A novel SNAP25-caveolin complex correlates with the onset of persistent synaptic potentiation

We have identified synaptic protein complexes in intact rat hippocampal sli ces using the rapid chemical cross-linking reagent paraformaldehyde. Cellul ar proteins were rapidly cross-linked, solubilized, separated electrophoret ically by SDS-PAGE, and then identified immunologically. Multiple complexes containing syntaxin, the synaptosomal-associated protein of 25 kDa (SNAP25 ), and vesicle-associated membrane protein (VAMP) were observed to coexist in a single hippocampal slice including a 100 kDa cross-linked protein comp lex that exhibited the same electrophoretic migration as a member of the pr eviously identified SDS-resistant soluble N-ethylmaleimide-sensitive fusion attachment protein receptor "core" of the 20 S complex. A VAMP-synaptophys in complex, reported previously in vitro, was also observed in the hippocam pal slices. This study links biochemical and physiological studies involvin g presynaptic proteins implicated in secretion and confirms that these prot eins that have been studied extensively previously in the presence of deter gent do form "bona fide" cellular complexes. Importantly, we have also dete cted additional novel protein complexes that do not correspond to complexes identified previously in vitro. After the induction of persistent synaptic potentiation, an abundant 40 kDa SNAP25-caveolin1 complex was observed. Th e SNAP25-caveolin1 complex was not abundant in control slices and, therefor e, represents the first demonstration of a reorganization of protein comple xes in intact hippocampal slices during the induction of synaptic potentiat ion. The interaction between caveolin1 and SNAP25 was confirmed biochemical ly by demonstration of the association of caveolin with recombinant-immobil ized SNAP25 and by the coimmunoprecipitation of SNAP25 using caveolin-speci fic antisera. Caveolin1, like SNAP25, was observed to be abundant in isolat ed hippocampal nerve terminals (synaptosomes). Immunofluorescent studies de monstrated that both SNAP25 and caveolin1 are present in neurons and coloca lize in axonal varicosities. These results suggest that a shortlasting SNAP 25-caveolin interaction may be involved in the early phase of synaptic pote ntiation.