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.
Journal
Science