In a joint effort, the groups of Heinrich Betz from the
Max Planck Institute for Brain Research (Frankfurt, Germany)
and George Augustine from the Duke University (Durham, NC)
have tested the hypothesis that NSF, a ubiquitous protein
required for a variety of general membrane fusion and
secretion events in eukaryotic cells, is also involved in
neurotransmitter release at synapses. Their results,
appearing in the February 20 issue of SCIENCE, provide the
first hints at molecular events that may control the time
course of neurotransmitter release.
Nerve cells release neurotransmitter by allowing transmitter containing organelles, so called synaptic vesicles, to fuse with the neuronal plasma membrane, thus secreting their content. This process has recently been found to share many similarities with general vesicle trafficking events, e.g. protein transport to the cell surface, as employed by cells from yeast to mammals. The major differences are speed - synaptic vesicles can perform secretion within 200 microseconds after arrival of a stimulus - and tight control by calcium levels, which must rise thousandfold to trigger vesicle fusion. Using a combination of molecular biological and physiological techniques, the groups of Betz and Augustine now have found evidence that the speed of transmitter release itself may be controlled, somewhat ironically, by the evolutionarily highly conserved general fusion protein NSF (for N-methylmaleimide sensitive fusion protein). The groups have synthesized NSF-fragments and screened these peptides for their potency to prevent the major activator of NSF, the soluble NSF-attachment protein SNAP, from interacting with NSF in vitro. To elucidate the consequences of perturbed NSF-function for an intact synapse, inhibitory peptides were then injected into the nerve terminal of a squid giant axon preparation, in a series of experiments performed at the Marine Biological Laboratories in Woods Hole (MA). The unusual size and robustness of this synapse preparation allows introduction of several electrophysiological recording electrodes, paired with injection of molecules directly into the presynaptic nerve terminal, where synaptic vesicles are located. Peptides that were inhibitory in vitro also inhibited synaptic transmission at the giant synapse, indicating that unperturbed NSF-function is essential for neurotransmitter release. Unexpectedly, the NSF peptides also delayed transmitter release, i.e. the postsynaptic response recorded was not only smaller in amplitude, but took longer to reach the peak and faded more slowly than in controls. Further experiments pinpointed the site of action of the NSF-peptides to a subset of synaptic vesicles that are located directly at the presynaptic plasma membrane and being prepared ("primed") for fusion.
While neuronal communication relies on both the extent and duration of synaptic transmission, only the molecular mechanisms regulating the amount of transmitter release are known in considerable detail. The observations reported in this paper are the first to directly implicate a defined molecule in regulation of the time course of transmitter release and thus are expected to have considerable implications for further unravelling the molecular mechanisms governing temporal aspects of synaptic communication. In addition, they provide another glimpse at the way cells recruit proteins that have long been used in evolution into the highly specialized machinery of neurotransmitter release.