A long term objective of this laboratory is to understand the structure, location and functions of spectrin isoforms within mammalian brain. In the last grant period we identified three brain spectrin isoforms: [1] brain spectrin(240/235) which is found in axons and neuronal soma, [2] brain spectrin(240/235E) which is an erythroid-like isoform found in neuronal soma and dendrites as well as oligodendrocytes, and [3] brain spectrin(240/235A) which is located exclusively in astrocyte soma and processes. We have also identified two beta spectrin mRNAs of 9 Kb and 11 Kb size in mouse brain. Through cloning and sequencing of cDNA, we have obtained greater than 50% of the deduced sequence of the 9 Kb mRNA (which we believe to be the beta subunit of 240/235E). During the next grant period, we will complete the sequence of the 9 Kb and 11 Kb transcripts; identify the spectrin isoform specificity for binding of synaptic vesicles (synapsin I) and ankyrin, utilizing bacterially expressed segments of the 9 Kb and 11 Kb beta spectrins; identify the minimal binding sequence and essential amino acids for binding synaptic vesicles and ankyrin by deletion mutagenesis and site specific mutagenesis of beta spectrin cDNA, and determine whether the interaction of synaptic vesicles with brain spectrin via synapsin I is regulated by Ca2+ and calmodulin (new specific aim). As small spherical synaptic vesicles appear to be attached to the active zone of the presynaptic plasma membrane by spectrin, the newly added specific aim is essential to our understanding of Ca2+ regulated exocytosis of neurotransmitter within the nerve terminal. The Principal Investigator and his coworkers published 60 scientific articles and abstracts on brain spectrin and related molecules during the previous grant period (1986- 1991).
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