Reuptake is the reabsorption of a
neurotransmitter by a
neurotransmitter transporter
Neurotransmitter transporters are a class of membrane transport proteins that span the cellular membranes of neurons. Their primary function is to carry neurotransmitters across these membranes and to direct their further transport to specific intr ...
located along the
plasma membrane of an
axon terminal (i.e., the
pre-synaptic neuron
A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. ...
at a synapse) or
glial cell
Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form myel ...
after it has performed its function of transmitting a
neural impulse.
Reuptake is necessary for normal synaptic physiology because it allows for the recycling of neurotransmitters and regulates the level of neurotransmitter present in the synapse, thereby controlling how long a signal resulting from neurotransmitter release lasts. Because neurotransmitters are too large and hydrophilic to diffuse through the membrane, specific
transport proteins
A transport protein (variously referred to as a transmembrane pump, transporter, escort protein, acid transport protein, cation transport protein, or anion transport protein) is a protein that serves the function of moving other materials within ...
are necessary for the reabsorption of neurotransmitters. Much research, both biochemical and structural, has been performed to obtain clues about the mechanism of reuptake.
Protein structure
The first primary sequence of a reuptake protein was published in 1990. The technique for protein sequence determination relied upon the purification, sequencing, and cloning of the transporter protein in question, or expression cloning strategies in which transport function was used as an assay for cDNA species coding for that transporter. After separation, it was realized that there were many similarities between the two DNA sequences. Further exploration in the field of reuptake proteins found that many of the transporters associated with important neurotransmitters within the body were also very similar in sequence to the GABA and norepinephrine transporters. The members of this new family include transporters for
dopamine,
norepinephrine
Norepinephrine (NE), also called noradrenaline (NA) or noradrenalin, is an organic chemical in the catecholamine family that functions in the brain and body as both a hormone and neurotransmitter. The name "noradrenaline" (from Latin '' ad' ...
,
serotonin,
glycine
Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid ( carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinog ...
,
proline and
GABA. They were called Na
+/Cl
− dependent neurotransmitter transporters.
Sodium
Sodium is a chemical element with the symbol Na (from Latin ''natrium'') and atomic number 11. It is a soft, silvery-white, highly reactive metal. Sodium is an alkali metal, being in group 1 of the periodic table. Its only stable ...
and
chloride
The chloride ion is the anion (negatively charged ion) Cl−. It is formed when the element chlorine (a halogen) gains an electron or when a compound such as hydrogen chloride is dissolved in water or other polar solvents. Chloride sa ...
ion dependence will be discussed later in the mechanism of action. Using the commonalities among sequences and hydropathy plot analyses, it was predicted that there are 12 hydrophobic membrane spanning regions in the 'Classical' transporter family.
In addition to this, the N- and C-termini exist in the
intracellular space
Intracellular space is the interior space of the plasma membrane. It contains about two-thirds of TBW. Cellular rupture may occur if the intracellular space becomes dehydrated, or if the opposite happens, where it becomes too bloated. Thus it i ...
. These proteins also all have an extended extracellular loop between the third and fourth transmembrane sequences. Site-directed chemical labeling experiments verified the predicted topological organization of the serotonin transporter.
In addition to neurotransmitter transporters, many other proteins in both animals and prokaryotes were found with similar sequences, indicating a larger family of Neurotransmitter:Sodium Symporters (NSS). One of these proteins, LeuT, from ''
Aquifex aeolicus
"''Aquifex aeolicus''" is a chemolithoautotrophic, Gram-negative, motile, hyperthermophilic bacterium. "''A. aeolicus"'' is generally rod-shaped with an approximate length of 2.0-6.0μm and a diameter of 0.4-0.5μm. "''A. aeolicus''" is neither v ...
'', was crystallized by Yamashita et al.
with very high resolution, revealing a molecule of leucine and two Na
+ ions bound near the center of the protein. They found that the transmembrane (TM) helices 1 and 6 contained unwound segments in the middle of the membrane. Along with these two helices, TM helices 3 and 8 and the areas surrounding the unwound sections of 1 and 6 formed the substrate and sodium ion binding sites. The crystal structure revealed pseudo-symmetry in LeuT, in which the structure of TM helices 1-5 is reflected in the structure of helices 6–10.
