Channels Flashcards
What facilitates the movement of ions across the membrane?
- Ion channels facilitate the movement of ions across the membrane
- However, membrane potential will influence direction of ion flow
Describe the movement of ions when the cell is subject to standard equilibrium conditions and at resting membrane potential and why this occurs
• When a cell is subject to standard equilibrium conditions and at resting membrane potential (about -60 mV)
o Large potassium gradient
High potassium concentration inside the cell (around 140 mM) vs low potassium concentration outside the cell (around 5 mM)
When a potassium channel is open, the potassium will flow from inside the cell to outside the cell
o Large sodium gradient
High sodium concentration outside the cell (around 130 mM) vs low potassium concentration inside the cell (around 10 mM)
When a sodium channel is open, the sodium will flow from outside the cell to inside the cell
o Calcium gradient
Calcium is dynamic inside the cell (nM when cell is resting, uM when cell is activated) vs higher calcium concentration outside the cell (1-2 mM)
When a calcium channel is open, the calcium will flow from outside the cell to inside the cell
o Large chloride gradient
High chloride concentration outside the cell (around 105 mM) vs low chloride concentration inside the cell (10 mM)
When a chloride channel is open, the chloride will flow from outside the cell to inside the cell
o There are large impermeant anions in the cytoplasm (these come from proteins and RNA)
o Sodium/Potassium ATPase channel will pump 2 K+ into the cell for each 3 Na+ pumped out of the cell
The energy used for this is derived from the hydrolysis of ATP
Sets up gradient that allows ions to move and respond to physiological processes
Describe what voltage gated ion channels (VGICs) are present in the dendrites of a neuron
• Dendrites
o Proton gated ion channels (HCN)
o Potassium gated ion channels (4.2, 3 and 2.1)
o Calcium channels
Describe what voltage gated ion channels (VGICs) are present in the axon hillock of a neuron
• Axon hillock
o Voltage dependent sodium channels
o Cyclic nucleotide-gated ion channels
o Acid-sensitive ions channels
Describe what voltage gated ion channels (VGICs) are present in the nodes of ranvier of a neuron
• Nodes of Ranvier o Voltage-dependent potassium channels Prior to the node and at the node o Voltage-dependent sodium channels At the node
Describe what voltage gated ion channels (VGICs) are present in the pre-synaptic of a neuron
• Pre-synaptic terminal o Voltage-dependent sodium channels o Voltage-dependent potassium channels o Calcium channels Trigger neurotransmitter release in the terminal to stimulate post-synaptic membrane
What are the roles of Kv and Nav channels in propagating the action potential?
• Nav opens in response to depolarization of the membrane potential
• Inward current due to the influx of Na+ causes further depolarization
o Nav will spontaneously inactivate after being open for 1-2 msec
• Depolarization also causes opening of Kv channels
• K+ ions flow out of the cell
o Speeds up the rate of depolarisation- causes the membrane potential to overshoot the resting state
o A diversity of Kv channels allows for variation in the duration of the action potential and the neuron’s rate of firing
Describe the format for voltage-gated ion channel nomenclature
• Formula: Xy0 o Where X is the ion that permeates the channel o Where y is the description of the channel/how the channel is activated o Where 0 is the subtype of the class of channel
Describe the voltage-gated ion channel family
• 143 members of the family in 7 major groups o Two major families Potassium channels TRP channels o Smaller families include Sodium channels • 9 major subtypes Calcium channels • 3 major subtypes CNG channels HCN channels TPC channels
What is the Nav1.7 channel, what is it expressed in, what is the impact of mutations in this channel, why is it an appealing drug target and what drugs are being developed for it?
• Mutations in Nav1.7 causes a complete loss of Na+ channel function and hence an inability to sense pain
• Nav1.7 is exclusively expressed by sensory neurones and so is a potential target for drugs to treat pain
• Selectivity for Nav 1.7 is critical because effects on related Nav channels would be fatal
• There are a number of pharmaceutical companies and academics working on developing selective Nav1.7 inhibitors but none of these drugs are on the market
o PF-05089771 (Pfizer)
o Monoclonal antibodies (Duke university)
o Tarantula toxins (peptides) are selective Nav1.7 inhibitors
University of Queensland and USYD
What is the purpose of Kv channels?
