FUNCTIONAL STATES OF THE RYANODINE RECEPTOR (RYR) & 3D STRUCTURE OF TRIAD

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。支持：NIH R01 R40615《兴奋-收缩耦合中的通道结构》，8/15/00-8/14/05。NIH R01 HL55438 - Ryanodine receptor adaptation的调节机制Hector Valdivia， University of Wisconsin， NIH HL76826 - heart E-C coupling的调节，4/1/04-3/31/09。RyRs是细胞内钙释放通道，在肌肉中尤为普遍，与肌浆网（SR）相关。ryr是由分子质量为565 Kda的亚基构成的同四聚体，使这些受体成为已知最大的离子通道（Samso和Wagenknecht, 1998； Fill和Copello, 2002； Wagenknecht和Samso， 2002）。在过去的12年里，Wagenknecht实验室一直致力于通过冷冻电子显微镜和三维重建技术对从骨骼和心脏肌肉中分离的ryr进行结构研究（Liu etal ., 2004; Radermacher etal ., 1992）。3D重建显示，RyR大约80%的质量位于细胞质中，是一个由10个不同结构域组成的复杂组装体（Radermacher et al., 1994）。除了释放钙（肌肉收缩的直接刺激）的功能外，RyRs在兴奋-收缩（E-C）耦合中起关键作用。E-C耦合是指神经元诱导的肌质膜（肌膜）去极化及其内陷（称为横(t)小管）导致Ca2+通过RyRs从SR释放的过程。也许肌膜/t小管组分中最重要的是一种被称为二氢吡啶受体的l型钙通道，它在e-c耦合中充当电压传感器（Rios和Pizarro, 1991;Jones, 2002）。二氢吡啶受体与RyR细胞质区之间的相互作用被认为介导了质膜/横管与sr中RyR的跨膜离子传导区之间的通信。RyR是一个配体门控通道。骨骼RyR的生理配体激活剂包括二氢吡啶受体（电压传感器）、微摩尔水平的Ca2+和毫摩尔ATP。抑制剂包括毫摩尔浓度的Ca2+和Mg2+。在一项采用时间分辨冷冻显微镜的开创性研究中，Unwin证明了乙酰胆碱受体，也是一个配体门控离子通道，可以在其两种功能状态下成像，一个关闭状态和一个开放状态（Unwin, 1995）。需要时间解决技术，因为当乙酰胆碱受体被生理激动剂（在本例中为乙酰胆碱）诱导打开时，打开状态是短暂的，在进入脱敏（失活）状态之前仅持续50-100毫秒。直到最近，人们还不清楚孤立的ryr是否失活，但现在有几项研究发现，从开放形式到被描述为“失活”或“适应”的状态的转变相当缓慢（时间常数约为100毫秒到秒，取决于实验条件）（Schiefer等人，1995；Laver和Lamb, 1998；Valdivia等人，1995）；Gyorke和Fill， 1993)；Lamb et al., 2000；Fill et al., 2000)。关于这些新发现的RyR状态的确切性质和生理意义，以及不同实验室获得的不同结果，目前存在争议。越来越多的证据表明，RyR的心脏异构体失活可以防止不受控制的钙诱导的钙释放（Wang et al., 2004）。兴奋-收缩（e-c）耦合机制是肌肉生理学家研究的主要问题。Ryanodine受体在该过程的信号转导事件中起关键作用。我们的研究将提供e-c耦合装置的结构细节，使用可能是获得该信息的唯一方法。已知几种肌肉疾病与ryanodine受体的突变有关，并且有证据表明ryanodine受体在心脏病和脑功能中也起作用。我们对ryr的整个研究计划取决于单粒子图像处理方法（TRD#3）和低温电镜技术（TRD#2），如上所述。冷冻水合亚细胞结构（TRD#1）的电子断层扫描对我们的项目（上面的项目3）的一个组成部分也是必不可少的。项目1：骨骼和心脏RyR的闭合、打开和失活/适应状态的三维重建，RyR的闭合状态很容易通过简单的保留激活剂来实现。通过快速添加活化剂，然后在规定的时间间隔足够短的时间内进行冷冻电镜冷冻，以诱导打开状态，从而不会形成失活/适应状态。在TRD#2中开发的技术对于进行拟议的实验是必要的。我们有充分的理由预测，在封闭状态和开放状态之间将会出现有趣的结构变化。我们自己的研究小组和贝勒大学的一个研究小组已经发表了RyRs在假设开放（但可能失活）状态下的冷冻电镜研究，这些研究是用适当水平的Ca2+和核苷酸处理受体获得的。Serysheva et al., 1999)。在两种状态之间检测到显著的结构差异，不仅在跨膜区域，而且令人惊讶的是，在距离离子通道相当远的细胞质区域（bbb100¿）也存在差异。为了确认和改进这些发现，应该在添加活化剂后的几个时间点以开放形式研究RyR。我们计划通过将其暴露于激活剂Ca2+，通过闪光光解笼中的Ca2+ （Ca2+-二硝基苯）来激活RyRs，就像在RyRs的电生理实验中所做的那样（Gyorke等人，1994）。或者，微滴喷雾方法也可用于将网格上的ryr暴露于喷雾中包含的活化剂。如果我们发现反应时间大于1秒足以捕获RyR的开态，则预混合方法可能是更可取的。除了防止RyR形成不希望的、缓慢形成的构象状态外，时间分辨方法可能被证明是必不可少的。迄今为止，所有关于RyR的低温电镜研究都是在纯化的、洗涤剂溶解的材料上进行的。在这些制备中，洗涤剂的存在和膜双分子层的缺乏可能导致对受体在其自然环境中无效的结论。我们发现，在低水平的洗涤剂中，受体的封闭形式在短时间内（几分钟）仍可溶解（Wagenknecht et al., 1997; Wagenknecht et al., 1994）。然而，我们最初在低浓度洗涤剂下表征开放形式的RyR的实验经常显示出EM网格上明显聚集的受体数量高得令人无法接受。通过使用时间分辨方法进行这些相同的实验，我们将能够将暴露于低洗涤剂水平的时间减少到1秒以下；这将大大减少溶解度问题，并允许使用更低水平的洗涤剂。快速稀释去除洗涤剂的优势可能是巨大的，并且普遍适用于膜蛋白，或者更普遍地适用于任何在溶液条件改变或添加配体时容易聚集的大分子(例如，确实发现了低ph诱导的塞姆利基森林病毒构象状态（Fuller等人，1995年）。最终，我们希望能够在脂质双分子层环境中研究ryr，在这种环境中，功能活动可以与结构工作并行监测。在将ryr重构成双层成像方面已经取得了一些初步的成功（Liu和Wagenknecht， 1999）。更一般地说，对于亚稳的大分子机器，如RyR，即使在非时间分辨实验中，减少暴露于EM网格碳表面的预防措施也可能有利于保留天然结构（Trujillo等，2004）。如上所述，越来越多的证据表明RyR至少可以存在三种主要构象：封闭、开放和失活（和/或适应）。可能在这些主要类别中有多个状态（Laver和Lamb， 1998）。随着我们对各种状态之间转换的理解的提高，通过冷冻电镜和图像重建来表征失活（或适应）状态的结构应该是可行的。它甚至可能被证明是可行的，以响应条件，模仿受体在体内所经历的结构转变的动力学。项目2:RyR复合物和蛋白质配体的三维结构，已知RyR与骨骼肌中的几种蛋白质直接相互作用，形成称为三元结的信号转导复合物。一个目标是使用分离的RyR作为“组装平台”来制备含有这些配体的复合物。我们已经在确定两种配体fk506结合蛋白和钙调蛋白在RyR表面的结合位置方面取得了进展（Wagenknecht et al., 1994; Wagenknecht et al., 1996; Wagenknecht et al., 1997; Samso and Wagenknecht, 2002; Sharma et al. 2004（提交））。特别令人感兴趣的是RyR与二氢吡啶受体的相互作用（Samso等人，1999），这被认为是e-c耦合信号传导装置的核心。序列特异性抗体是另一类配体，用于通过体外组装和3D冷冻显微镜探测RyR的结构。