Molecular and kinetic gating mechanisms of SK channels

SK通道的分子和动力学门控机制

• 批准号: 7275559
• 负责人: Xixi Chen
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7275559
• 关键词: Action Potentials; Address; Affect; Amino Acids; Ammonium; Behavior; Binding; Biophysics; Brain; Brain region; C-terminal; Calcium; Calcium Channel; Calcium Channel Binding; Calcium Signaling; Calcium-Activated Potassium Channel; Calmodulin; Cyclic Nucleotides; Cysteine; Data; Dependence; Dimerization; Dose; Drug Design; Family; Fire - disasters; Hand; Helix (Snails); Hydrophobic Interactions; Hydrophobicity; Ion Channel; Kinetics; Knowledge; Lead; Link; Lobe; Location; Mediating; Modification; Molecular; Mutate; Mutation; Neuraxis; Neurologic; Neurons; Numbers; Parkinson Disease; Pattern; Probability; Property; Protocols documentation; Range; Roentgen Rays; Schizophrenia; Series; Shapes; Side; Signal Transduction; Site; Structure; Synapses; Synaptic plasticity; Testing; Therapeutic; Transmembrane Domain; base; calcium-activated potassium channel small-conductance; crosslink; cyclic-nucleotide gated ion channels; design; dimer; ear helix; extracellular; large-conductance calcium-activated potassium channels; motor control; nervous system disorder; research study; size; voltage

DESCRIPTION (provided by applicant): The overall objective of this application is to understand the molecular and kinetic gating mechanisms of small-conductance, calcium-activated potassium channels (SK channels). The SK channels participate in calcium signaling, electrical signaling and synaptic plasticity in the brain. They are potential molecular substrates for treating diseases of the nervous system such as Parkinson's disease and Schizophrenia. Understanding the gating mechanisms of the SK channels will provide specific knowledge about how these molecules can be manipulated in a therapeutic setting. I propose experiments for two specific aims. The first aim is to probe the inner pore properties and gate location of SK channels with intracellular blockers. I will characterize in detail dose-dependent activation of SK channels by calcium and dose- and voltage-dependent blockade by a series of quaternary ammonium blockers. Comparisons between blockers of different sizes and hydrophobicity will provide information about the properties and size of the SK channel pore. Correlation between doses of calcium (or open probability in single-channel recordings) and block efficiency will reveal possible links between the state of the channel (open or closed) and accessibility of the pore to the blockers. State-dependence of such accessibility would suggest an intracellular gate location. On the other hand, if the accessibility of the pore is state-independent, this would suggest a gate location above the blocking site. The second aim is to test the hypothesis that dimerization of the C termini of SK channels by calcium-bound calmodulin stabilizes the open state. I plan to make mutations of the SK channel with predictable results based on structural data. One set of mutations will be cysteine substitutions for residues with short inter-subunit distance in the dimmer structure. If the dimer structure is associated with an open channel, cross-linking these residues should lock the channel in the open state. Another set of mutations will target the hydrophobic interactions between the SK channel and the N-lobe of calmodulin in the dimer structure. Hydrophobic residues in the SK channel will be mutated to polar residues, which are expected to weaken the interaction and destabilize the dimer. This should lead to a reduced efficiency of calcium activation of the channel.

描述（由申请人提供）：本应用的总体目的是了解小导，钙激活的钾通道（SK通道）的分子和动力学门控机制。 SK通道参与大脑中的钙信号传导，电信号传导和突触可塑性。它们是治疗神经系统疾病（例如帕金森氏病和精神分裂症）的潜在分子底物。了解SK通道的门控机制将提供有关在治疗环境中如何操纵这些分子的特定知识。 我提出了两个特定目标的实验。第一个目的是探测具有细胞内阻滞剂SK通道的内部孔特性和门位置。我将通过一系列第四纪铵阻滞剂来详细表征SK通道对SK通道的剂量依赖性激活。不同大小和疏水性的阻滞剂之间的比较将提供有关SK通道孔的性质和大小的信息。钙剂量（或单渠道记录中的开放概率）与阻滞效率之间的相关性将揭示通道状态（开放或封闭）之间的可能链接以及孔与阻滞剂的可及性。这种可及性的状态依赖性将表明细胞内门位置。另一方面，如果孔的可访问性是与州无关的，这将表明阻塞位点上方的门位置。第二个目的是检验以下假设：通过钙结合的钙调蛋白稳定开放状态，通过钙结合的钙调蛋白二聚化。我计划根据结构数据进行可预测结果的SK通道突变。一组突变将是对昏暗者结构中产生距离短的残基的半胱氨酸取代。如果二聚体结构与开放通道关联，则将这些残基的交联应以开放状态锁定通道。另一组突变将针对二聚体结构中SK通道和钙调蛋白的N-叶片之间的疏水相互作用。 SK通道中的疏水残基将被突变为极性残基，这些残基有望削弱相互作用并破坏二聚体的稳定。这应该导致通道的钙激活效率降低。