Objective: To understand the metabolic processes underlying the

synthesis & metabolism of amino acid & peptide neurotransmitters.

Major points to be covered:

-regulation of metabolism by enzymes
-metabolic processes neurons share with other cells and organs
-properties and functions of enzymes and pumps (transporters).
-metabolic contingencies imposed by the existence of a
blood-brain-barrier, i.e. the central role of glucose

-synthesis and metabolism of amino acid transmitters and

GABA.
-glutamate
-aspartate
-glycine

-neuropeptide synthesis and the pathway to regulated release

 Neuronal Metabolism
• Neurons share with other cells the need and
ability to synthesize nucleic acids, proteins,
carbohydrates and lipids.
• Likewise they share the metabolic
processes required to generate chemical
energy for these processes:
– EMP, HMP Shunt, TCA, oxid phos.
• Neurons must be able to synthesize and
metabolize neurotransmitters.
• Neurons must also synthesize second
messenger molecules needed to mediate
signal transduction.
 The brain makes use
of general metabolism
to find precursors
and in some cases the
finished products for
synaptic physiology.
glycine
 Enzymes
• Help processes within neurons overcome
activation energy, and provide a site of
regulation.
• Essentially all chemical reactions in cells are
mediated by enzyme, protein catalysts.
• A catalyst acts by bringing together the
reactants, and thereby increasing the rate of
a chemical reaction, without being
permanently changed in the reaction.
• Enzymes also allow the coupling of
energetically unfavourable reactions with
reactions that release free energy. If together
the two reactions result in a negative DG, the
coupled reaction can occur.
Enzymes lower
activation energy
for reactions.

Mol. Biol. of the Cell

Enzymes permit coupled reactions, for example falling rocks
turn wheel to raise water for a different type of work.

Mol. Biol. of the Cell

ATP is a useful energy currency
since it can form high-energy
intermediates permitting
the coupling of energetically
unfavorable reactions to
favorable ones.

Mol. Biol. of the Cell

General Properties of Enzymes
• Enzymes are highly specific due to the
specific structure of the active site
• Substrate specificity
• Reaction specificity
• Enzymes bind substrates in specific ways that
stabilize a reactive conformation, known as
the TRANSITION STATE
• Some enzymes require cofactors for
complete activity (vitamin B6, active
coenzyme form deficiency can impact
GABA synthesis).
Velocity (V) as a function of
substrate (S) plot.
Saturation

pseudo
1st order

Km
Michaelis-Menton Equation, describes saturable enzyme
kinetics, also applicable to binding of ligands to receptors.

know this, it describes many

interactions: enzymes, receptors,
protein-protein.

V=Vmax* [S]/([S]+Km)

