The 25 kDa synaptosome-associated protein (SNAP-25) is a key component of the plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) which regulates neurotransmitter release at presynaptic terminals. Resolution of the crystal structure of a SNARE complex has shown that SNAP-25 interacts with syntaxin primarily via an amino terminal site and VAMP/synaptobrevin via a carboxyl terminal site (Sutton et al. 1998). Truncation of the SNAP-25 carboxyl terminal (SNAP-25-CT) inhibits SNAP-25 binding to VAMP in vitro (Chapman et al. 1994). SNAP-25 also binds to the subtypes of voltage-dependent Ca2+ channels implicated in presynaptic neuro-transmitter release (Sheng et al. 1996).
To investigate the physiological consequences of SNAP-25-CT on the disassembly of preformed SNARE complexes, we examined the role of SNAP-25-CT in synaptic transmission at cholinergic synapses formed between rat superior cervical ganglion neurons (SCGNs) in culture. SCGNs were prepared from 7-day-old rats using standard, humane, procedures. Following a stable period of recording evoked excitatory postsynaptic potentials (EPSPs), a synthetic peptide of the carboxyl terminal 13 amino acids of SNAP-25 (aa 194-206) was diffused (at t = 0 for 2-3 min) into the presynaptic neuron from a suction pipette containing a 1:1 solution of 2.5 mM peptide and 5 % Fast Green FCF.
EPSP amplitude gradually decreased after peptide injection. At a stimulation frequency of 0.05 Hz, the maximum EPSP decrease was observed at 40-50 min; the reduction in EPSPs was 20 ± 12 % (mean ± S.E.M.) 50 min after injection (P ≤ 0.05, Student’s t test). No obvious change in the time course of the EPSPs was observed with SNAP-25 peptide injection. A control peptide containing 26 amino acids of SNAP-25 (aa 146-171), which does not interact with presynaptic SNAREs, showed no significant effect on synaptic transmission (0.05 Hz, n = 4). We next examined the effects of stimulation frequency. The maximum EPSP decrease was observed at 40-50 min for frequencies of 0.1 and 0.2 Hz; the reduction in EPSPs was 24 ± 6.4 % (0.1 Hz, at 40 min, n = 6) and 31 ± 9.3 % (0.2 Hz, at 50 min, n = 5).
Although these data show a correlation between frequency and inhibition of mean EPSPs, a similar trend was seen in control conditions, consistent with the SNAP-25 effect not being dependent on synaptic activity. However, it is clear that the SNAP-25-CT peptide inhibits excitatory synaptic transmission in SCGNs, possibly by acting as a competitor with SNAP-25 for SNARE complex assembly, implicating the SNAP-25 C terminal as an important molecular determinant of SNARE function.
This work was supported by The Wellcome Trust and The Japanese Ministry of Education, Science, Sports and Culture.