RyRs还以载子和Ca2+结合的形式结合调节分子钙调素（Meissner, 2002; Tang et al., 2002）。因此，钙调素可以被认为是RyR的常驻调节成分；钙调素在其他离子通道中也有类似的作用（Budde et al., 2002）。有趣的是，我们通过单粒子低温电镜进行的结构分析显示，两种钙调素有两个离散的结合位点，相距约30 μ m （Samso和Wagenknecht, 2002； Wagenknecht等人，1997）。我们假设钙调素在这两个位点之间切换而不与ryanodine受体分离。我们建议使用时间分辨低温电镜来验证这一假设，以诱导在缺乏外源性钙调素的情况下结合位点的假定开关。由于配体的平衡常数变化很大，通常需要在高于EM的最佳浓度（RyR为~10-7 M）下使用RyR和配体进行组装反应，以最大化结合配体的RyR的比例。时间分辨冷冻显微镜（TRD2）快速稀释技术的发展，将使具有较低亲和力（Kd >0 -7）的RyR配体能够在适当的浓度下孵育，以获得高收率（>90%），然后在比配体解离速率慢得多的时间框架内快速稀释用于冷冻电镜。使用快速稀释技术来获得高产量的体外冷冻显微镜组装产品可能是其最强大的应用。项目3。其他实验室的工作表明，从包含功能完整的三联的骨骼肌中分离膜组分是可行的（Ikemoto etal ., 1994）。这些制剂的低分辨率显微镜显示存在完整的三联体（即肌浆网状来源的囊泡连接到横向小管来源的囊泡，在连接区域的膜间空间中可以看到ryr） （Kim等人，1990），冷冻水合三联体的冷冻电子显微镜产生的图像中可以直接看到桥接的ryr。在目前的资助期内，我们确定了冷冻水合分离三联结的第一批断层图（Wagenknecht et al.）。2002)重建了一些单独的ryanodine受体，并揭示了它们与钙螯合蛋白（在sr的ryr富集区域的基础上）引起的腔内密度致密垫的关系。冷冻水合三元组应该很容易适用于高精度免疫电镜的应用，以识别在断层扫描中分解的蛋白质成分。时间分辨断层扫描的一个潜在有趣的应用将是寻找钙螯合蛋白引起的密度变化，因为钙储备通过ryr释放Ca2+而耗尽。引用1。Budde T, Meuth S, Pape HC（2002）钙依赖性神经元钙通道失活[综述]。自然评论神经科学3:873-883。2. 黄晓明，黄晓明（2002）Ryanodine受体钙离子释放通道。物理Rev 82: 893-922。3. 张建军，张建军，张建军，张建军。（2000）Ryanodine受体适应机制。[J] .中国生物医学工程学报（英文版），32(3):873-882。4. 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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. SUPPORT: NIH R01 R40615 ¿Structure of channels in excitation-contraction coupling¿, 8/15/00-8/14/05. Terence Wagenknecht, Wadsworth Center NIH R01 HL55438 ¿Modulatory mechanisms of Ryanodine receptor adaptation¿, 3/8/96-8/31/04 Hector Valdivia, Department of Physiology, University of Wisconsin NIH HL76826 ¿Modulation of cardiac E-C coupling¿, 4/1/04-3/31/09. Hector Valdivia, Department of Physiology, University of Wisconsin ABSTRACT RyRs function as intracellular calcium release channels that are particularly prevalent in muscle where they are associated with the sarcoplasmic reticulum (SR). RyRs are homotetramers constructed from a subunit of molecular mass 565 Kda, making these receptors the largest known ion channels (Samso and Wagenknecht, 1998; Fill and Copello, 2002; Wagenknecht and Samso, 2002). The Wagenknecht laboratory has been engaged in structural studies of isolated RyRs from skeletal and heart muscle by cryoelectron microscopy and three-dimensional reconstruction for the past 12 years (Liu et al., 2004; Radermacher et al., 1992). 3D reconstructions have shown that about 80% of the mass of the RyR is located in the cytoplasm as a complex assembly of ~10 distinct domains (Radermacher et al., 1994). Besides their function in releasing calcium, which is the immediate stimulus for muscle contraction, RyRs play a key role in excitation-contraction (E-C) coupling. E-C coupling refers to the process by which neuron-induced depolarization of the muscle plasma membrane (sarcolemma) and it invaginations known as transverse (t)-tubules) leads to release of Ca2+ from the SR via the RyRs. Perhaps the most important of the sarcolemma/t-tubule components is an L-type calcium channel known as the dihydropyridine receptor, which serves as the voltage sensor in e-c coupling (Rios and Pizarro, 1991;Jones, 2002). Interactions between the dihydropyridine receptor and the cytoplasmic region of the RyR are thought to mediate communication between the plasma membranes/transverse tubules and the transmembrane, ion-conducting region of the RyR in the SR. The RyR is a ligand-gated channel. Physiologic ligand activators of the skeletal RyR include the dihydropyridine receptor (the voltage-sensor), Ca2+ at micromolar levels, and millimolar ATP. Inhibitors include Ca2+ and Mg2+ at millimolar concentrations. In a pioneering study that employed time-resolved cryo-microscopy, Unwin demonstrated that the acetylcholine receptor, also a ligand-gated ion channel, could be imaged in two of its functional states, a closed and an open state (Unwin, 1995). Time-resolved techniques were required because when the acetylcholine receptor is induced to open by physiologic agonists (in this case acetylcholine), the open state is short-lived, lasting only 50-100 ms before assuming a desensitized (inactivated) state. Until recently it was not clear whether isolated RyRs inactivated, but now several studies have uncovered rather slow transitions (time constants ~100 ms to seconds, depending on experimental conditions) from the open form to states that have been described as either ¿inactivated¿or ¿adapted¿ (Schiefer et al., 1995; Laver and Lamb, 1998;Valdivia et al., 1995) Gyorke and Fill, 1993) ; Lamb et al., 2000; Fill et al., 2000). Controversy currently exists regarding the precise nature and physiologic significance of these newly identified states of the RyR, and also regarding the disparate results that have been obtained in different laboratories. Evidence is mounting for a physiological role for inactivation of the heart isoform of the RyR to prevent uncontrolled calcium-induced calcium release (Wang et al., 2004). The mechanism of excitation-contraction (e-c) coupling is a major research problem among muscle physiologists. Ryanodine receptors play a key role in the signal-transducing events of the process. Our research will provide structural details of the e-c coupling apparatus using what are perhaps the only methods available for obtaining this information. Several muscle diseases are known to involve mutations of the ryanodine receptor, and there is evidence that RyRs play roles in heart disease and brain function as well. Our entire research program on RyRs depends on single-particle image processing methodology (TRD#3) and cryo-EM technology (TRD#2), as described in detail above. Electron tomography of frozen-hydrated subcellular structures (TRD#1) is also essential to one component of our program (Project 3 above). Project 1: Three-dimensional reconstruction of closed, open and inactivated/adapted states of skeletal and cardiac RyR Closed states of the RyR are easy to achieve by simply withholding activators. Open states will be induced by rapid addition of activators followed by freezing for cryo-EM at defined time intervals of sufficiently short duration that inactivated/adapted states will not form. The technology developed in TRD#2 is necessary to carry out the proposed experiments. There is good reason to anticipate that interesting structural changes will be observable between the closed and open states. Our own group and a group at Baylor have published cryo-EM studies of RyRs in putatively open (but possibly inactivated) states that were obtained treating the receptor with appropriate levels of Ca2+ and nucleotide Sharma et al., 2000; Serysheva et al., 1999). Signficant structural differences were detected between the two states, not only in the transmembrane regions as might be expected, but, surprisingly, also in the cytoplasmic regions at distances quite distant (>100 ¿) from the ion channel. To confirm and improve upon these findings, the RyR should be