Content area
Full Text
ABSTRACT The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCI solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K^sup +^ over Cl^sup -^ (P^sub kappa+^/P^sub Cl^- > 8) and preferred K^sup +^ over Li^sup +^, Na^sup +^, Rb^sup +^, Cs^sup +^, or choline^sup +^. The synaptophysin channel is impermeable to Ca^sup 2+^, which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.
INTRODUCTION
Synaptic transmission involves the regulated release of transmitter molecules to the synaptic cleft, where they interact with postsynaptic receptors, which subsequently transduce the information. Synaptic vesicles accumulate neurotransmitters and release them during exocytosis to the synaptic cleft. In examining the exocytosis process many proteins from the synaptic vesicles have been studied, and several conserved family proteins in synaptic vesicles were shown (De Camilli and Jahn, 1990). One such family is the synaptophysin family, accounting for ~6-8% of the total synaptic vesicle protein (Jahn et al., 1985). Synaptophysin is a hexameric protein consisting of 38 kDa monomers and is a major integral membrane protein of transmitter-containing vesicles found in neurons and endocrine cells (Wiedenmann and Franke, 1985). Based on amino acid sequences deduced from rat and human cDNA and genomic clones, the predicted synaptophysin structure consists of four transmembrane domains, two intravesicular domains (Sidhof et al., 1987; Buckley et al., 1987; Leube et al., 1987), and a cytoplasmic carboxyl tail containing a unique Ca^sup 2+^-binding repeat (Rehm et al., 1986). Based on the predicated structure, it was suggested that synaptophysin forms a channel in the synaptic...