• Kv channels- large family with diverse functions
o Stabilising force
Setting the cells resting membrane potential
Repolarizing the cell after an action potential
Controlling the cell’s rate of firing and shape of the action potential
Do Kv channels always have 6 transmembrane domains?
No.
6 transmembrane K+ channels and 2 transmembrane K+ channels
What are the 4 main classes of Kv channels?
- Delayed rectifiers
- A-type channels
- Ca2+-activated K+ channels (KCa)
- Inward rectifiers
What is the purpose of Kv delayed rectifier channels?
• Delayed rectifiers- delayed activation after depolarization and inactivate slowly- facilitate repolarization
What is the purpose of A type channels?
• A-type channels transiently activated when a cell is depolarized after a period of hyperpolarization- decreases firing frequency
What is the purpose of Ca2+-activated K+ channels (KCa)?
• Ca2+-activated K+ channels (KCa)- respond to Ca2+, remain open for prolonged period- prolong hyperpolarization
o Open in response to binding of Ca2+- after depolarization induced Ca2+ entry
o Remain open for a long period (about a few seconds)
o Cause “long after hyperpolarization”- hyperpolarization after an action potential-can slow the rate of firing of action potentials
What is the purpose of inward rectifier channels?
• Inward rectifiers- G proteins can regulate their activity (GIRKs)
What is the molarity of extracellular neuron calcium?
1.5mM
What is the molarity of intracellular neuron calcium?
• Intracellular Ca2+ is 0.1-0.2 uM but may rise to 100 uM after opening of Ca2+ channels
What can calcium entry in a neuron trigger?
• Ca2+ entry can trigger many intracellular processes o Muscle contraction o Neurotransmitter release o Activation of second messenger systems o Alteration in gene expression o Apoptosis (cell death) o Depolarization- Ca2+ spikes
What calcium channels are classified as L type?
Cav1.1- 1.4
What calcium channels are classified as P/Q type?
Cav 2.1
What calcium channels are classified as N type?
Cav 2.2
What calcium channels are classified as R type?
Cav 2.3
What calcium channels are classified as T type?
Cav 3.1-3.3
Where is Cav1.1 found?
Cav 1.1- skeletal muscle
Where is Cav1.2 found and what is it inhibited by?
Cav 1.2- cardiac muscle, smooth muscle, brain
• In neurones, these channels are found in the cell body and in proximal dendrite (they are not involved in neurotransmitter release at the synaptic terminal)
• Inhibited by verapamil, nifedipine
What is the function of Cav2.1-Cav2.3 channels?
• Regulation of neurotransmitter release- Ca2+ influx through these channels causes release of neurotransmitter
Why are synthetic peptide blockers being developed for Cav2.2 channels?
• Synthetic peptide blocker of N type channels is being developed for the treatment of chronic pain
What is the function of Cav3.1-3.3 channels?
• Repetitive firing of neurons
What do TRP channels respond to and what are their major roles?
- TRP channels respond to a wide variety of sensory stimuli- temperature, touch, pain osmolarity, pheromones, taste and other stimuli
- Major roles in pain perception- heat, cold, sensitive to capsaicin
What are the two major TRP channels involved in pain perception?
o Major members involved in pain perception are TRPV1 and TRPA1
Describe what TRPV1 channels are sensitive to and what they are activated by
TRPV1 is sensitive to different physical and chemical stimuli, including
• Heat
• Acidic pH
• Mechanical stimuli
TRPV1 is also activated by a variety of ligands:
• Vanilloids, such as capsaicin, the major pungent constituent of chilli
• Cannabinoids
• Ginsenosides
• Various animal-derived toxins, such as VaTx1, VaTx2 and VaTx3 found in the venom of the tarantula
• Endovanilloids, including leukotriene B4 and 12-S-HPETE and anandamide
What does activation of nociceptive TRP channels result in?
o Activation of nociceptive TRP ion channels in dorsal root ganglion neurons leads to the influx of Na+ and Ca2+ resulting in membrane depolarization that can trigger voltage-gated ion channel-dependent action potentials that transmit the information to the spinal cord and the higher nerve centres
What are channelopathies?
• Channelopathies- diseases caused by disturbed function of ion channel subunits or the proteins that regulate them. These diseases may be either congenital (often resulting from a mutation or mutations in the encoding genes) or acquired (often resulting from autoimmune attack on an ion channel).
What ion channels affect episodic ataxia?
