chapter 7 - vesicular and plasma membrane nt transporters Flashcards
7.1. [vesicular and plasma membrane nt transporters] name them
SV transporters for NTs (SVNTTs)
> SVs fill with NT through a process driven by the vacuolar-type H-ATPase, which uses the energy released by ATP hydrolysis to pump protons in the SV lumen
> there are 3 major determinants of SV filling with NT:
* cytosolic concentration of the NT
* electrochemical driving force across the SV membrane (determined by the activity of the vacuolar-type H-ATPase)
* intrinsic properties of the SVNTT
classical SVNTTs:
VGLUT (1-3): vesicular glutamate transporter: exchanges H* for glutamate
VMAT: vesicular monoamine transporter: recognizes multiple monoamines as substrates; exhanges 2 lumenal H* for one protonated monoamine
VAchT: vesicular acetylcholine transporter: exchanges H* for Ach
VGAT: vesicular GABA transporter: recognizes both inhibitory NTs
GABA and glycine; co-transports GABA and 2 Cl
- monoamine NTs include dopamine, noradrenaline, adrenaline and serotonin
7.2. [vesicular and plasma membrane nt transporters] explain their function (how) and include their driving forces and their role in nt cycles
Vesicular and plasma membrane NT transporters
on SVs:
* load SVs with NT
or
on plasma membrane:
* terminate synaptic transmission
* recycle NT
1-SV exocytosis leads to secretion of acetylcholine (ACh) into the synaptic cleft, allowing ACh to activate postsynaptic ACh receptors (AChR).
2-ACh in the synaptic cleft is hydrolized by acetylcholinesterase (AChE), resulting in the production of acetate and choline (Ch).
3-choline is taken up in the presynaptic neuron by the high-affinity choline transporter (ChT).
4-in the presynaptic nerve terminal, choline is converted into ACh again by choline acetyl transferase (ChAT).
5-ACh is subsequently transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT).
example for glu
glutamine-glutamate cycle
> Glutamate is taken up by astrocytes using the excitatory amino acid transporters 1 and 2 (EAAT1, 2).
> In astrocytes, glutamate is converted into glutamine by glutamine synthase
> Glutamine is transferred back to neurons through system N transporters expressed by glia and system A transporters expressed by neurons. System N transporters (SN1, 2) exchange 1 Na* and 1 glutamine for 1 H. System A transporters (SA1-3) couple the movement of neutral amino acids (e.g. glutamine) to the flux of Na.
> Within neurons, glutamine is converted to glutamate (and ammonia) by phosphate-activated glutaminase (PAG).
> NT is also transported back into the presynaptic terminal by Na* and
CI-dependent plasma membrane transporters
(exproins previous suite)
7.3. [vesicular and plasma membrane nt transporters] infer how they can modulate synaptic strenght
> regulation of NT transporter activity contributes to synaptic plasticity:
* increased activity of SVNTTs -
-> increased NT concentration in
SVs, and simultaneously reduced NT concentration in the cytoplasm
* reduced NT uptake at the plasma membrane increases postsynaptic receptor activation but depletes NT stores
How is activity of PMNTTs regulated?
> gene expression regulation: regulation of transcription and splicing of pR
> trafficking: PMNTTs undergo both constitutive and regulated trafficking to and from the cell surface, a process influenced by multiple kinases (e.g. PKC, CaMKII, MAPK, ERK, PKG).
> Phosphorylation-dependent catalytic activation
> PMNTT-associated proteins:
* SNARE proteins, in particular syntaxin 1A (SYN1A): association of SYN1A with PMNTTs typically inhibits PMNTT activity
* kinases and phosphatases, e.g. PKC, PKG, CaMKIl, protein phosphatase 2A (PP2A)
* cell surface receptors, e.g. integrinß3-containing adhesion receptors (ITGB3), D2 DA receptors which associate with DAT, leading to enhanced DAT surface expression and DA uptake
Activity of PMNTTs is regulated at four levels:
* Gene expression regulation: Regulation of the transcription of genes encoding for transporters and of the splicing of pre-mRNAs. If you generate more mature mRNA for a given transporter, there will be a higher level of expression of the transporter on the presynaptic plasma membrane and thus the total activity will increase.
* Trafficking: The PMNTTs undergo trafficking to and from the plasma membrane. PMNTTs are synthesised in the rough endoplasmic reticulum (RER) and then follow the secretory pathway through the Golgi apparatus to the plasma membrane. Once a PMNTT is at the plasma membrane, it can be re-endocytosed, i.e., removed from the cell surface. An endocytosed receptor can be exocytosed again. This endo- and exocytosis determines the number of transporters present at the presynaptic plasma membrane and therefore also the activity of the transporters. This process of trafficking can be influenced by multiple kinases (PKC, CaMKII, MAPK, ERK, PKG, etc.).
