Munc 18a binding to syntaxin 1A and 1B isoforms defines its localization at the plasma membrane and blocks SNARE assembly in a three-hybrid system assay

Syntaxin 1 and synaptobrevin/VAMP play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxins 1A and 1B and synaptobrevin/VAMPs 1 and 2, have been found in nerve endings. Morphological and biochemical studies have revealed a characteristic colocalization and selective interactions patterns of syntaxin 1 and synaptobrevin/VAMP isoforms in nervous and endocrine systems. Moreover, studies in vitro with recombinant proteins have shown characteristic interaction patterns for each syntaxin 1-synaptobrevin/VAMP pair. The cytosolic protein Munc-18a modulates neurotransmission by inhibiting the binding of synaptobrevin/VAMP and SNAP-25 to syntaxin 1A. In the present study, several binding assays were used to demonstrate that Munc-18a significantly binds both isoforms of syntaxin I (syntaxins 1A and 1B). Moreover, the coexpression of Munc-18a and syntaxin 1A or syntaxin I B in 29.3 T cells revealed syntaxin 1-dependent localization of Munc-18a in the plasma membrane. By using the three-hybrid system, we showed the inhibitory role of Munc-18a in the formation of syntaxin 1-synaptobrevin/VAMP complexes regardless of the isoforms. Since Munc-18a can bind both isoforms of syntaxin 1, the present data suggest that this protein is a general modulator of the formation of different SNARE complexes in the nerve endings.