Molecular Mechanisms of Synaptic SK2 Channel Function

描述（申请人提供）：阿帕胺敏感的小电导钙活化钾通道（SK通道）在大脑中广泛表达。在三个SK通道基因（SK1、SK2和SK3）中，SK2表达量最高。在海马CA1锥体神经元中，SK2定位于突触后膜，其功能是减少突触反应并影响学习和记忆。SK2基因编码该蛋白的两种亚型，一种是长亚型（SK2- l），另一种是短亚型（SK2- s），后者完全包含在长亚型中。SK2- l n端独特的氨基酸是SK2通道突触表达所必需的。然而，突触含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SK2通道是由SK2- l异构体组成的同质通道还是由SK2- s和SK2- l组成的异质通道，将表达SK2- l和GFP的重组腺相关病毒（rAAV）注射到SK2缺失小鼠海马CA1区。在CA1荧光神经元中，突触SK2通道的活性将通过分析在缺乏和存在维生素a的情况下诱发epsp的振幅来测量。SK2-L n端结构域的序列分析揭示了一个候选SH3蛋白-蛋白相互作用基序。为了验证该基元是否为SK2通道的突触表达所必需，我们将在缺乏SK2- l的小鼠的CA1锥体神经元中表达缺乏SH3结构域的SK2- l的rAAV，并评估突触诱发的epsp对apamin的敏感性。最近的蛋白质组学实验已经确定了两个MAGUK支架蛋白，MPP2和SAP-97，作为潜在的SK2相互作用蛋白。为了确定MPP2和SAP-97是否与SK2通道相互作用以及是否为突触SK通道表达所必需，我们首先将MPP2和SAP-97与SK2- s和SK2- l一起在HEK293细胞中表达，并通过共免疫沉淀来确定相互作用。接下来，通过子宫内电穿孔将shrna引入CA1锥体神经元，敲低MPP2和SAP-97的表达，并测量apamin对突触诱发epsp的影响。这些实验将提供突触SK2通道的组成和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.