* Phosphorylation-dependent catalytic activation: The transporter can be phosphorylated, which can directly affect its function (either making it more or less efficient).
* PMNTT-associated proteins:
o Syntaxin 1A (and other SNARE proteins): It can associate with PMNTTs and typically results in the inhibition of the activity of PMNTTs.
o Kinases and phosphatases: e.g., PKC, PKG, CaMKII, protein phosphatase 2A (PP2A).
o Cell surface receptors: E.g., D2 dopamine receptors that associate with dopamine transporters (DATs), which leads to enhanced DAT cell surface expression and thus enhanced DAT activity and increased dopamine uptake.
7.4. [vesicular and plasma membrane nt transporters] understand/explain the effects of drugs that act on the plasma membrane nt transporters (PMNTTs) -a- and how genetic mutations in PMNTTs may lead to disease -b-
A
Plasma membrane NT transporters (PMNTTs)
> PMNTTs use transmembrane ion or voltage gradients to energize the reuptake transport. Thus, PMNTTs rely on Na* and K* gradient across the plasma membrane, established by the ubiquitous Na/K ATPase
> PMNTTs are the targets of numerous psychoactive drugs (e.g. cocaine, amphetamine, …) and of drugs used for the treatment of psychiatric disorders, including schizophrenia, depression, attention-deficit hyperactivity disorder (ADHD).
OP
è.g. cocaine blocks the dopamine PMNTT; amphetamines bind to PMNTTs responsible for reuptake of NA, DA and 5-HT, with uptake of amphetamine resulting in efflux of these NTs from the cytoplasm to the synaptic cleft; also act on VMAT.
GABA, glycine, NE, DA and 5-HT transporters belong to a larger family of Na* and Cl-coupled transporters (SLC6 family) with 12 transmembrane domains and cytosolic N- and C-termini EAATs belong to a distinct family (SLC1) with 8-10 transmembrane domains
Members of the NE, DA, SERT monoamine transporter family bind extracellularly a Na* ion, the neurotransmitter, and a Cl ion. The transporter then flips to the inside of the cell, where the Na* ion, the neurotransmitter, and a Clion are released. After binding of a K* ion, the transporter flips back to the outside of the cell. Thus, the function of the transporter is dependent on the Na/K ATPase.
B
Diseases linked to PMNTT dysfunction
> A457P mutation in NET: leads to orthostatic intolerance (Ol), a.k.a. postural orthostatic tachycardia syndrome (POTS), characterized by syncope on standing. The A457P mutation strongly reduces NET surface expression and hence NE uptake. Interestingly, Ol subjects also show cognitive impairments, presumably due to reduced NET activity and increased NE signaling in the brain
> a promoter variant in the NET gene that results in recruitment of a transcriptional repressor leading to reduced NET expression, influences the risk for ADHD, as well as systolic blood pressure
> genetic variation in SERT contributes to obsessive-compulsive disorder (OCD) and autism-spectrum disorders (ASD)
> A559V variant in DAT is linked to ADHD and bipolar disorder
Clinical significance
PMNTTs are of high significance to pharmacologists. They are the targets of numerous psychoactive drugs (like cocaine and amphetamine) and of drugs used for the treatment of psychiatric disorders (like schizophrenia, depression, and ADHD). Examples:
* Cocaine: blocks the dopamine PMNTT. Therefore, dopamine concentrations are increased in the synaptic cleft and dopaminergic neurotransmitter is increased. However, because reuptake is also blocked, the cytosolic concentration of dopamine is reduced, which counteracts the filling of SVs with dopamine. This results in lower amounts of dopamine in SVs and thus a reduced amount of dopamine being secreted with exocytosis.
* Amphetamines: bind to the PMNTTs responsible for the reuptake of noradrenaline, dopamine, and serotonin. The amphetamines are then taken up into the presynaptic nerve terminal, which results in an efflux of the neurotransmitters from the cytoplasm to the synaptic cleft. Thus, the cytosolic concentrations of the neurotransmitters is reduced and the concentration inside the synaptic cleft is increased. Amphetamines also act on VMAT and thus also get imported into the SVs and cause the neurotransmitters to get exported from the SVs.
* Selective serotonin reuptake inhibitors (SSRIs): inhibit the serotonin PMNTTs. These are used for the treatment of patients with depression.
