Structural rearrangements in SK channels during CaM-mediated gating

CaM 介导的门控过程中 SK 通道的结构重排

• 批准号: 8003397
• 负责人: Riina Maie Luik
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003397
• 关键词: Address; Binding; Biochemical; Calcium; Calmodulin; Cardiac; Cell membrane; Complex; DNA Sequence Rearrangement; Dependence; Development; Dyes; EF Hand Motifs; EF-Hand Domain; Energy Transfer; Feedback; Genetic Transcription; Glean; Ion Channel; Ion Channel Gating; Ions; Label; Location; Mediating; Methodology; Monitor; Mutation; N-terminal; Neurons; Optical Methods; Physiology; Proteins; Reporting; Role; SK potassium channel; Site; Smooth Muscle; Structure; Techniques; Tryptophan; base; cell motility; computerized data processing; driving force; fluorophore; patch clamp; public health relevance; research study; sensor; therapeutic target

DESCRIPTION (provided by applicant): Calmodulin (CaM) acts as the primary cytosolic [Ca2+] sensor for Ca2+ signaling processes as diverse as secretion, motility, transcription, and proliferation. The binding of Ca2+ to EF-hand domains within CaM initiates a conformational change that promotes the interaction of CaM with a number of target proteins, including ion channels, gated pores that conduct ions across cell membranes. CaM modulates the activity of both Ca2+-permeable channels and channels that control the electrochemical driving force for Ca2+ entry, providing a critical feedback mechanism for CaM activity through changes in intracellular [Ca2+]. The Ca2+-dependent opening of small conductance, Ca2+-activated potassium (SK) channels is conferred by the constitutive association of CaM with a binding domain (CaMBD) within each pore- forming subunit of the SK tetramer. As with other ion channels, the structural features of CaM-mediated gating have been gleaned from biochemical and crystallographic studies of CaM bound to a channel fragment including the CaMBD.Acentral question surrounding these studies is whether CaM/CaMBD structures represent functional states in intact channels. To address this issue, we have combined inside-out patch-clamp recordings with optical methods to monitor gating dynamics in functional SK channels. We have site-specifically attached small organic dyes to CaM, and we have incorporated these labeled CaMs into SK channels by taking advantage of a recent finding that SK channels cannot form stable interactions with co-expressed CaMs bearing mutations in the N-terminal EF-hands, allowing the rescue of channel activity in excised patches with exogenously applied CaM. In Specific Aim 1, we will use Fvrster resonance energy transfer (FRET) between fluorescent CaMs co-assembled within the same SK tetramer to determine the structural configuration of CaMs in open and closed SK channels. Because energy transfer between CaMs labeled with donor fluorophores (D-CaMs) and CaMs labeled with acceptor fluorophores (A-CaMs) depends on the distance and orientation between D-CaMs and A-CaMs, FRET reports the spatial arrangement of CaMs within the channel complex. In Specific Aim 2, we will determine the location and state-dependence of CaM/SK contacts by monitoring the quenching of bimane attached to CaM by proximal tryptophans introduced into SK channels. Because the site-specific labeling of CaM and SK residues is based upon closely apposed residues in the CaM/CaMBD x-ray structure, quenching between these residues provides a direct comparison of the CaM/CaMBD and SK channel structures. The techniques developed here provide a general methodology for probing the functional interactions between CaM and its ion channel targets. PUBLIC HEALTH RELEVANCE: Small conductance calcium-activated potassium (SK) channels are regarded as attractive therapeutic targets because of their essential roles in neuronal, smooth muscle, and cardiac physiology. Detailed functional and structural studies of SK channels are critical for the development of potent and selective modulators of SK function. The experiments described within this proposal will interrogate SK structure in functional channels using an approach that can be extended to structural studies in other ion channels.

描述（由申请人提供）： 钙调素（CaM）作为主要的胞质[Ca 2 +]传感器的Ca 2+信号转导过程，如分泌，运动，转录和增殖。Ca 2+与CaM内EF-手结构域的结合引发构象变化，其促进CaM与许多靶蛋白的相互作用，包括离子通道、将离子传导穿过细胞膜的门控孔。CaM调节Ca 2+渗透性通道和控制Ca 2+进入的电化学驱动力的通道的活性，通过细胞内[Ca 2 +]的变化为CaM活性提供关键的反馈机制。Ca 2+依赖性的小电导、Ca 2+激活的钾（SK）通道的开放由CaM与SK四聚体的每个成孔亚基内的结合结构域（CaMBD）的组成性缔合赋予。与其他离子通道一样，钙调素介导的门控的结构特征已经从钙调素结合到通道片段（包括CaMBD）的生物化学和晶体学研究中收集到。围绕这些研究的一个中心问题是，钙调素/CaMBD结构是否代表完整通道的功能状态。为了解决这个问题，我们结合了由内而外膜片钳记录与光学方法来监测功能SK通道的门控动力学。我们有网站特异性连接的小有机染料钙调素，我们已纳入这些标记的钙调素到SK通道，利用最近的发现，SK通道不能形成稳定的相互作用与共表达的钙调素轴承突变的N-末端EF-手，允许救援的通道活动切除补丁外源性应用钙调素。在具体目标1中，我们将使用荧光钙调素之间的Fvrster共振能量转移（FRET）共组装在同一SK四聚体，以确定钙调素在开放和封闭SK通道的结构配置。由于供体荧光团标记的钙调素（D-CaMs）和受体荧光团标记的钙调素（A-CaMs）之间的能量转移取决于D-CaMs和A-CaMs之间的距离和取向，FRET报告了通道复合物内钙调素的空间排列。在具体目标2中，我们将确定的位置和状态依赖性的钙调素/SK接触，通过监测淬火的bimane连接到钙调素的近端的双金属引入SK通道。由于CaM和SK残基的位点特异性标记是基于CaM/CaMBD X射线结构中紧密并列的残基，因此这些残基之间的淬灭提供了CaM/CaMBD和SK通道结构的直接比较。这里开发的技术提供了一个通用的方法来探测钙调素和它的离子通道目标之间的功能相互作用。 公共卫生相关性： 小电导钙激活钾（SK）通道被认为是有吸引力的治疗目标，因为它们在神经元，平滑肌和心脏生理学中的重要作用。详细的SK通道的功能和结构的研究是至关重要的，有效的和选择性的SK功能调节剂的发展。本提案中描述的实验将使用可以扩展到其他离子通道中的结构研究的方法来询问功能通道中的SK结构。