PCOL3022 Lecture 7: Transporters

Difference between receptors, ion channels, and transporters

Receptors - Ligand interacts with the extracellular domain and does not enter the cell

Ion channels - Allows ions to passively diffuse into the cell (with electrochemical gradient)

Transporters - Moves substrates/everything into the cell against an electrochemical gradient

Difference between passive and active transport

Passive - Facilitated diffusion / The movement of molecules across the cell membrane via the aid of a membrane protein
(e.g. Na+-independent glucose transporter)

Active - Requires energy, moves substrate against a concentration gradient
(a) Primary - Energy comes from light/ Direct hydrolysis of ATP
(e.g. Na+K+ ATPase)
(b) Secondary - Uses pre-existing ion gradients / Indirectly coupling transport with another molecule that is moving along its gradient
(e.g. plasma membrane glutamate transporter)

Passive transport example

- Na+ independent glucose transporters

Primary active transporters examples

- Pumps
- Na/K ATPase

Secondary active transporters example

- Plasma membrane glutamate transporter
- Uses pre-existing ion gradients

Neurotransmitter Transporters location

- Post- and pre-synaptic neurons
- Glial cells

Neurotransmitter Transporters Function

- To clear neurotransmitters from the synaptic cleft

Neurotransmitter transporter type of transport

Secondary active

What is the role of glutamate and NSS family transporters?

- Glutamate family transporters regulate extracellular glutamate concentrations to maintain dynamic synaptic signaling processes

- NSS family transporters regulate termination of neurotransmission by rapid removal of neurotransmitters from the synaptic cleft

Glutamate transporter family responsibility

- Only responsible for glutamate and aspartate

What are glutamate transporters called?

- VLGUTs (not studied)
- EAATs (excitatory amino acid transporters)

Glutamate transporters co-transports...

1 glu-
3 Na+
1 H+
Counter transport of 1 K+

- Can maintain a 10^6 fold gradient across the membrane

How many subtypes of glutamate transporters are there?

- EAATs have 5 subtypes, with 50-60% amino acid identity
- EAATs are ~1% of total brain membrane proteins

EAAT Subtype 1

- Cell type: Glia
- Found in astrocytes
- CNS Location: Widely expressed

EAAT Subtype 2

- Cell type: Glia
- Found in astrocytes
- CNS Location: Widely expressed

EAAT Subtype 3

- Cell type: Neuron
- Found on post-synaptic neurons
- CNS Location: Widely expressed, kidneys

EAAT Subtype 4

- Cell type: Neuron
- Found on post-synaptic neurons
- CNS Location: Cerebellum

EAAT Subtype 5

- Cell type: Neuron
- CNS Location: Retina *can be on presynaptic neuron

Where can you find EAAT Subtypes 1 and 2?

Astrocytes

Where can you find EAAT Subtypes 3 and 4?

- Post-synaptic neurons
- To "mop up" excess glutamate

What do EAATs transport?

- Coupled to Na+, H+ with K+ counterion
- IN: Glu-, 3x Na+, H+
- OUT: K+
- Net transfer of 2+

- Can maintain a million-fold gradient across the membrane

EAAT common substrates, substrate inhibitors, and blockers

- Substrate = L-glutamate/L-aspartate
- Substrate inhibitors = D-aspartate
- Blocker = TBOA

What is the substrate inhibitor for EAATs 1 and 3, but blocker for EAATs 2 and 4?

4-MG

What is the Na+-dependent aspartate transporter from archaea?

Glt(Ph)

What is the competitive inhibitor (vs L-Asp) of Glt(Ph)? Benzene ring opened the ring

TBOA

What does TBOA's benzene ring prevent?

