Molecular Mechanisms of Synaptic SK2 Channel Function

突触SK2通道功能的分子机制

• 批准号: 8592945
• 负责人: Melissa Hernandez
• 金额: $ 5.22万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8592945
• 关键词: Affect; Amino Acids; Apamin; Area; Behavior; Brain; Calcium; Cells; Co-Immunoprecipitations; DLG1 gene; Data; Dendritic Spines; Disabled Persons; Disease; Electroporation; Endocytosis; Genes; Hippocampus (Brain); Injection of therapeutic agent; Ions; Ischemia; Knockout Mice; Laboratories; Learning; Ligands; MPP2 gene; Maps; Measures; Mediating; Membrane Potentials; Memory; Modification; Molecular; Mus; N-terminal; Neurons; Palmitoylated Membrane Protein 2; Plasmids; Play; Postsynaptic Membrane; Protein Binding Domain; Protein Isoforms; Proteins; Proteomics; Recombinant adeno-associated virus (rAAV); Regulation; Role; SH3 Domains; Scaffolding Protein; Scientist; Sequence Analysis; Site; Solid; Synapses; Synaptic Membranes; Synaptic Potentials; Synaptic Transmission; Synaptic plasticity; Techniques; Testing; Training; Transgenic Mice; Vertebral column; Wild Type Mouse; calcium-activated potassium channel small-conductance; career; hippocampal pyramidal neuron; in utero; in vivo; insight; knock-down; memory encoding; mutant; neuronal excitability; novel; protein protein interaction; public health relevance; research study; response; small hairpin RNA; trafficking

DESCRIPTION (provided by applicant): Apamin-sensitive small conductance calcium activated potassium channels (SK channels) are widely expressed in the brain. Among the three SK channel genes (SK1, SK2 and SK3) SK2 is the most highly expressed. In hippocampal CA1 pyramidal neurons SK2 is localized to the post-synaptic membrane where it functions to reduce synaptic responses and influence learning and memory. The SK2 gene encodes two isoforms of the protein, a long form (SK2-L) and a short form (SK2-S) that is entirely contained within the long form. The unique amino acids on the N-terminal of SK2-L are necessary for synaptic expression of SK2 channels. However, the subunit composition of synaptic SK2-containing channels and the mechanisms that govern synaptic targeting are unknown. To determine whether synaptic SK2 channels are homomeric channels composed of the SK2-L isoform or are heteromeric channels composed of both SK2-S and SK2-L, a recombinant adeno-associated virus (rAAV) that expresses SK2-L and GFP will be injected into area CA1 of the hippocampus of SK2 null mice. Synaptic SK2 channel activity will be measured in fluorescent CA1 neurons by analyzing the amplitudes of evoked EPSPs in the absence and presence of apamin. Sequence analysis of the SK2-L N-terminal domain reveals a candidate SH3 protein-protein interaction motif. To test whether this motif is necessary for synaptic expression of SK2 channels, a rAAV that expresses SK2-L lacking the SH3 domain will be expressed in CA1 pyramidal neurons of mice that lack SK2-L, and synaptically evoked EPSPs will be assessed for apamin sensitivity. Recent proteomic experiments have identified two MAGUK scaffolding proteins, MPP2 and SAP-97, as potential SK2 interacting proteins. To determine whether MPP2 and SAP-97 interact with SK2 channels and are required for synaptic SK channel expression, MPP2 and SAP-97 will first be expressed in HEK293 cells along with SK2-S and SK2-L and interactions will be determined by co- immunoprecipitations. Next, MPP2 and SAP-97 expression will be knocked down using shRNAs introduced into CA1 pyramidal neurons by in utero electroporation, and the effects of apamin on synaptically evoked EPSPs will be measured. These experiments will provide important information regarding the composition of synaptic SK2 channels and the protein network responsible for their synaptic localization and function, and they will provide novel insights into the molecular mechanisms underlying learning and memory.

描述（由申请人提供）：阿帕明敏感性小电导钙激活钾通道（SK通道）在大脑中广泛表达。在三种SK通道基因（SK 1、SK 2和SK 3）中，SK 2是最高表达的。在海马CA 1区锥体神经元中，SK 2定位于突触后膜，在那里它的功能是减少突触反应并影响学习和记忆。SK2基因编码蛋白质的两种同种型，长型（SK2-L）和完全包含在长型内的短型（SK2-S）。SK2-L的N-末端上的独特氨基酸对于SK2通道的突触表达是必需的。然而，突触SK2通道的亚基组成和控制突触靶向的机制尚不清楚。为了确定突触SK 2通道是由SK 2-L同种型组成的同源通道还是由SK 2-S和SK 2-L组成的异源通道，将表达SK 2-L和GFP的重组腺相关病毒（rAAV）注射到SK 2缺失小鼠海马的CA 1区。在荧光CA 1神经元中，通过分析在缺乏和存在蜂毒肽的情况下诱发的EPSP的幅度来测量突触SK 2通道活性。SK2-L N-末端结构域的序列分析揭示了一个候选的SH 3蛋白-蛋白相互作用基序。为了测试该基序是否是SK 2通道的突触表达所必需的，表达缺乏SH 3结构域的SK 2-L的rAAV将在缺乏SK 2-L的小鼠的CA 1锥体神经元中表达，并且将评估突触诱发的EPSP的apamin敏感性。最近的蛋白质组学实验已经确定了两个MAGUK支架蛋白，MPP 2和SAP-97，作为潜在的SK2相互作用蛋白。为了确定MPP 2和SAP-97是否与SK 2通道相互作用并且是突触SK通道表达所需的，MPP 2和SAP-97将首先在HEK 293细胞中沿着SK 2-S和SK 2-L表达，并且相互作用将通过共免疫沉淀来确定。接下来，将使用通过子宫内电穿孔引入CA 1锥体神经元的shRNA敲低MPP 2和SAP-97表达，并测量蜂毒肽对突触诱发的EPSP的影响。这些实验将提供有关突触SK2通道的组成和负责其突触定位和功能的蛋白质网络的重要信息，并将为学习和记忆的分子机制提供新的见解。