Molecular Mechanisms of Synaptic SK2 Channel Function

描述(申请人提供)：阿帕明敏感的小电导钙激活钾通道(SK通道)在大脑中广泛表达。在SK通道的三个基因(SK1、SK2和SK3)中，SK2的表达最高。在海马CA1区，锥体神经元SK2定位于突触后膜，其功能是减少突触反应，影响学习和记忆。SK2基因编码蛋白质的两种异构体，一种是长的(SK2-L)，另一种是完全包含在长的蛋白质中的短的(SK2-S)。SK2-L N端的独特氨基酸是SK2通道突触表达所必需的。然而，突触SK2通道的亚单位组成和支配突触靶向的机制尚不清楚。将表达SK2-L和绿色荧光蛋白的重组腺相关病毒注射到SK2基因缺失小鼠的海马区，以确定突触通道是由SK2-L亚型组成的同质通道还是由SK2-S和SK2-L共同组成的异构体通道。通过分析在没有和存在阿帕明的情况下诱发的EPSP的幅度，将在荧光CA1神经元中测量突触SK2通道的活动。对SK2-L N-末端结构域的序列分析表明，可能存在一个SH3蛋白-蛋白质相互作用基序。为了测试这个基序是否对SK2通道的突触表达是必需的，我们将在缺乏SK2-L结构域的小鼠的CA1区锥体神经元中表达一个表达SK2-L结构域的重组腺相关病毒，并对突触诱发的EPSP进行凋亡素敏感性评估。最近的蛋白质组学实验已经确定两个Maguk支架蛋白，MPP2和SAP-97，是潜在的SK2相互作用蛋白。为了确定MPP2和SAP-97是否与SK2通道相互作用以及突触SK通道的表达是否需要，首先将MPP2和SAP-97与SK2-S和SK2-L一起在HEK293细胞中表达，并通过免疫共沉淀法确定相互作用。接下来，通过宫内电穿孔将shRNAs导入CA1区锥体神经元，下调MPP2和SAP-97的表达，并观察apamin对突触诱发的EPSP的影响。这些实验将提供有关突触SK2通道的组成以及负责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。

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.