-state structure that is consistent with channel opening being an asynchronous conformational change that starts at the extracellular agonist-binding sites and propagates towards the intracellular end of the pore. In this paper, I characterize the gating transition state of unliganded AChRs, and report a remarkable difference: unlike that of diliganded gating, the unliganded transition state is not a hybrid of the closed- and open-state structures but, rather, is almost indistinguishable from the open state itself. This displacement of the transition state along the reaction coordinate obscures the mechanism underlying the unliganded closed ⇌ open reaction but brings to light the malleable nature of free-energy landscapes of ion-channel gating.The muscle acetylcholine receptor channel (AChR) is the neurotransmitter-gated ion channel that mediates neuromuscular synaptic transmission in vertebrates (). Although the structure of this large pentameric transmembrane protein (∼470 residues per subunit) is not known with atomic resolution, a wealth of structural information exists, mainly from mutational studies, affinity labeling, chemical modification of specific residues, electron microscopy, and crystallography (reviewed in ref. ). As is the case of any other allosteric protein, the dynamic behavior of this receptor-channel can be understood in the framework of thermodynamic cycles, with conformational changes and ligand-binding events as the elementary steps (-). Thus, the AChR can adopt a variety of different conformations that can interconvert (closed, open, and desensitized ‘states’), and each conformation has a distinct ligand-binding affinity (low affinity in the closed state and high affinity in the open and desensitized states) and a particular ‘catalytic efficiency’ (ion-impermeable in the closed and desensitized states, and ion-permeable in the open state). To meet the physiological requirement of a small closed ⇌ open (‘gating’) equilibrium constant for the unliganded receptor, and a large gating equilibrium constant for the ACh-diliganded receptor, the affinity of the AChR for ACh must be higher in the open than in the closed conformation (-). This follows from the notion that the equilibrium constants governing the different reaction steps (ligand binding and gating) of these cyclic reaction schemes are constrained by the principle of detailed balance.Hence, irrespective of whether the receptor is diliganded, monoliganded or unliganded, two changes must take place in going from the closed state (low ligand affinity and ion-impermeable) to the open state (high ligand affinity and ion-permeable): a) the pore becomes permeable to ions, and b) the transmitter-binding sites, some 50 Å away from the pore domain (), increase their affinity for the ligand (with the reverse changes taking place during closing). The apparent lack of stable intermediates between the closed and open conformations, inferred from kinetic modeling of the diliganded-gating reaction (), suggests that these two changes occur as a result of a one-step, global conformational change. The question, then, arises as to whether this concerted conformational change proceeds synchronously (i.e., every residue of the protein moves ‘in unison’) or asynchronously (i.e., following a sequence of events; ref. ) and, if the latter were the case, whether multiple, few, or just one sequence of events is actually traversed by the channel to ‘connect’ the end states.Analysis of the correlation between rate and equilibrium constants of gating in diliganded AChRs has allowed us to address some of these issues by probing the structure of the transition state (, -), that is, the intermediate species between the end states of a one-step reaction that can be most easily studied. Interpretation of these results in the framework of the classical rate-equilibrium free-energy relationships of physical organic chemistry (, ), revealed that AChR diliganded gating is a highly asynchronous reaction, and suggested that the transition-state ensemble is quite homogeneous, as if the crossing of the energy barrier were confined to a narrow pass at the top of the energy landscape. In the opening direction, the conformational rearrangement that leads to the low-to-high affinity change at the extracellular binding sites precedes the conformational rearrangement of the pore that renders the channel ion-permeable. This propagated global conformational change, which we have referred to as a ‘conformational wave’ (), must reverse during channel closing so that closing starts at the pore and propagates all the way to the binding sites.It is not at all obvious why the diliganded-gating conformational change starts at the binding sites when the channel opens, nor even why the conformational change propagates at all through the receptor, instead of taking place synchronously throughout the protein. Is there any correlation between the location of the domain that binds agonist and the location of the initiation site for the opening conformational change? Could the latter have started from the intracellular end of the pore, for