The long term goal is to understand the mechanisms by which synaptic components are assembled into a fully functional synapse. In this project it is proposed to carry out molecular, genetic, and physiological analyses of a group of proteins, MAGUKs, which are involved in the clustering and targeting of channels and receptors to their synaptic locations. This family of proteins is highly conserved between mammals and flies, from both structural and functional perspectives. The investigators propose to perform a deletion analysis of a fly MAGUK member (DLG) to determine the exact domains with which it targets ShakerK+ channels to glutamatergic neuromuscular synapses. Analysis of the DLG mutant proteins generated in this project will be performed in an in vivo model system, the fly neuromuscular junction, using targeted expression of these mutant proteins to postsynaptic muscle cells. They will also use the yeast two-hybrid system, coimmunoprecipitation, and genetic experiments to identify the proteins that directly interact with this DLG targeting site. This investigation will allow them to ascertain how ion channels are anchored to synapses, and to uncover additional functions of the proteins identified. Studies in mammals have identified a novel protein, GKAP, which interacts with the guanylate kinase domain of the vertebrate MAGUK, PSD-95. However, the function of this protein is unknown. The investigators will clone the Drosophila GKAP homolog to initiate a genetic analysis of its synaptic function. Finally, they propose to use anatomical and electrophysiological techniques to determine the ontogeny of Shaker K+ channel clustering, whether glutamate receptor clustering is also dependent on DLG, and if the clustering of Shaker channels is dependent on motor neuron innervation. These studies will contribute in a fundamental way to our knowledge of the mechanisms of synapse formation. This knowledge will be essential to understand the etiology of many neural disorders, as well as to design strategies to repair damage after stroke, trauma, or disease.