studied in the open form at several time points following addition of activators. We plan to activate RyRs by exposing them to the activator, Ca2+, by flash photolysis of caged Ca2+ (Ca2+-Dinitrophen) as has been done for electrophysiologyl experiments on RyRs (Gyorke et al., 1994). Alternatively, the microdroplet spray approach could also be used to expose RyRs on the grid to activators contained in the spray. The pre-mix approach may be preferable if we find that reaction times greater than 1 second are sufficiently short to trap the open state of RyR. The time-resolved approach may prove to be indispensable for reasons other than preventing formation of undesirable, slow-forming conformational states of the RyR. All of the cryo-EM studies on the RyR to date have been conducted on purified, detergent-solubilized material. The presence of detergent and the lack of a membrane bilayer in these preparations could lead to conclusions that are not valid for the receptor in its natural environment. We have found that at reduced levels of detergents, the closed form of the receptor remains soluble for short periods (minutes) of time (Wagenknecht et al., 1997; Wagenknecht et al., 1994). However, our initial experiments to characterize the open form of the RyR at low concentrations of detergent often showed unacceptably high amounts of apparently aggregated receptors on the EM grid. By performing these same experiments using time-resolved methods we will be able time to reduce the time of exposure to low detergent levels to less than 1 second; this should greatly reduce solubility problems, and allow even lower levels of detergent to be used. The advantages of rapid-dilution to remove detergents could be substantial and of general applicability to membrane proteins or, more generally to any macromolecule that becomes prone to aggregation upon a change in solution conditions or addition of a ligand (e.g. as was indeed found for a low pH-induced conformational state of Semliki Forest virus (Fuller et al., 1995). Eventually, we expect to be able to study the RyRs in a lipid bilayer environment where functional activities can be monitored in parallel with structural work. Some preliminary success has already been achieved in imaging RyRs reconstituted into bilayers (Liu and Wagenknecht, 1999). More generally, for macromolecular machines that are metastable, such as RyR, precautions to reduce exposure to the carbon surface of the EM grid might be beneficial to retention of native structure even in non-time-resolved experiments (Trujillo et al., 2004) As discussed above, evidence is accumulating that RyRs can exist in at least three major conformations: closed, open, and inactivated (and/or adapted). Probably there are multiple states within each of these major categories (Laver and Lamb, 1998). As our understanding of the transitions between the various states improves, it should become feasible to characterize the structure of the inactivated (or adapted) state by cryo-EM and image reconstruction. It may even prove feasible to follow the kinetics of structural transitions undergone by the receptor in response to conditions that mimic those experienced by the receptor in vivo. Project 2: 3D structure of complexes of RyR and protein ligands RyRs are known to interact directly in with several proteins in skeletal muscle to form a signal-transducing complex known as the triad