With a competitive inhibitor, the Km is increased but

the Vmax is not effected.
Km’=Km*(1+[I]/Ki), note when I= Ki the Km doubles
With a noncompetitive inhibitor only the Vmax is reduced.
Vmax’=Vmax*(1-[I]/([I]+Ki)), note when I= Ki the Vmax halves
 Km and Vmax
• The activity of enzymes can be discussed in terms
of their Km, a measure of the affinity of the enzyme
for its substrate, and the Vmax, which is the
maximal velocity of the enzymatic reaction.
• Km has two meanings: 1) the concentration of
substrate at which 1/2 the active sites on an
enzyme are filled. 2) the ratio of dissociation to
association rates for enzyme substrate interactions.
Km=kdissoc/kassoc. Since the association rates
of many reactions at going the speed of diffusion,
the strength of binding and rates of reaction are
often determined by the dissociation rate.
• Although these terms are associated with enzymes
they are related to other saturable systems such
as transporters (Kt, Vmax) and receptors (Kd,
Bmax).
 Competitive inhibitors.
• Action: at the catalytic site, where it
competes with substrate for binding in a
dynamic equilibrium- like process.
Inhibition is reversible by substrate.
• Effect: Vmax is unchanged; Km, as
defined by [S] required for 1/2 maximal
activity, is increased.
 Noncompetitive inhibitors.
• Action:Binds E or ES complex other than at
the catalytic site. Substrate binding unaltered,
but ESI complex cannot form products.
Inhibition cannot be reversed by substrate. .
• Effect: Vmax is reduced; Km, as defined by
[S] required for 1/2 maximal activity, is
unchanged.
• Knowing if something is competitive or
non-competitive is important since it
determines how much inhibitor you need
relative to substrate (practical
implication!!)
Substrate or ligand concentration
Transport can be saturable.
Relative
scales, simple
diffusion rates
will be low for
polar
substances.
Channels and carriers.
Since many transported compounds are charged their
movement is governed by electrical and chemical
gradients just like small ions such as K+, Na+, Cl-, and
Ca2+.
Uniports-facilitative or uncoupled
transport
• Molecules or ions move down their
concentration gradient via a specific carrier.
• In contrast to a channel which will allow
movement of thousands of ions per
millisecond and whose specificity is primarily
mediated by pore size, a facilitative carrier
requires binding of a specific substrate which
induces conformational changes in the carrier
through which the substrate is moved, and
then released, restoring the carrier to its
original conformation.
Carrier-Mediated Transport,
Uniporters.
• Carrier types at the blood brain barrier:
hexose, monocarboxylic acid, large
neutral amino acid, basic amino acid,
acidic amino acid, choline, purine, and
nucleoside carriers.
• These substances serve as building
blocks for all brain macromolecules and
neurochemicals.
 Symports and antiports
• Couple movement of one molecule with that
of one or more other substrates. Energy is
derived from concentration gradients no ATP
needed (directly) although indirectly to
establish gradient.
• The high-affinity pumps for amino acids,
and neurotransmitters are principally Na+-
symporters, i.e. the movement of Na+ down
its electrochemical gradient provides the free-
energy required to move another substrate
(neurotransmitter) up its concentration
gradient
• Na+/Ca2+ antiporters, and Na+/H+ antiporters
move these ions out of cells as Na+ enters.
 Na+, Ca2+
protons
Glutamate exchange
The Na+ gradient
can be used to
pump glucose
uphill.
 Primary active transport
• Systems utilize the free-energy obtained by
ATP hydrolysis to move ions against
concentration gradients (uphill), i.e. Na+-, K+-
ATPase or the Ca2+ ATPase.
• Estimated to require up to half the brain
ATP, while other biochemical processes
including protein, lipid and neurotransmitter
synthesis together use perhaps 10%.
• Other primary pumps, such as Ca2+-ATPases
and proton pumps probably account for the
rest. The brain uses 20% of total body
oxygen consumption, thus 10% of total is
used primarily to maintain neuronal ionic
gradients via this pump.
 +
Na / K + ATPase
 Na+/ K+ ATPase
• Energy is directed into the pumping
process by the 3Na+-dependent
phosphorylation, followed by the 2K+-
dependent dephosphorylation.
Phosphorylation induces a
conformational change that moves 3Na+
to the outside of the cell.
• Pump stoichiometry is 3/2 making it
electrogenic.
Fundamental Neurosci.
2002 Zigmond et al.
 Role of the pump in resting
membrane potential.
• If pump is blocked with ouabain (blocks
binding of K+) an immediate small
depolarization occurs (only a few mV),
however membrane will remain relatively
constant as it is largely determined by K+
permeability, however the membrane is also
slightly permeable to Na+ and over time the
membrane potential will depolarize if Na+
diffuses in unchecked by the pump.
 Glucose
• Is the major fuel of the brain because it is
the only fuel which enters in sufficient
amounts to support the energy requirements.
• Glucose gains access to brain and into cells
by specific carriers - blood levels much higher
than brain levels, thus glucose moves down
its concentration gradient via facilitative
transport.
• Glucose utilization is tied to neuronal
activity and increased blood flow, basis of
PET functional imaging with 2-deoxyglucose.
• Isolated neurons can use other fuels such as
pyruvate and lactate, but they normally are
not BBB permeable.
 Blood
(~6 mM
CSF glucose).
(~4 mM
glucose).