Kv1.1, Cav2.1
What ion channels affect paralysis?
Cav1.1, Nav1.4
What ion channels affect myotonia?
Kv1.1, Nav1.4, CLC1
What ion channels affect long qt syndrome?
Kv1.7, MinK, MIRP1, Nav1.5
What ion channels affect seizures?
Kv7.2, Kv7.3, KCa, Nav1.1, Nav1.2, Cav (β subunit), CLC2
What is the structure of the voltage-dependent sodium channel? What structure is critical for the function of this channel?
• Voltage-dependent sodium channel (Nav)
o Large α subunit that contains 4 groups of 6 transmembrane domain (s1-6) within itself
s4 domain contains a series of positively charged residues which are critical for the function of the voltage-dependent channels
This is common to ALL VGICs
o β subunits (β 2/4 and β 1/3 for voltage-dependent sodium channels)
What is the structure of the voltage-dependent calcium channel? What structure is critical for the function of this channel?
• Voltage-dependent calcium channel (Cav)
o Large α subunit that contains 4 groups of 6 transmembrane domain (s1-6) within itself
s4 domain contains a series of positively charged residues which are critical for the function of the voltage-dependent channels
o Has α, β and γ subunits which modulate function and affect trafficking
What is the structure of the voltage-dependent potassium channel? What structure is critical for the function of this channel?
• Voltage-dependent potassium channels (Kv) and TRP channels
o The α subunit has 1 group of 6 transmembrane domains (s1-6)
s4 domain contains a series of positively charged residues which are critical for the function of the voltage-dependent channels
However, 4 SEPARATE proteins (4 SEPARATE α subunits) associate to form a single ion channel
Describe the commonalities of alpha subunits in all VGICs
o α subunits
The α subunits of the Na+, K+ and Ca2+ channels show considerable structural similarity
The α subunits can function on their own and contain all the proteins required for function of the VGIC
What is the function of beta subunits in voltage-gated ion channels?
o β subunits
The accessory or β subunits are more diverse and modulate the function of the α subunit
β subunit can regulate expression levels, location and trafficking
β subunit can alter voltage dependence of activation or inactivation
β subunit can bind drugs that modulate function
Phosphorylation of β subunit can regulate VGIC functions
What molecular/structural properties of VGICs are responsible for voltage-gated ion channel ion selectivity?
o Contain an aqueous pore that controls selectivity for Na+/K+/Ca2+ ions
o All VGICs have similar pore structure
o The selectivity filters of VGICs
What is the role of the aqueous cavity of VGICs?
Aqueous cavity- where water molecules and ions can flow through (no selectivity)
• This is the part of the channel that closes or opens
What is the role of the selectivity filter in VGICs and how does it achieve this role? Where is it found?
Selectivity filter- filter found at the end of the aqueous channel that selects for a specific ion
• Specific interactions of ions with the amino acids side chains in this selectivity filter make it selective
• This part of the channel is static
• Found near the extracellular edge of the pore
How much more sensitive are potassium channels to potassium over sodium?
K+ channels are 100-1000-fold selective over Na+
How much more sensitive are sodium channels to sodium over potassium?
Na+ channels are 10-fold selective for Na+ over K+
How much more sensitive are calcium channels to calcium over other cations?
Ca2+ channels are 1000-fold selective for Ca2+ over other cations
How do voltage gated ion channels open in response to changes in membrane potential? How does this work and what allows this to happen?
o VGIC open in response to changes in membrane potential
VGICs contain a voltage sensor, which moves in response to changes in membrane potential
• 25 degree tilt and 3A shift in S4 transmembrane domain in response to a change in membrane potential will pull open the channel
o Directional movement depends on how attracted the positive charges on S4 are to different sides of the membrane
Opposite charges attract
o S4 domain is linked through linker region to the bottom of the aqueous cavity of the channel
This means that movement of the S4 domain will pull the linker region and hence the aqueous cavity of the channel
Regulatory domains in some related channels can regulate opening of channels- e.g. Ca2+ activated K+ channels, cAMP regulated K+ channels
What does it mean when a voltage gated ion channel is deactivated and how does it happen? Is it a slow or fast process?