example, and have propagated to the (extracellular) transmitter-binding sites? What difference does it make to be liganded or unliganded as far as the mechanism of the gating conformational change is concerned? To address these issues, I set out to explore the mechanism of gating in unliganded AChRs by probing the structure of the corresponding transition state using kinetic measurements, site-directed mutagenesis, and the concepts of rate-equilibrium free-energy relationships and Φ-value analysis.Briefly, a Φ-value can be assigned to any position in the protein by estimating the slope of a ‘Brönsted plot’href="#FN3" rid="FN3" class=" fn">2 [log (gating rate constant) versus log (gating equilibrium constant)] where each point corresponds to a different amino-acid substitution at that given position. More coarsegrained Φ-values can also be obtained by using different agonists or different transmembrane potentials, for example, as a means of altering the rate and equilibrium constants of gating. Very often, rate-equilibrium plots are linear, and 0 < Φ < 1. A value of Φ = 0 suggests that the position in question (in the case of a mutation series) experiences a closed-state-like environment at the transition state whereas a value of Φ = 1 suggests an open-state-like environment. A fractional Φ-value suggests an environment that is intermediate between those experienced in the closed and open states (href="#R16" rid="R16" class=" bibr popnode">16).Earlier results indicated that the Φ-values obtained by varying the transmembrane potential are different in diliganded and unliganded AChRs. These Φ-values, which are a measure of the closed-state-like versus open-state-like character of the channel’s voltage-sensing elements at the transition state, are 0.070 ± 0.060 in diliganded receptors (href="#R17" rid="R17" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403675">17), and 1.025 ± 0.053 in unliganded AChRs (href="#R11" rid="R11" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403647">11, href="#R18" rid="R18" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403651">18). The present study reveals that residues at the transmitter-binding sites (href="/pmc/articles/PMC1463891/figure/F1/" target="figure" class="fig-table-link figpopup" rid-figpopup="F1" rid-ob="ob-F1" co-legend-rid="lgnd_F1">Figure 1), the extracellular loop that links the second (M2) and third (M3) transmembrane segments (M2-M3 linker), and the upper and lower half of M2, which during diliganded gating have Φ-values of ∼1 (ref. href="#R11" rid="R11" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403676">11), ∼0.7 (ref. href="#R10" rid="R10" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403618">10), ∼0.35 (refs href="#R8" rid="R8" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403666">8, href="#R11" rid="R11" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403673">11, href="#R12" rid="R12" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_786536501">12), and ∼0 (ref. href="#R12" rid="R12" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_786536498">12), respectively, have also Φ-values very close to 1 during unliganded gating. This generalized shift in Φ-values suggests that the diliganded → unliganded perturbation deforms the energy landscape of gating in such a way that the ‘new’ transition state occurs very close to the open state, to such an extent that all tested positions experience an open-state-like environment at the transition state of unliganded gating. Thus, the transition state occurs so ‘late’ (i.e., so close to the open state) that its inferred structure does not provide any clues as to the intermediate stages of this reaction.href="/pmc/articles/PMC1463891/figure/F1/" target="figure" rid-figpopup="F1" rid-ob="ob-F1">target="object" href="/pmc/articles/PMC1463891/figure/F1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC1463891/figure/F1/" target="figure" rid-figpopup="F1" rid-ob="ob-F1">Figure 1:Putative threading pattern of an AChR subunit through the membrane. This pattern is thought to be conserved throughout the superfamily of cys-loop receptor subunits (which includes, in vertebrates, the subunits that form acetylcholine nicotinic receptors, serotonin type 3 receptors, glycine receptors, and γ-aminobutyric acid type A and type C receptors). The M2 domain is thought to be a transmembrane, mostly α-helical segment with 19 intramembrane residues (referred to as 1’ to 19’, from the intracellular to the extracellular end). Five M2 segments, each contributed by a different subunit of the pentameric receptor-channel, form most of the lining of the membrane pore (href="#R2" rid="R2" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_311112051">2). An alternative threading pattern with M1 having as many as three non-helical transmembrane passes has been recently proposed (href="#R19" rid="R19" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403667">19). This discrepancy, however, does not compromise the conclusions of this work. The adult form of the muscle-type AChR is formed by four different subunits (α, β, δ, and ε) in an α2βδε stoichiometry.