4X Glut-1
expressed on the
ab-lumenal side
Farrell and Pardridge
1991

Fundamental Neurosci. 2002 Zigmond et al.

 Glucose transport
• The Km of the BBB glucose transporter is
about 7 mM, which is about the level of
plasma glucose, thus brain glucose varies
directly with changes in blood levels. The
blood brain barrier transporter is Glut-1.
• Neurons possess a carrier of higher affinity,
Glut-3 Km = 200 mM, allowing them to extract
glucose from the extracellular space. Within
neurons, glucose is immediately
phosphorylated to a charged, impermeant
metabolite, glucose-6-PO4, thus the
intracellular glucose concentration is
effectively zero. Why is it advantageous to
reduce the apparent free concentration of
glucose?
 Fundamental Neurosci.
Used in 2002 Zigmond et al.
PET scanning.
 Glycolysis and TCA cycle
• Within the cell, glucose enters the glycolysis
pathway in the cytoplasm, and via pyruvate
and acetyl-CoA, in the mitochondrial tri-
carboxylic acid cycle (TCA) or Krebs cycle. In
these systems, reducing equivalents are
generated and via oxidative phosphorylation
they generate ATP, the chemical fuel for the
brain.
• Glycolysis and the TCA cycle are also the
source of non-essential amino acid
precursors used to synthesize the
neurotransmitters glutamate, aspartate,
GABA, and glycine.
TCA Cycle
& ETC
 Blood brain barrier.
• What is the blood brain barrier (BBB)?
• The existence of a BBB prevents molecules
in the circulation from freely entering the
brain.
• Prevents constant fluctuations in circulating
metabolites, ions, and hormones from directly
influencing neuronal activity.
• Diffusion allows passage of gases, i.e. (O2
and CO2) and lipid soluble compounds, i.e.
psychoactive drugs.
Endothelium

The BBB largely

occurs at capillaries
through astrocyte
endfeet and
endothelium tight
junctions.

Transport across is
selective. Carrier types at
the BBB: hexose,
monocarboxylic acid, large
neutral amino acid, basic
amino acid, acidic amino
acid, choline, purine, and
nucleoside carriers. Drewes
LR. Adv Exp Med Biol.
1999;474:111-22.
Iadecola and Nedergaard 2007 Nat. Neurosci.
Perivascular glia contain high levels of the antioxidant
tripeptide glutathione (Sun et al. 2006.)
Paulson, European Neuropsychopharmacology
12, 2002, Pg. 495
 Brain Activity & Blood Supply
are Tightly Linked.
• It has been known for over 100 years that
increased neuronal activity is associated
with increases in blood flow. Roy CS,
Sherrington CS (January 1890). "On the
Regulation of the Blood-supply of the Brain".
J. of Physiol. 11 (1-2): 85–158.17.
• Changes in blood flow or oxygenation are
used as a surrogate measure of neuronal
activity.
Glial and neuronal control of brain blood flow

Glial and neuronal control of brain blood flow David Attwell1, Alastair M. Buchan2, Serge
Charpak3, Martin Lauritzen4, Brian A. MacVicar5 & Eric A. Newman6 Nature 2010 468:231
Neurotransmitters:
small molecule & neuropeptide.
 Small molecule
Neurotransmitters (MW<300)
are synthesized in the terminal.

precursor uptake

Fundamental Neurosci.
2002 Zigmond et al.

