Molecular Mechanisms of Synaptic SK2 Channel Function

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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.