机译：乙酰胆碱受体通道（AChR）是变构膜蛋白，通过交替打开和关闭（“门控”）阳离子选择性跨膜孔来介导突触传递。尽管打开通道并不需要配体结合，但激动剂（例如，乙酰胆碱）的结合会增加闭合的⇌开放平衡常数，因为离子通道的离子不渗透→离子渗透转变伴随有低→高转变激动剂结合位点的亲和力变化。肌肉AChRs的门控构象变化可以动力学建模为两态反应，这一事实为相应过渡态的实验表征铺平了道路，该过渡态代表了分离闭合和开放分子事件的连续序列的快照。状态。以前对完全（二）配体AChRs的研究，结合单通道动力学测量，定点诱变和物理有机化学线性自由能关系框架中的数据分析，提出了一种过渡态结构，即与通道开放一致的是异步构象变化，该构象变化始于细胞外激动剂结合位点并向孔的细胞内端传播。在本文中，我描述了非配体AChR的门控跃迁状态，并报告了一个显着差异：与双配体门控的跃迁状态不同，非配体跃迁状态不是闭环和开环结构的混合，而是几乎无法区分的。从开放状态本身。过渡态沿反应坐标的这种位移掩盖了未配体的闭合⇌开放反应的机理，但揭示了离子通道门控自由能态的可塑性。肌肉乙酰胆碱受体通道（AChR） < / sup>是介导脊椎动物（ ）的神经肌肉突触传递的神经递质门控离子通道。尽管这种大五聚体跨膜蛋白的结构（每个亚基约470个残基）尚不清楚，但仍存在大量结构信息，主要来自突变研究，亲和标记，特定残基的化学修饰，电子显微镜和晶体学（参阅参考资料）。与其他变构蛋白一样，该受体通道的动态行为可以在热力学循环的框架中理解，构象变化和配体结合事件是基本步骤（ -< sup> ）。因此，AChR可以采用多种不同的构象，这些构象可以相互转换（闭合，开放和脱敏的“状态”），并且每种构象具有独特的配体结合亲和力（闭合状态下的亲和力低，开放状态下的亲和力高）脱敏状态）和特定的“催化效率”（在关闭和脱敏状态下离子不可渗透，在打开状态下离子可渗透）。为了满足生理要求，对于未配体的受体，其封闭的⇌敞开（“门控”）平衡常数较小，对于ACh配体的受体，其门控平衡常数较大，在开放状态下，AChR对ACh的亲和力必须高于处于闭合构型（ - ）。这是基于这样的观念，即控制这些循环反应方案的不同反应步骤（配体结合和门控）的平衡常数受详细平衡原理的约束。因此，无论受体是配位的，单配位的还是未配位的，都有两个变化必须在从封闭状态（低配体亲和力和离子不可渗透性）到开放状态（高配体亲和力和离子可渗透性）的过程中进行：a）孔对离子可渗透，b）递质结合位点，距孔区域（ ）约50Å，增加了它们对配体的亲和力（在闭合过程中发生了相反的变化）。从二配位门控反应的动力学模型（ ）推断，闭合构象和开放构象之间显然缺乏稳定的中间体，这表明这两个变化是由于一步一步全局构象而产生的更改。于是，问题就产生了：这种一致的构象变化是同步进行（即蛋白质的每个残基“一致”移动）还是异步进行（即遵循一系列事件；参考），如果后者是案例，是否多个，或者实际上只有一个事件序列被通道遍历以“连接”最终状态。对已配位AChRs的门控速率和平衡常数之间的相关性的分析使我们能够通过探究AChR的结构来解决其中的一些问题过渡状态（ ， - ），即最容易研究的一步反应的最终状态。在物理有机化学的经典速率-平衡自由能关系（ ， ）框架内对这些结果的解释表明，AChR进行了配位门控是一个高度异步的反应，表明过渡态集合非常均匀，好像能量屏障的穿越被限制在能量景观顶部的狭窄通道。在打开方向上，导致细胞外结合位点低至高亲和力变化的构象重排先于使通道离子可渗透的孔的构象重排。这种传播的整体构象变化（我们称为``构象波''（ ））必须在通道闭合期间反转，以便闭合从孔开始并一直传播到结合位点。根本不清楚为什么通道打开时配位门构象改变始于结合位点，甚至为什么构象改变根本通过受体传播，而不是在整个蛋白质中同步发生。结合激动剂的结构域的位置与开放构象变化的起始位点的位置之间是否有任何相关性？后者是否可以从例如孔的细胞内末端开始，并已经传播到（细胞外）递质结合位点？就门控构象变化的机制而言，配体或非配体有什么区别？为了解决这些问题，我着手通过动力学测量，定点诱变以及速率平衡自由能关系和Φ值的概念来探究相应过渡态的结构，从而探索了非配位AChR的门控机理。简而言之，可以通过估计“Brönsted图”的斜率将Φ值分配给蛋白质中的任何位置。 href="#FN3" rid="FN3" class=" fn"> 2 [ log （门控速率常数） log log （门控平衡常数）]，其中每个点对应于该点的不同氨基酸取代给定位置。也可以通过使用不同的激动剂或不同的跨膜电位来获得更粗糙的Φ值，例如，作为改变门控速率和平衡常数的一种手段。速率平衡图通常是线性的，并且0 <Φ<1。Φ= 0的值表明，所讨论的位置（在突变系列的情况下）在过渡状态下经历类似于封闭状态的环境Φ= 1则表示类似开状态的环境。分数Φ值表示介于处于关闭状态和打开状态的环境之间的环境（ href="#R16" rid="R16" class=" bibr popnode"> 16 ）。较早的结果表明，通过改变跨膜电位获得的Φ值在已配体和未配体AChR中均不同。