Transporters/carriers are
required to transit the
vesicle and plasma membrane.
Neurotransmitter transporters:

• Plasma membrane forms terminate

neurotransmission, replenish
neurotransmitter pool, and may have a
signaling function.
• Vesicular transporters use both concentration
gradient and protons to concentrate
transmitter in vesicles: these transporters
make neurons transmitter specific.
Molecular Structure of Plasma Membrane
Neurotransmitter Transporters

• Norepinephrine, GABA, serotonin, dopamine,

glycine, choline, and proline transporters
• Homologous in their 12 transmembrane
spanning domains.
• In the case of GABA, transport results from
the co-transport of Na+ and Cl- ions
 Glutamate Plasma Membrane
Transporters Form a Distinct Family.
• Structure different from other transmitter transporters - 8
transmembrane domains not related to other existing
mammalian transporter clones.
• Homologous to each other (~50%) and to a bacterial
proton-coupled glutamate transporter.
• Glast1 (EAAT1), Expressed in glial cells. Storck et al.
PNAS 89, 10955-10959 (1992).
• GLT-1 (EAAT2), Localized to glial cells. Pines et al.
Nature 360, 464-467 (1992).
• EAAC1 (EAAT3), Relatively neuron specific in brain, also
expressed in intestinal tissues, and kidney. Kanai and
Hediger Nature 360, 467-471 (1992).
Transporter is
electrogenic allowing
its current to be
measured and
studied with the
patch clamp method.
 Vesicle glutamate transporters.

• Several members including VGLUT1 and

VGLUT2, and VGLUT3 isolated by R.
Edwards lab (Science;289:957-60, 2000
first publication).
• Defines glutamatergic neuron classes,
although all neurons contain glutamate, only
those expressing VGLUT’s can package it
at high concentrations for synaptic release.
• Transport mediated by a combination of H+
and ionic gradients.
 Vesicular accumulation
of amino acids results
from both a gradient of
membrane potential and
pH.

Chaudhry et al. 2002 JCB.

 Neurotransmitter glycine.
• Non-essential amino acid derived from glycolysis
and TCA cycle intermediates.
• Glycine made from glucose via amino acid
serine.
• High-affinity uptake system removes glycine from
synapse. Shares a vesicular pump with
GABA, VGAT
• Glycine and its pump found in high levels in
spinal cord, in neurons presynaptic to strychnine-
sensitive glycine receptor-chloride channel.
• Receptors mainly found in the spinal cord.
 Neurotransmitter glutamate
• Na+-dependent, high-affinity uptake system has
been well characterized, and occurs principally
in glutamate nerve terminals (EAAC-1/EAAT3).
• Glutamate uptake into glial cells allows
metabolism via glutamine synthetase.
Glutamine formed in glia then enters neurons to
provide a precursor for glutamate synthesis via
glutaminase activity.
• Since glutamate transport is determined by ion
concentration gradients it is described by the
Nernst potential. At positive voltages the
transporter can reverse (may occur during a
stroke).
 Astro Gln efflux
through system N.

Neuronal Gln uptake

by system A.

see Chaudhry et al.

2002 JCB.

Fundamental Neurosci.
2002 Zigmond et al.
Fundamental Neurosci.
2002 Zigmond et al.
Fundamental Neurosci.
2002 Zigmond et al.
 Regeneration of the Glutamate
Neurotransmitter Pool in Neurons
 Glu Metabolism, 4 Synthetic Pathways
1) From a-KG (2-oxoglutarate) & ammonia via GDH. This pathway is of
fundamental importance in the synthesis of all amino acids, since it is the key mechanism
for the formation of a-amino groups directly from ammonia. Transamination of a-keto acids
with glutamate as amino group donor then allows the introduction of a-amino groups into
the synthesis of other amino acids.

2) From a-KG & Asp by AST; antibodies to this enzyme stain many presumed
glutamate neurons

3) From Gln by glutaminase; antibodies to this enzyme also stain some

presumed glutamate neurons. Glutaminase removes the NH2 from the glutamine.

4) From a-KG by ornithine-aminotransferase or via proline oxidase.