There is an extracellular cavity in the protein, into which protrudes a helical hairpin formed by extracellular loop EL4. In TM1, an
aspartate distinguishes
monoamine
Monoamine neurotransmitters are neurotransmitters and neuromodulators that contain one amino group connected to an aromatic ring by a two-carbon chain (such as -CH2-CH2-). Examples are dopamine, norepinephrine and serotonin.
All monoamines ar ...
NSS transporters from
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...
transporters which contain a
glycine
Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid ( carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinog ...
at the same position. External and internal "gates" were assigned to pairs of negatively and positively charged residues in the extracellular cavity and near the cytoplasmic ends of TM helices 1 and 8.
Mechanism of action
The classic
transporter protein
A transport protein (variously referred to as a transmembrane pump, transporter, escort protein, acid transport protein, cation transport protein, or anion transport protein) is a protein that serves the function of moving other materials within ...
s use transmembrane ion gradients and electrical potential to transport neurotransmitter across the membrane of the presynaptic neuron. Typical neurotransmitter sodium symport (NSS) transporters, which are Na
+ and Cl
− ion dependent, take advantage of both Na
+ and Cl
− gradients, inwardly directed across the membrane. The ions flow down their concentration gradients, in many cases leading to transmembrane charge movement that is enhanced by the membrane potential. These forces pull the neurotransmitter substrate into the cell, even against its own concentration gradient. At a molecular level, Na
+ ions stabilize amino acid binding at the substrate site and also hold the transporter in an outward-open conformation that allows substrate binding. The role of the Cl
− ion in the
symport
A symporter is an integral membrane protein that is involved in the transport of two (or more) different molecules across the cell membrane in the same direction. The symporter works in the plasma membrane and molecules are transported across the ...
mechanism has been proposed to be for stabilizing the charge of the symported Na
+.
After ion and substrate binding have taken place, some conformational change must occur. From the conformational differences between the structure of TMs 1-5 and that of TMs 6–10, and from the identification of a substrate permeation pathway between the binding site of
SERT and the
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...
, a mechanism for conformational change was proposed in which a four-helix bundle composed of TMs 1, 2, 6 and 7 changes its orientation within the rest of the protein. A structure of LeuT in the inward-open conformation subsequently demonstrated that the major component of the conformational change represents movement of the bundle relative to the rest of the protein.
Mechanism of reuptake inhibition
The main objective of a
reuptake inhibitor
Reuptake is the reabsorption of a neurotransmitter by a neurotransmitter transporter located along the plasma membrane of an axon terminal (i.e., the pre-synaptic neuron at a synapse) or glial cell after it has performed its function of transmi ...
is to substantially decrease the rate by which neurotransmitters are reabsorbed into the presynaptic neuron, increasing the concentration of neurotransmitter in the synapse. This increases neurotransmitter binding to pre- and postsynaptic neurotransmitter receptors. Depending on the neuronal system in question, a reuptake inhibitor can have drastic effects on cognition and behavior. Non-competitive inhibition of the bacterial homologue LeuT by tricyclic antidepressants resulted from binding of these inhibitors in the extracellular permeation pathway.
However, the competitive nature of serotonin transport inhibition by antidepressants suggests that in neurotransmitter transporters, they bind in a site overlapping the substrate site.
Human systems
Horschitz et al.
examined reuptake inhibitor selectivity among the rat serotonin reuptake protein (SERT) expressed in human embryonic kidney cells (HEK-SERT). They presented SERT with varying doses of either
citalopram
Citalopram, sold under the brand name Celexa among others, is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. It is used to treat major depressive disorder, obsessive compulsive disorder, panic disorder, and so ...
(an
SSRI
Selective serotonin reuptake inhibitors (SSRIs) are a class of drugs that are typically used as antidepressants in the treatment of major depressive disorder, anxiety disorders, and other psychological conditions.