o Deactivated state
When the membrane potential reverses back to its resting state the channel may close (opposite of activation)
Slow process
What does it mean when a voltage gated ion channel is inactivated and how does it happen? Is it a slow or fast process? Give an example of such a channel
Voltage dependent Na+ channels will close immediately after being activated (even when there is still depolarization)
• Influx of sodium ions through sodium channel will induce a change in conformation of the intracellular region of the protein
Ball and chain model for inactivation- influx of positive charge will cause an intracellular domain to swing into the open aqueous pore of the channel to prevent ion flow
• However, the aqueous pore itself is still in the same conformation as when the channel is open
• Connection between voltage sensor and aqueous pore of the channel is mediated by a linker region
Rapid process
Blocks activation of channel until the ball is released from channel pore
Would blocking all Nav channels have a therapeutic effect?
• Blocking all Nav channels would have major unwanted side effects
o Complete loss of sensory system, sedations, coma, possible death
In general, how are therapeutic effects produced by blocking Nav channels and what determines their benefits? Why are these channels good to modulate with drugs?
• Therapeutic effects can be achieved through selective modulation of Nav channel subtypes
o Many Nav blocking drugs gain access to the channel by binding to the open state of the pore- that is, only active neurones are affected
o Rates of activation and inactivation are key to determining selectivity of effects
o There are 9 main types of Na+ channel α subunits
These are in a variety of different tissues, and hence different tissues can be targeted by targeting different α subunits
What are examples of drugs that modulate Nav activity?
- Tetrodotoxins
- Phenytoin and carbamazepine
- Local anaesthetics (cocaine, lidocaine, procaine)
What is the mechanism through which tetrodotoxin works on Nav channels and what is its use?
Mechanism:
- Binds to the external surface of the α subunit of the channel in the S5-S6 loop region and blocks the pore
- Variation in this region generate channel with differing sensitivity
Use:
- Different sensitivities to tetrodotoxin: a toxin found in marine species such as puffer fish, globe fish and blue ringed octopus
- Used mainly as an experimental tool to isolate the effects of Na+ channels in vitro
What is the mechanism through which Phenytoin and carbamazepine works on Nav channels and what is its use?
Mechanism: Slow the recovery from the inactivated state and stabilise it in inactivated state- limits the firing rates of neurons
Use: Therapeutic- Used for the treatment of epilepsy by preventing seizures
What is the mechanism through which Local anaesthetics (cocaine, lidocaine, procaine) works on Nav channels and what is its use?
Mechanism:
- Non-ionized form crosses the membrane
- Binds to the Na+ channel at sites exposed to the lipid membrane (fenestrations) and blocks the channel
- Causes drug to bind to the inactivated state of the channel creating a use dependent blocker
Use: Loss of sensation, awareness or pain numbing
Are drugs that modulate voltage-gated potassium channels therapeutic or experimental? Give two examples
- Wide variety of K+ channel blockers- not many that are used to treat neurological disorders (more experimental tools than therapeuticals)
- Tetraethylamonium (TEA) inhibits most K+ channels and is a very useful research tool
- Cs+ inhibits delayed rectifiers: KCa, KIR, KATP
What are two examples of drugs that modulate calcium voltage-dependent ion channels and how do they work? What are they used for?
• Gabapentin and pregabalin are used for the treatment of chronic pain and epilepsy
o Bind to the α2δ subunit and disrupt trafficking of the channel to the membrane
Reduced expression of this channel at the cell surface-> means there is a smaller response
o Selective for CaV2.2 (N-type) that regulate neurotransmitter release in sensory neurons
What are the two main classes of ligand-gated ion channels (LGICs)?
- Pentameric LGIC superfamily (also called Cys-loop receptors and nicotinoid receptors)
- Tetrameric- Excitatory ionotropic glutamate receptors (excitatory)
What are types of pentameric ligand-gated ion channels?
o nACh receptors (excitatory)
o 5-HT3 receptors (excitatory)
o GABAA receptors (inhibitory)
o Strychnine-sensitive glycine receptors (inhibitory)
What are types of tetrameric ligand-gated ion channels?
o AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) o Kainate o NMDA (N-methyl-D-aspartate)
Describe the typical structure and construction of pentameric LGICs
o 5 separate subunits- two alpha subunits, two beta subunits and one γ subunit
Each subunit has 4 transmembrane domains, an intracellular loop and an extracellular region/cys loop (where there is a disulfide bond)
When 5 subunits come together to form the single ion channel, the 2nd transmembrane domain of each of the 5 subunits come together to form the pore through which the ions will flow
Describe the second transmembrane domain of acetylcholine/serotonin receptors
o Acetylcholine/5HT3 receptors-
Second transmembrane domain contains negatively charged residues at the top and the bottom to select for positively charged ions
Describe the second transmembrane domain of GABA/glycine receptors
o GABA/Glycine receptors-
Second transmembrane domain contains positively charged residues at the top and the bottom to select for negatively charged ions
Describe how the second transmembrane domain confers ion selectivity in ligand-gated ion channels and why. Include how the composition of the second transmembrane protein influences the open/closed conformation in ligand-gated ion channels.
o Second transmembrane domain (M2) pore lining helices contain charged residues that confer ion selectivity
o In closed conformation, alpha helices bend towards the centre of the pore, leading to a narrow region that is too narrow and too hydrophobic to allow ion passage through the channel
Hydrophobic residues in the closed conformation are directed inwards towards the centre of the channel-> block passing of molecules through channel
o Open state- Pore opening occurs via rotation and tilting of the pore lining helices
Hydrophobic residues are pulled apart- allows for water molecules to pass through the channel
Ligand binding to extracellular domain provides energy to cause conformational change in protein, which causes opening of the channel
Describe the typical structure of excitatory glutamate receptors (tetrameric) and how these relate to function
From extracellular to intracellular units-
o Amino terminal domain
o Ligand binding domain
Where ligands bind- when ligands do bind, this structure closes down and causes amino terminal domain rotation, causing channel activation
• 4 subunits- two GluNR1 subunits and two GluNR2 subunits
o Glutamate binds to the GluNR2 subunits
o Glycine and D-serine bind to the GluNR1 subunits
o Transmembrane domains (M1-4)
Residues in transmembrane domain 2 control cation permeability- RNA editing
Flip/Flop region yields 2 splice variants for each subunit
What are the electrical properties of AMPA receptors compared to NMDA receptors in terms of:
- Activation speed
- Desensitisation speed
- Part of the excitatory postsynaptic current component it is responsible for
- Ion selectivity
- Expression pattern
AMPA:
- Fast activation
- Fast desensitisation
- First part of the excitatory postsynaptic current component
- Most selective for sodium
- Some allow calcium (dependent on RNA editing)
- Widely expressed
NMDA:
- Slow activation
- Slow desensitisation
- Second part of the excitatory postsynaptic current component
- Allows sodium and calcium permeation
- Widely expressed
Are AMPA and NMDA receptors often separate or co-localised?
• AMPA and NMDA receptors are often co-localised
How is the NMDA receptor activated?
• Voltage-dependent block by magnesium:
o AMPA receptor needs to be activated by glutamate for the neuron to become depolarised
o Magnesium block (that is sitting in pore of channel) is only released when the cell is depolarised (above positive membrane potential) and when BOTH glutamate and glycine are bound
o Once magnesium block is released, the glutamate and glycine can activate the NMDA receptor
• Unlike other glutamate receptors that only need glutamate for activation, NMDA receptors require both glutamate and glycine for activation
o May also use D-serine instead of glycine
What are the endogenous ligands of pentameric LGICs and what ions do they allow?
o Endogenous ligands GABA (inhibitory) • Allow anions/chlorine to flow through Glycine (inhibitory) • Allow anions/chlorine to flow through Acetylcholine (excitatory) • Allow cations/sodium to flow through 5HT3 (excitatory) • Allow cations/sodium to flow through
Where does GABA bind in pentameric LGIC receptors?
• Binds at interface between α and β subunits
Where does the ligand generally bind in pentameric LGICs? What is the consequence of this?
o Ligand binds to extracellular domain, which changes the conformation of the protein such that the gap between the subunits (in transmembrane domain) is opened-causes activation of the channel
Allows for ion flow
Describe NMDA receptors in terms of:
- Subunits
- Ions
- Agonists
- Antagonists
- Subunits: NR1, NR2A, NR2B, NR2C, NR2D
- Ions: Na+, Ca2+, K+
- Agonists: Glutamate, NMDA, Aspartate, Glycine
- Antagonists: D-AP5/D-APV, MK-801, Ketamine, Phencyclidine
Describe AMPA receptors in terms of:
- Subunits
- Ions
- Agonists
- Antagonists
- Subunits: Glu1-4
- Ions: Na+, (Ca2+)
- Agonists: Glutamate, AMPA
- Antagonists: CNQX, NBQX, GYK153655
Describe kainate receptors in terms of:
- Subunits
- Ions
- Agonists
- Antagonists
- Subunits: Glu5-7, KA1-2
- Ions: Na+, (Ca2+)
- Agonists: Glutamate, Kainate
- Antagonists: CNQX, Ly294486
What kinds of subunits can acetylcholine LGICs contain?