Both these pathways form P5C (pyrroline 5-carboxylic acid), which via P5C dehydrogenase
can yield glutamate. There is no evidence yet that these are neuronal enzymes. However,
a high-affinity proline uptake system has recently been found that appears to be associated
with glutamate pathways.
(astrocyte)

Fundamental Neurosci.
2002 Zigmond et al.
 Neuropeptide neurotransmitters.
• History i.e. regulated release of enzymes
from exocrine cells, and hormones such as
insulin from endocrine cells
• The discovery of vasopressin release from
posterior pituitary in the 1940s by du
Vigneaud demonstrated that neurons could
secrete peptides for intercellular
communication
• This was followed by the discovery of
hypothalamic factors regulating the anterior
pituitary by Guillemin and Schally
• The discovery, in the mid-seventies, of
enkephalins as endogenous ligands for
discovered opiate receptors.
Fundamental Neurosci.
2002 Zigmond et al.
 Synthesis & Processing
of Neuropeptides, RNA.
• mRNA splicing to generate different bioactive
peptides, selective usage of some exons. A
mechansim by which a single gene encodes
polypeptides of varied function. Splicing
occurs in the nucleus. Substance P and
substance K are encoded by the same gene
but are only found together in mature mRNA
in some tissues. Calcitonin and CGRP are
formed in different neurons by alternative
splicing of introns.
• mRNA moves through nuclear pores and into
cytoplasm.
 Peptide synthesis.
• Proteolytic maturation then occurs in acidic,
clathrin-coated secretory vesicles. Involves
endopeptidases, which often cleave C-
terminal to the paired dibasic amino acids, i.e.
Lys-Arg, Arg-Arg. POMC can be processed
into at least 6 different peptide hormones
through proteolytic cleavage (ACTH, b-
endorphin, Clip, a-MSH, g-MSH, b-LPH, etc).
• Processing can be specific to different brain
or pituitary regions.
• The dibasic residues are then removed by
carboxypeptidase.
 Peptide synthesis.
• Some prohormones, i.e. somatostatin, are
cleaved by other endopeptidases, N-terminal
to dibasic pairs, which are then removed by
aminopeptidases.
• Many peptides end in a modified C-terminal
amide. This is formed by the action of
peptidyl-glycine-a-amidating monooxygenase
(PAM) which converts the C terminal Gly to a
amide group. Amidation is critical for the
function of some peptides (such as substance
P).
• Vesicles containing peptides are moved via
fast axonal transport to release sites
 Degradation
• specific uptake systems have not been
identified
• presumably, diffusion from synapses,
and proteases of various sorts on the
surface of neurons and glia cleave the
peptides to their constitutive amino
acids, which can then be reutilized
 Methods of study

• Purification via bioassay, chemical assay,

molecular cloning
• Synthesis allows antibody production, RIA,
immunohistochemistry, radioligand binding,
electrophysiology
• Most peptides act via G protein-coupled
receptors modulate K+ channels and Ca++
channels and can be studied
electrophysiologically.
 Anatomy, localization
• Found in most, if not all neurons, can coexist
with other peptides or with amine and amino
acid transmitters, present in dense core large
vesicles.
• Made in the cell body on ribosomes and
transported to terminals.
• If a prohormone is cleaved prior to packaging
in vesicles, it is possible to sort the mature
peptides to different vesicles. In fact, work in
Aplysia indicates that peptides in distinct
vesicles can be sorted to different neuronal
compartments Cell, 54:813-822 1988. This
would appear to contravene Dales Law: 1) a
neuron has only one transmitter and 2) a
neuron is only excitatory or inhibitory.
 Sources
• Fundamental Neuroscience
Fundamental Neuroscience 3rd Edition.
• Cooper, Bloom & Roth, The
Biochemical Basis of
Neuropharmacology, 7th Ed.
• Molecular Biology of the Cell by Alberts,
4th ed.
• Mark’s Basic Medical Biochemistry, 4th
Ed.