SSRIs increase the extracellul ...
) or
desipramine
Desipramine, sold under the brand name Norpramin among others, is a tricyclic antidepressant (TCA) used in the treatment of depression. It acts as a relatively selective norepinephrine reuptake inhibitor, though it does also have other activiti ...
(an inhibitor of norepinephrine reuptake protein, NET). By examining the dose-response curves (using a normal medium as control), they were able to quantify that citalopram acted on SERT as an SSRI, and that desipramine had no effect on SERT. In a separate experiment, Horschitz et al. exposed HEK-SERT with citalopram on a long-term basis. They noticed that long-term exposure led to a down-regulation of binding sites. These results suggest some mechanism for long-term changes in the pre-synaptic neuron after drug therapy. Horschitz et al. found that after removing citalopram from the system, normal levels of SERT binding site expression returned.
Depression has been suggested to be a result of a decrease of serotonin found in the synapse, although this hypothesis has been challenged since as early as the 1980s. It was initially supported by the successful reduction of depressive symptoms after administration of
tricyclic antidepressant
Tricyclic antidepressants (TCAs) are a class of medications that are used primarily as antidepressants, which is important for the management of depression. They are second-line drugs next to SSRIs. TCAs were discovered in the early 1950s and wer ...
s (such as desipramine) and SSRIs. Tricyclic antidepressants inhibit the reuptake of both serotonin and norepinephrine by acting upon both the SERT and NET. SSRIs selectively inhibit the reuptake of serotonin by acting upon SERT. The net result is an increased amount of serotonin in the synapse, thus increasing the probability that serotonin will interact with a serotonin receptor of the postsynaptic neuron. There are additional mechanisms by which serotonin
autoreceptor
An autoreceptor is a type of receptor located in the membranes of nerve cells. It serves as part of a negative feedback loop in signal transduction. It is only sensitive to the neurotransmitters or hormones released by the neuron on which the au ...
desensitization must occur, but the net result is the same. This increases serotonin signaling, which according to the hypothesis is believed to elevate mood and thus relieve depressive symptoms. This proposal for the antidepressant mechanism of serotonin reuptake inhibitors does not account for the time course of the therapeutic effect, which takes weeks to months, while transporter inhibition is essentially immediate.
The net effect of
amphetamine (AMPH) use is an increase of dopamine, norepinephrine and serotonin in the synapse. It has been shown that AMPH acts upon
trace amine-associated receptor 1
Trace amine-associated receptor 1 (TAAR1) is a trace amine-associated receptor (TAAR) protein that in humans is encoded by the ''TAAR1'' gene. TAAR1 is an intracellular amine-activated and G protein-coupled receptor (GPCR) that is primarily ex ...
(TAAR1) to induce efflux and reuptake inhibition in the serotonin, norepinephrine, and
dopamine transporter
The dopamine transporter (also dopamine active transporter, DAT, SLC6A3) is a membrane-spanning protein that pumps the neurotransmitter dopamine out of the synaptic cleft back into cytosol. In the cytosol, other transporters sequester the dop ...
s. This effect requires the transporter and TAAR1 to be co-localized (occur together) within the same neuron.
Neuroprotective role
Astrocytes
Astrocytes (from Ancient Greek , , "star" + , , "cavity", "cell"), also known collectively as astroglia, are characteristic star-shaped glial cells in the brain and spinal cord. They perform many functions, including biochemical control of endo ...
seem to utilize reuptake mechanisms for a neuroprotective role. Astrocytes use
excitatory amino acid transporter 2 (EAAT2, aka GLT-1) to remove
glutamate from the synapse. EAAT2
knockout mice were more prone to lethal and spontaneous seizures and acute brain injuries among the cortex. These effects could be linked to increased concentrations of glutamate in the brains of EAAT2 knockout mice, analyzed post-mortem.
References
{{reflist, 33em
Neurotransmitters
Neurotransmitter transporters