α1-9
β1-4
What kinds of subunits can serotonin LGICs contain?
A
B-E
What kinds of subunits can GABA LGICs contain?
α1-6 β1-4 γ 1-3 δ,ε,π,θ p
What kinds of subunits can Glycine LGICs contain?
α1-3
β
What percentage of amino acid sequence identity is there between subunits of different types?
• 30% amino acid sequence identity between α, β etc.
What percentage of amino acid sequence identity is there between subunits of the same type?
• 70% identity between α1-6 etc.
Why is there not larger diversity in the arrangement of subtypes in LGICs?
• Potential for a large number of receptor subtypes
o But there are number of preferred arrangements of subunits, so diversity is not as large as it could potentially be
What properties do channels vary in depending on their subunit combinations?
• Channels vary in agonist and antagonist sensitivity, ion conductance properties, rates of channel activation and desensitisation due to subunit diversity
What are common subunit combinations in acetylcholine LGICs?
o nAChR: homomeric α1 or α7, or a ααβββ heteromeric subunit combination
What are common subunit combinations in serotonin LGICs?
o 5HT3R homomeric A, or A +1 other subunit
What are common subunit combinations in GABA LGICs?
o GABAAR heteromeric ααββ + 1 other subunit or homomeric p
What are common subunit combinations in Glycine LGICs?
o GlyR homomeric α1 or ααα, ββ or ααββ
What is a homomeric receptor?
• Homomeric receptor- contains 5 copies of the same subunit
What is a heteromeric receptor?
• Heteromeric receptor- contains different subunits
o Although it is rare that each subunit class (α, β…) is of a different type (as in they are most likely all, for example, α1 or such in a heteromeric receptor)
What drugs can modulate the activity of GABAa receptors?
- Bicuculline
- Benzodiazepines (diazepam, temazepam)
- Barbituates (such as pentobarbitones)
- Ethanol
- General anaesthetics (e.g. propofol)
- Neurosteroids (e.g. allopregnanolone)
What is bicuculline and what is it used for?
• Bicuculline
o Competitive antagonist of most GABAA receptors
o Block inhibitory neurotransmission- cause massive excitation and convulsion
Can cause death
o Not therapeutic, but experimental-> inactivates GABAA receptors
What is the effect of benzodiazepines (diazepam, temazepam) and how does it act?
o Enhances the effect of GABA- has to work in conjunction with GABA
o Positive allosteric modulators of GABAA receptors
Channel opens at higher frequency but channel open time remains the same
What are the therapeutic effects of benzodiazepines mediated by?
o Therapeutic effects of benzodiazepines (BZ) are mediated by different α subunits
What are the therapeutic effects of benzodiazepines?
Benzodiazepines cause sedation, muscle relaxation, anxiolysis
How do benzodiazepines work? Why is their effect specific to certain GABA receptors and what are these GABA receptors? Describe
Benzodiazepine binding site contains a crucial histidine residue, which is present on α1, α3, α5- but not α4 and α6
• Benzodiazepines bind at the interface between α and γ subunits to cause activation
• Therefore, benzodiazepines are selective for GABAA receptors containing α1, α3, α5 and γ subunits
How can the effects of benzodiazepines on individual alpha subunits of GABAa receptors be elucidated experimentally?
Can knockout the effects of benzodiazepines on individual subunits by creating a histidine to arginine (H to R) mutation for experimental procedures
• Express in mice either a single subunit H to R mutant to knockout function of that particular subunit OR leave on the 4 α subunit as wild type and the other three as mutants to see which pharmacological effect is retained
What alpha subunit mediates the sedative actions of benzodiazepines when it acts on GABA receptors?
Sedative actions are mediated by α1
What alpha subunit mediates the anxiolysis actions of benzodiazepines when it acts on GABA receptors?
