Mechanisms of Neurotransmitter Storage 
and Release 
Reinhard John, Ph.D. — Associate Investigator 
Dr. Jahn is also Associate Professor of Pharmacology at Yale University School of Medicine. He received his 
Ph.D. degree at the University of Gottingen, Germany. His postdoctoral training was with Hans Dieter 
Soling at the University of Gottingen and later with Paul Greengard at Yale University and the Rockefeller 
University. He was Assistant Professor at the Rockefeller University and subsequently headed a research 
group at the Max Planck Institute for Psychiatry, Martinsried. 
NERVE cells, or neurons, communicate with 
each other and with other cells by means of 
small molecules, the neurotransmitters. This 
communication occurs at specialized contact 
zones, the synapses. Upon arrival of incoming ac- 
tion potentials, the sender cell releases its neuro- 
transmitters, which cross the synaptic cleft. The 
plasma membrane of the receiving cell has spe- 
cific receptor molecules that, in turn, translate 
the signals into functional changes. 
In the resting state, the presynaptic nerve end- 
ings of the sender cell store their neurotransmit- 
ters in small membrane-enclosed compartments, 
the synaptic vesicles. When an action potential 
arrives, voltage-gated calcium channels open and 
calcium ions (Ca^"^) enter the terminal from the 
extracellular space. Within a fraction of a milli- 
second, synaptic vesicles fuse with the plasma 
membrane, releasing their contents. The vesicle 
membrane protein is then retrieved by endocyto- 
sis for use in regenerating fusion-competent syn- 
aptic vesicles. 
The details of this membrane recycling are still 
largely unclear, and the enzymes catalyzing the 
major steps have not been identified. As a starting 
point for detailed functional analysis, we and 
others have characterized the major membrane 
proteins of synaptic vesicles. This was facilitated 
by the fact that synaptic vesicles are abundant and 
can easily be purified in large amounts. Due to 
the smallness of the vesicles, the number of pro- 
tein species per individual vesicle is inherently 
limited. Therefore it should be possible to iden- 
tify most, if not all, of the major protein constitu- 
ents. To date, several families of unique vesicle 
proteins have been characterized, and advanced 
tools for their study are available. We are 
currently involved in a systematic chemical analy- 
sis of the vesicle membrane to identify the re- 
maining proteins of this organelle. 
Parallel to this protein analysis, we have re- 
cently begun to establish assay systems for indi- 
vidual steps of the vesicle cycle. One of the 
model systems accessible for biochemical analy- 
sis is the isolated nerve terminal. Upon homogen- 
ization of nervous tissue, nerve terminals, though 
sheared off their axons, reseal and remain func- 
tional for several hours after isolation. Applica- 
tion of depolarizing stimuli or Ca^"^ ionophores 
causes a massive exocytotic transmitter release 
and a parallel increase in membrane turnover. 
We found that during exo-endocytosis, one of 
the vesicle proteins, the GTP-binding protein 
rab3A, dissociates from the vesicle membrane 
and reassociates again at a later stage of the mem- 
brane cycle. This dissociation-association cycle is 
probably necessary for an orderly and sequential 
processing of the vesicle membrane. Thus small 
GTP-binding proteins may serve in the nerve ter- 
minal, as in other organelles, as "status indica- 
tors" for the recycling membrane, being asso- 
ciated only with a specific step in the life cycle of 
the vesicle membrane. The biochemical events 
leading to GTPase activation and membrane disso- 
ciation and reassociation are presently under 
study. In addition, the individual G proteins 
should allow an isolation of the compartments 
representing separate steps in the membrane 
cycle. 
Thus we have isolated clathrin-coated vesicles 
from nerve terminals by conventional procedures 
and have analyzed their membrane and coat com- 
position. We have also developed immunoisola- 
tion procedures for synaptic vesicle-derived 
membrane populations, using monoclonal anti- 
bodies directed against individual membrane 
components. We hope these studies will aid in 
the functional definition of the compartments in- 
volved in synaptic vesicle recycling and will form 
the basis for the reconstitution of individual steps 
of the membrane cycle in cell-free systems. 
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