Structural rearrangements in SK channels during CaM-mediated gating

描述(由申请人提供)： 钙调素(CaM)是细胞内钙离子的主要感受器，参与钙离子信号的分泌、运动、转录和增殖等多种过程。钙调素中钙离子与EF-Hand结构域的结合启动了构象变化，促进了钙调素与许多靶蛋白的相互作用，包括离子通道，即引导离子穿过细胞膜的门控小孔。钙调素调节钙离子通透通道和控制钙离子进入的电化学驱动力的通道的活性，通过细胞内[钙离子]的变化为钙调素的活性提供关键的反馈机制。钙离子激活的小电导钾(SK)通道的开放依赖于CaM与SK四聚体每个成孔亚基中的结合结构域(CaMBD)的构造性结合。与其他离子通道一样，CaM介导的门控的结构特征是从CaM与包括CaMBD在内的通道片段结合的生化和结晶学研究中获得的。围绕这些研究的一个中心问题是，CaM/CaMBD结构是否代表完整通道中的功能状态。为了解决这个问题，我们将内向外膜片钳记录与光学方法相结合，以监控正常SK通道中的门控动态。我们将小分子有机染料定点连接到CaM上，并利用最近的一项发现将这些标记的CaM整合到SK通道中，该发现表明SK通道不能与N端EF-Hand中带有突变的共表达CAM形成稳定的相互作用，从而允许在外源应用CaM的情况下挽救切除斑块中的通道活动。在具体目标1中，我们将使用在同一SK四聚体中共同组装的荧光凸轮之间的Fvrster共振能量转移(FRET)来确定打开和关闭SK通道中的凸轮的结构构型。由于供体荧光标记的凸轮(D-CAM)和受体荧光标记的凸轮(A-CAM)之间的能量传递依赖于D-CAM和A-CAM之间的距离和方向，FRET报告了凸轮在通道复合体中的空间排列。在具体目标2中，我们将通过监测近端色氨酸进入SK通道对连接到CaM上的Bimane的猝灭来确定CaM/SK接触的位置和状态依赖性。由于CaM和SK残基的定点标记是基于CaM/CaMBD X射线结构中紧密相对的残基，这些残基之间的猝灭提供了CaM/CaMBD和SK通道结构的直接比较。所开发的技术为探索CaM与其离子通道靶标之间的功能相互作用提供了一种通用的方法。 公共卫生相关性： 小电导钙激活钾通道(SK)因其在神经元、平滑肌和心脏生理学中的重要作用而被认为是有吸引力的治疗靶点。对SK通道进行详细的功能和结构研究，对于开发高效、选择性的SK功能调节剂是至关重要的。本方案中描述的实验将使用一种可以扩展到其他离子通道的结构研究的方法来询问功能通道中的SK结构。

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.