Anxiolysis actions are mediated by α2
What alpha subunit mediates the muscle relaxation actions of benzodiazepines when it acts on GABA receptors?
Muscle relaxation is mediated by α2 and α4
What alpha subunit mediates the impaired motor coordination actions of benzodiazepines when it acts on GABA receptors?
Impaired motor coordination is mediated by α1 and α3
What is the effect of barbituates on GABA receptors, how do they act and what is their use?
• Barbituates (such as pentobarbitones)
o Enhance activity of GABAA receptors
o May act alone to stimulate receptor directly or enhance effect of GABA to stimulate receptor
o Act on all GABAA receptors and makes them open for longer periods of time
o Not as therapeutically useful
What is the effect of ethanol on GABA receptors, how do they act and what is their use?
• Ethanol
o Enhances the actions of GABA
o Prolongs the open time of the GABAA receptor channel
o Binds within the transmembrane domain of the receptor
What is the effect of general anaesthetics on GABA receptors and how do they act?
• General anaesthetics (e.g. propofol)
o Enhances the actions of GABA and GABAA receptors
o May directly stimulate receptors at high concentration
o Bind at the interface between α and β subunits within the transmembrane region of the channel
Between transmembrane domains 1 and 4 and coming close to transmembrane domain 2
• Alters conformation of transmembrane domain 2 to stabilise membrane in its open state, facilitating more chlorine ions moving through the channel
What is the effect of neurosteroids on GABA receptors and how do they act?
• Neurosteroids (e.g. allopregnanolone)
o Promote channel opening
Enhance activity of GABAA receptor
o δ subunits are most sensitive
What drugs can modulate glycine receptors?
- Strychnine
- Ivermectin
- Picrotoxin
What is the effect of strychnine on glycine receptors and how does it work?
• Strychnine-
o Antagonist poison that causes paralysis
o Bind at subunit interface (as does glycine)
Binds between two α subunits
What is the effect of ivermectin on glycine receptors and how does it work?
• Ivermectin-
o Allosteric enhancer of glycine receptors
o Binds to the transmembrane domain to keep channel in open state
What is the effect of picrotoxins on glycine receptors and how does it work?
• Picrotoxin
o Not therapeutic drug
o Inhibitor of homomeric glycine receptors
Stabilised by residues proline (position 250) and threonine (position 258) on α subunits
o Heteromeric glycine receptors is not affected much by picrotoxin
Does not have the required residues for stabilisation as only α subunits have the required residues- if there are not 5 α subunits, then the picrotoxin cannot be stabilised
What drugs can modulate serotonin receptors?
- Alosetron
* Ondansetron
What is the drug alosetron used for?
o Irritable bowel syndrome
What is ondansetron used for and how does it bind to serotonin receptors?
• Ondansetron
o Anti-emetic (nausea associated with chemotherapy)
o Competitive antagonist
o Likely to bind at the 5HT3 binding site at the interface between subunits to inhibit activity at the serotonin receptor
Describe drugs that can bind to the amino terminal domain of AMPA and kainite receptors
o ATD
Lectins
Describe drugs that can bind to the ligand binding binding pocket of AMPA and kainite receptors
o LBD agonist binding pocket
Partial and full agonists
Competitive antagonists
Describe drugs that can bind to the ion channel pore of AMPA and kainite receptors
o Ion channel pore
Uncompetitive antagonists
Polyamines
Cations
Describe drugs that can bind to the ligand binding domain dimer interface of AMPA and kainite receptors
o LBD dimer interface Cyclothiazide (AMPA) Aniractetam (AMPA) CX614 (AMPA) Cl- and K+ (kainate) Ca2+ (GluD2)
Describe drugs that can bind to the ligand binding domain-TMD linkers of AMPA and kainite receptors
o LBD-TMD linkers
GYKI-53655 (AMPA)
CP-465,022 (AMPA)
What are the four basic classes of receptors? Describe their basic mechanism and the timescale they act on
• Ligand-gated ion channels (ionotropic receptors)
o Time scale: milliseconds
o Binding of ligand results in ion flux-> hyperpolarisation or depolarisation-> cellular effects
• G-protein coupled receptors (metabotropic)
o Operate through coupling to other signalling elements inside the cell
Signal to ion channels-> change in excitability-> cellular effects
Signal to second messengers-> calcium release OR protein phosphorylation OR other-> cellular effects
• Kinase-linked receptors
o Time scale: hours
o Protein phosphorylation-> gene transcription-> protein synthesis-> cellular effects
• Nuclear receptors
o Ligand binds to receptors in nucleus-> gene transcription-> protein synthesis-> cellular effects
o Time scale: hours
Describe the basic structure of all G-protein coupled receptors
o All G-protein coupled receptors possess a homologous organised 7 α-helix transmembrane domain that interact with G protein
7 sequence stretches of about 25-35 consecutive residues that show a relatively high degree of calculated hydrophobicity
These sequences represent 7 alpha-helices that span the plasma membrane in a counter-clockwise manner forming a receptor
What is the function of GPCRs? Describe
o Sense extracellular signals
Sensory: light, taste, odours etc.