junction. One goal is to use the isolated RyR as an ¿assembly platform¿ to prepare complexes containing each of these ligands. Already, we have made progress in determining the binding locations of two ligands, FK506-binding protein and calmodulin, on the surface of the RyR (Wagenknecht et al., 1994; Wagenknecht et al., 1996; Wagenknecht et al., 1997; Samso and Wagenknecht, 2002; Sharma et al. 2004 (submitted)). Of particular interest is the interaction of the RyR with the dihydropyridine receptor (Samso et al., 1999), which is thought to represent the core of the signal-tranducing apparatus of e-c coupling. Sequence-specific antibodies represent another class of ligand that is being used to probe the structure of the RyR by means of in vitro assembly and 3D cryo-microscopy. RyRs also exhibit the somewhat unusual property of binding the regulator molecule, calmodulin, in both its apo and Ca2+-bound forms (Meissner, 2002; Tang et al., 2002). Thus, calmodulin could be considered to be a resident regulatory component of the RyR; a similar role for calmodulin has been postulated for other ion channels (Budde et al., 2002). Interestingly, our structural analyses by single-particle cryo-EM have shown two discrete binding sites, separated by about 30 ¿ngstroms, for the two forms of calmodulin (Samso and Wagenknecht, 2002; Wagenknecht et al., 1997). We have hypothesized that calmodulin swithches between these two sites without dissociating from the ryanodine receptor. We propose to test this hypothesis using time-resolved cryo-EM to induce the putative switch of binding sites in the absence of exogenous calmodulin. Since the equilibrium constants for ligands vary widely, it is frequently desirable to perform the assembly reactions using RyR and ligand at concentrations that are higher than optimal for EM (~10-7 M for RyR) so as to maximize the fraction of RyR that has bound ligand. The development of rapid dilution techniques of time-resolved cryo-microscopy (TRD2) will allow RyR ligands with lower affintities (Kd>10-7) to be incubated at concentrations appropriate for obtaining high yields (>90%), and then to be rapidly diluted for cryo-EM within a time frame that is much slower than the rate of ligand dissociation. The use of the rapid dilution technique for obtaining high yields of in vitro assembly products for cryo-microscopy may well prove to be its most powerful application. Project 3. Cryo-tomography of the skeletal muscle triad junction Work in other laboratories has shown that it is feasible to isolate membrane fractions from skeletal muscles that contain functionally intact triads (Ikemoto et al., 1994). Low resolution microscopy on these preparations shows the presence of intact triads (i.e. sarcoplasmic reticulum-derived vesicles joined to transverse-tubule-derived vesicles with RyRs visible in the intermembrane space in the junctional regions) (Kim et al., 1990), and cryo-EM of frozen-hydrated triads yields images in which the bridging RyRs are directly visible. During the current grant period we determined the first tomograms from frozen-hydrated isolated triad junctions (Wagenknecht et al., 2002) The reconstruction resolved some of the individual ryanodine receptors and revealed their relationship to the dense mat of luminal density attributed to calsequestrin that underlies the RyR-enriched regions of the SR. 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