- Prevent HP2 from closing
- Thus preventing necessary conformational change

Structure of Glt(Ph)

- From Pyrococcus horikoshii (archaea -- easier to crystallise, stable under lab conditions)

- Trimer of 3 identical subunits, each capable of transport
- 37% identity to human EAAT 2

EAAT Excitotoxicity Process

- Ischaemia (lack of oxygen in the brain)
- Depletion of glucose
- Reduction of ATP

- Causes failure of Na+/K+ ATPase
- Causes rundown of membrane potential, uncoordinated action potential generation

- Causes excessive Glu release + Glutamate Transporter failure
- Cause excessive stimulation of Glutamate Receptors
- Causes excessive Ca2+ influx
- Causes activation of protease, lipases, NO synthase, endonucleases
- Causes cell death (necrosis/apoptosis)

Glutamate Transporters as Drug Targets

- For neurodegenerative conditions: Alzheimer's, Parkinson's, Motor neurone disease

NSS Family

Neurotransmitter sodium symporter

What NTs are the transporters for in NSS Family?

- Gly, DA, GABA, NA, 5HT

NSS Family coupled to...

- Co-transport of Na+ and Cl-

What are transporter/s for Neurotransmitter Gly? Function? Therapeutics?

- GlyT1 and GlyT2

- Function: Transport glycine

- Therapeutics: Pain, antipsychotics

Gly T1

- 2x Na+, Cl-
- Slightly less effective
- Found in excitatory neurones where some low level of Gly is required as a co-agonist of NMDA-Rs

Gly T2

- 3x Na+, Cl-
- More effective
- Found in inhibitory neurones where no Gly is required as an agonist

Pharmacological relevance of Gly T1

- Gly T1 inhibitors: sarcosine, NFPS
- Elevates [Gly] at excitatory synapses to increase NMDA-R stimulation
- Treats schizophrenia (thought to be linked to reduced NMDA-R activity)

Pharmacological relevance of Gly T2

- Enhance glycinergic inhibition in spinal cord
- Reduces excitatory transmissions of pain signals to brain
- Reduces perception of pain
- Could be useful in sp

What are transporter/s for Neurotransmitter GABA? Function? Therapeutics?

- GAT 1-4
- Function: Transport ABA
- In neurons/glial cells throughout CNS
- Therapeutics: Anticonvulsants drugs

GATs

- Found in neurons and glial cells in CNS
- Coupled to 2x Na+, Cl-
- Transporter inhibition increases inhibitory GABA transmission
- Used as a anticonvulsant
- E.g. tigabine, nipecotic acid (GAT1 selective)

Catecho

- DAT (DA): 2x Na+, Cl-
- Drugs of abuse: Amphertamine, meth, cocaine, MTPT

- NET (NA): Na+, Cl-
- Drugs of abuse: Cocaine, meth, amphetamines

- SERT (5HT): Na+, Cl- (potentially K+ as counterion)
- Drugs of abuse: MDMA, cocaine

Methamphetamine effects

- Meth has more exaggerated effects and higher addiction risk
- Effects: Alertness, euphoria, agitation, energy, increased libido, nausea, irritability (more intense than amphetamine)

Metham

- Cravings, withdrawal-depression, weight loss, psychosis, meth mouth

Methamphetamine overdose

- Brain damage, paranoia, delusions, hallucinations, death by stroke, heart failure, cardiac arrest

Amphetamine mechanism of action (AMP MOP)

- AMP is a false substrate of DAT
- AMP reduces DA uptake AND causes reverse transport of DA

- Competes with DAT/NET AMP accumulates in the cytoplasm, DA is elevated in synapse
- Depletes cystolic DA - reverse transport
- AMP inhibits MAO to prevent NT breakdown
- Competes with VMAT to deplete DA storage in vesicle

Cocaine Pharmacology

- Stimulant and appetite suppressant
- Na+ channel blocker (Used as a topical anaesthetic)

- Inhibits DAT, SERT, and NET
(Competitive blocker of NT transport -- molecule is too large to pass through transporter)

dDAT

- Drosophila dopamine transporter
- 12 x TMD, "shot glass" shape
- Substrate buried within
- Co-transport of 2 x Na+, Cl-
- Substrate and Na+ binding sites are similar to all of the NSS family