Several diseases have been linked to the dysfunction of PMNTTs:
* A457P mutation in NET: Orthostatic intolerance (OI) aka postural orthostatic tachycardia syndrome (POTS) are characterised by syncope (i.e., fainting) on standing. The norepinephrine transporter (NET) is dysfunctional due to a missense mutation (A457P: amino acid 457 is changed from A to P) in the gene encoding for this transporter. This mutation reduces the surface expression of the transporter -> reduced reuptake of norepinephrine -> increased concentration in the synaptic cleft. These patients also show cognitive impairment, presumably due to reduced NET activity and increased norepinephrine signalling in the brain.
* Promotor variant in NET gene: A promotor variant in the NET gene results in the recruitment of a transcriptional repressor, which leads to reduced transcription of the NET gene and thus reduced NET expression. This influences the risk for ADHD as well as systolic blood pressure.
* Genetic variation in SERT: Contributes to OCD and ASD.
* A559V variant in DAT: Is linked to ADHD and bipolar disorder.
7.5. [vesicular and plasma membrane nt transporters] sv vs plasma membrane transporters
——————-SV transporters for nt (SVNTTs)
> cytosolic NT concentrations depend on:
* NT biosynthesis by specific enzymes: can be rate-limiting, e.g. tyrosine hydroxylase (TH) in catecholamine biosynthesis. TH activity is regulated by phosphorylation and regulation at the transcriptional and posttranscriptional level
* NT reuptake by plasma membrane transporters
Note: NT biosynthetic enzyme and NT
tryptophan hydroxylase -serotonin (=5-HT)
choline acetyltransferase (ChAT) -acetylcholine (Ach)
_glutamic acid decarboxylase (GAD) -GABA
catecholamine NTs: dopamine and noradrenaline
5-HT= 5-hydroxytryptamine
——————-Plasma membrane NT transporters (PMNTTs)
Plasma membrane NT transporters (PMNTTs) use transmembrane ion or voltage gradients to energise the reuptake transport. Thus, PMNTTs rely on the sodium and potassium gradients across the plasma membrane, which is established by the ubiquitous Na+/K+ ATPase.
There are different families of PMNTTs based on structure:
* Sodium and chloride coupled transporters (SLC6) family: GABA, glycine, noradrenaline, dopamine, and serotonin. These transporters have 12 transmembrane domains and cytosolic N and C terminals.
* SLC1 family: EAATs. This small distinct family has 8-10 transmembrane domains.
> PMNTTs use transmembrane ion or voltage gradients to energize the reuptake transport. Thus, PMNTTs rely on Na* and K* gradient across the plasma membrane, established by the ubiquitous Na/K ATPase
PMNTTS are the targets of numerous psychoactive drugs (e.g. cocaine, amphetamine, …) and of drugs used for the treatment of psychiatric disorders, including schizophrenia, depression, and attention-deficit hyperactivity disorder (ADHD).
e.g. cocaine blocks the dopamine PlINTT, amphetamines bind to
PMINTTs responsible for reuptake of NA, DA and 5-HT, with uptake of amphetamine resulting in efflux of these NTs from the cytoplasm to the synaptic cleft; also act on VMAT.
GABA, glycine, NE, DA and 5-HT transporters belong to a larger family of Na* and Cl-coupled transporters (SLC6 family) with 12 transmembrane domains and cytosolic N- and C-termini
EAATs belong to a distinct family (SLC1) with 8-10 transmembrane domains
Members of the NE, DA, SERT monoamine transporter family bind extracellularly a Na* ion, the neurotransmitter, and a Cl ion. The transporter then flips to the inside of the cell, where the Na* ion, the neurotransmitter, and a Cl ion are released. After binding of a K* ion, the transporter flips back to the outside of the cell. Thus, the function of the transporter is dependent on the Na/K ATPase.
7.6. [vesicular and plasma membrane nt transporters] How is it possible that vesicular neurotransmitter transporters are able to pump neurotransmitters from the cytoplasm (where neurotransmitter concentration is relatively low) into the synaptic vesicles (where neurotransmitter concentration is high)? What fuels this process?
Vesicular neurotransmitter transporters exchange protons for neurotransmitter, and H+ concentration is much higher in the SVs as compared to the cytoplasm (the SV interior is acidic). This H+ electrochemical driving force fuels the import of neurotransmitter into the SVs. This H+ electrochemical gradient is established by the vacuolar-type H+-ATPase, which uses the energy released by ATP hydrolysis to pump protons in the SV lumen.
