Molecular Mechanisms in the Regulation 
of Synaptic Transmission 
I 
Richard L. Huganir, Ph.D. — Associate Investigator 
Dr. Huganir is also Associate Professor of Neuroscience at the Johns Hopkins University School of Medi- 
cine. He completed his undergraduate work in biochemistry at Vassar College and received his Ph.D. de- 
gree in biochemistry and molecular and cell biology from Cornell University, where he performed his 
thesis research in the laboratory of Efraim Packer. After completing a postdoctoral fellowship with Paul 
Greengard at Yale University School of Medicine, Dr. Huganir moved to the Rockefeller University, where 
he was Assistant Professor of Molecular and Cellular Neurobiology. 
INFORMATION processing in the brain de- 
pends on the transmission of signals between 
neurons at specialized areas of contact, called 
synapses. At synapses, ion channel proteins in the 
neuronal cell membrane generate an electrical 
current, which triggers the release of chemical 
signals from the first neuron, called the presynap- 
tic neuron. These chemical signals, or neurotrans- 
mitters, bind to specific receptor proteins in the 
membrane of the second neuron, called the post- 
synaptic neuron. The neurotransmitter receptors 
then generate electrical currents in the second 
neuron, thereby completing the process of syn- 
aptic transmission. 
Both the amount of neurotransmitter released 
from the presynaptic neuron in response to a 
given electrical signal and the sensitivity of the 
postsynaptic receptor system for a given amount 
of neurotransmitter can be modulated by extra- 
cellular factors. The molecular mechanisms that 
underlie this modulation have only begun to 
be defined. Recent studies have provided evi- 
dence that protein phosphorylation is an import- 
ant mechanism in the regulation of synaptic 
transmission. 
Protein phosphorylation systems consist of 
three primary components, a protein kinase, a 
substrate protein, and a phosphoprotein phos- 
phatase. Protein kinases are enzymes that catalyze 
the chemical transfer of phosphate molecules 
from ATP to specific substrate proteins. The activ- 
ities of many protein kinases are regulated by neu- 
rotransmitters and hormones through the actions 
of substances called second messengers, such as 
cAMP, calcium, and diacylglycerol. Substrate 
proteins include many cellular components, 
among them enzymes, ion channels, and neuro- 
transmitter receptors. The addition of the nega- 
tively charged phosphate group alters the struc- 
ture of these substrate proteins, thereby 
regulating their functional properties. Phospho- 
protein phosphatases are enzymes that reverse 
the process of protein phosphorylation, remove 
the phosphate group from the substrate protein, 
and return it to its basal state. 
My laboratory is concerned with the structure 
and function of neurotransmitter receptors and 
the role of protein phosphorylation in the regula- 
tion of the properties of the neurotransmitter re- 
ceptors. We have chosen the best-characterized 
neurotransmitter receptor and ion channel in neu- 
robiology today, the nicotinic acetylcholine re- 
ceptor, as a model system. In addition, we have 
been studying the major excitatory neurotransmit- 
ter receptor in the brain, the glutamate receptor, 
and the major inhibitory receptor in the brain, 
the GABA^ receptor. These receptors are neuro- 
transmitter-dependent ion channels that generate 
electrical currents in the postsynaptic mem- 
brane of the synapse in response to their 
neurotransmitter. 
To study the molecular mechanisms involved 
in neurotransmitter receptor and ion channel 
function, it is essential to identify chemically the 
specific proteins required for this activity. We 
began by defining the molecular components re- 
quired for the functioning of the nicotinic acetyl- 
choline receptor ion channel. Using membrane 
reconstitution techniques, we solubilized the nic- 
otinic receptor and its ion channel from isolated 
postsynaptic membranes, purified it, and recon- 
stituted it into phospholipid vesicles. These stud- 
ies demonstrated that the purified receptor, con- 
sisting of four types of protein subunits (a, /3, 7, 
6) , contains the ion channel and has all the biolog- 
ical properties of the nicotinic receptor in the 
intact cell. 
We next began to characterize the protein 
phosphorylation of these structural components. 
Postsynaptic membranes isolated from synapses 
highly enriched in the nicotinic acetylcholine re- 
ceptor contain at least three different types of 
protein kinases that phosphorylate the nicotinic 
receptor on six different phosphorylation sites: 
cAMP-dependent protein kinase phosphorylates 
the 7- and 6-subunits of the receptor; a calcium- 
and diacylglycerol-dependent protein kinase 
phosphorylates the 5-subunit; a protein tyrosine 
kinase phosphorylates the 0-, 7-, and 6-subunits. 
These postsynaptic membranes also contain 
phosphoprotein phosphatase activity that de- 
phosphorylates the phosphorylated nicotinic 
acetylcholine receptor. 
203 