这些Φ值是对过渡受体中通道电压感测元件在过渡状态下的闭合状态（ vs 闭合状态）的量度，为（0.070±0.060）（ href="#R17" rid="R17" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403675"> 17 ）和1.025 ±0.053在未配位的AChR中（ href="#R11" rid="R11" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403647"> 11 ， href="#R18" rid="R18" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403651"> 18 ） 。本研究揭示了在递质结合位点的残基（href =“ / pmc / articles / PMC1463891 / figure / F1 /” target =“ figure” class =“ fig-table-link figpopup” rid-figpopup =“ F1“ rid-ob =” ob-F1“ co-legend-rid =” lgnd_F1“>图1 ），是连接第二个（M2）和第三个（M3）跨膜片段（M2-M3链接器）以及M2的上半部分和下半部分，在经过选通的门控中，其Φ值约为1（参考 href =“＃R11” rid =“ R11” class =“ bibr popnode tag_hotlink tag_tooltip” id =“ __ tag_143403676”> 11 ），约0.7（ref。 href="#R10" rid="R10" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403618"> 10 ），约0.35（ refs href="#R8" rid="R8" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403666"> 8 ， href="#R11" rid="R11" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_143403673"> 11 ， href="#R12" rid="R12" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_786536501"> 12 ）和〜0（参考< em> href="#R12" rid="R12" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_786536498"> 12 ），它们的Φ值也非常接近在非配体门控中为1。 Φ值的这种普遍变化表明，经配位的→未配位的扰动使门的能态变形，从而“新”过渡态非常接近于打开状态，从而使所有测试位置都经历了打开非配体门控过渡状态的类似状态的环境。因此，过渡态发生的时间“太晚”（即非常接近打开状态），以至于其推断的结构无法提供有关此反应的中间阶段的任何线索。 ft0-> <！-fig模式=文章f1-> href =“ / pmc / articles / PMC1463891 / figure / F1 /” target =“ figure” rid-figpopup =“ F1” rid-ob =“ ob-F1“> <！-fig / graphic | fig / alternatives / graphic mode =” anchored“ m1-> target =” object“ href =” / pmc / articles / PMC1463891 / figure / F1 /？report = objectonly“>在单独的窗口中打开 class =” figpopup“ href =” / pmc / articles / PMC1463891 / figure / F1 /“ target =” figure“ rid-figpopup =” F1图1： <！-说明a7-> AChR亚基穿过膜的推测穿线样式。这种模式被认为在整个cys-loop受体亚基的整个家族中都是保守的（在脊椎动物中，该家族包括形成乙酰胆碱烟碱样受体，5-羟色胺3型受体，甘氨酸受体以及γ-氨基丁酸A型和C型受体的亚基。 ）。 M2结构域被认为是一个跨膜，主要是带有19个膜内残基（从细胞内到细胞外端称为1'至19'）的α螺旋片段。五个M2节段，每个节段由五聚体受体通道的不同亚基组成，形成了膜孔的大部分内衬（ href =“＃R2” rid =“ R2” class =“ bibr popnode tag_hotlink tag_tooltip“ id =” __ tag_311112051“> 2 ）。最近有人提出了一种替代的线程模式，其中M1具有多达三个非螺旋跨膜通道（ href =“＃R19” rid =“ R19” class =“ bibr popnode tag_hotlink tag_tooltip” id = “ __tag_143403667”> 19 ）。但是，这种差异并不影响这项工作的结论。肌肉型AChR的成年形式由化学计量为α2βδε的四个不同的亚基（α，β，δ和ε）形成。

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    CLAUDIO GROSMAN;

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