This is a new application to study the role of brain spectrin in synaptic transmission. We will determine whether spectrin plays a direct role in depolarization-induced release of neurotransmitter from the presynaptic terminal. The specific hypothesis to be tested is that spectrin is an essential docking protein and Ca2+ sensor protein in the Ca2+-regulated vesicle fusion and neurotransmitter release from 50 nm diameter spherical synaptic vesicles.
The aims are to (1) Determine the rate of spectrin synthesis, turnover, and assembly in hippocampal neurons in the resting and depolarized state (+/-Ca2+). (2) Determine whether the proposed binding site for synapsin I on brain beta spectrin (res. 207-445 of beta(Sp)IIsigma1) can bind synaptic vesicles, and define the smallest functional subdomain and essential amino acids for attachment. Also determine whether synaptic vesicle binding displaces the attachment of spectrin and f-actin as proposed in the """"""""casting the line"""""""" hypothesis. Finally determine whether the interaction of synaptic vesicles with brain beta spectrin is directly regulated by free Ca2+ levels. (3) Determine whether microinjection of beta spectrin recombinant peptides and synthetic peptides which contain the synaptic vesicle binding domain (and FAB fragments against these synthetic peptides) alters neurotransmitter release from paired hippocampal neurons in culture. Peptides and antibodies will be injected into the presynaptic neurons and we will use patch clamp techniques and quantal analysis to study changes in transmitter release from small spherical synaptic vesicles. Dr. Goodman and his collaborators are in a unique position to successfully conduct these studies because they have developed the necessary spectrin DNA constructs, recombinant peptides, synthetic peptides and antibodies to perform the proposed studies. They also have the requisite expertise in neuronal cell culture, molecular biology, protein chemistry, electrophysiology and a long history of working with the spectrin molecule.