Neurotransmitters (nearly all)
• Glycine does not interact with G-protein coupled receptors
Hormones
Growth factors
o Transduce signal to intracellular side of cell from extracellular side
Induce G-protein activation and other intracellular signals
What is the size of the GPCR family? What percentage of the genome do they compose?
• Large diverse family: consist of more than 800 proteins in humans (more than 4% of the genome)
What are major drug targets of GPCRs?
More than 60% of all drugs • Monoamines (NA DA 5HT) • Other amines (e.g. histamines) • Amino acids • Other small molecules (ACh etc) • Lipids (e.g. cannabinoids) • Peptides • Chemokines • Etc.
What technological advances propel GPCR research and how?
• Radioligand binding techniques (1970s)
o Identification of receptors
• Second messenger neurochemistry (1970s)
o Demonstrated interaction with G-proteins and GTP
• GPCR protein purification (early 80s)
o Identification of relatives and families
• GPCR molecular cloning (mid 80s)
o Changing GPCRs for experimental purposes to understand their function
• GPCR crystal structure elucidation (from 2010-)
o Crystal structure of GPCRs
• Cryo-EM structure elucidation (from about 2015-)
o Identification of how the GPCRs signal
What are the different types of GPCR receptors?
- Glutamate receptors
- Rhodopsin
- Adhesion
- Frizzled/Taste2
- Secretin
Describe the structure of GPCR glutamate receptors and how these receptors work. Describe their substrate.
o Venus flytrap domain where the ligand binds
Binding domain of receptor is open until ligand binds, which causes it to close
Causes conformational shift in the transmembrane regions/intracellular regions
o The N-terminus of mGluR forms two distinct lobes separated by a cavity
o Function not as single proteins, but as obligate dimers in the membrane (two proteins, usually of the same type, combine)
Associations between the two are fairly loose and can be promiscuous
o Glutamate binds in the cavity, causing the lobes to close around the ligand (Venus fly trap model)
o GABAB has a similar mechanism
o Restricted to very small molecule neurotransmitters (including glutamate and gamma aminobutyric acid)
Describe the structure of GPCR rhodopsin receptors and how these receptors work/interact with the substrate
o Ligand binds in the membrane (in barrel of alpha helices) and transduces the signal
Ligand can also be permanently bound in the receptor with high affinity to keep the ligand trapped, and the interaction with light causes a cys-trans isomerism of the ligand in the receptor-> activates the receptor
o The ligand activation site is deep in extracellular mouth of most of the rhodopsin class GPRCs
Ligands can either be loosely bound (e.g. u-OR receptor) or extremely tightly and permanently bound with high affinity to the binding site of the receptor (e.g. M3 receptor)
Why is the glycoprotein hormone family an exception to the general rules of the rhodopsin GPCR family?
Glycoprotein hormone family (e.g. growth hormone)
• Large peptide receptors
• Extracellular domains bind to the peptides with extremely high affinity-> binding produces transformation
Why are protease-activated receptors an exception to the general rules of the rhodopsin GPCR family?
For protease activated receptors (PARs), the ligand is embedded in the N-terminal of the GPCR but can only be activated by a protease binding to the N terminal domain to cause hydrolysis
Describe the structure, workings and function of the adhesion GPCR family?
• Adhesion
o Two main extracellular domains that, through a torsion mechanism, transduces the signal into the rest of the protein
Adhesion domains
Gain domain
o Have large N-terminals: some bind to ECM proteins
Some of these are mechanosensors
Describe the main component of the frizzled GPCR family of receptors
o Cystein-rich domain
Describe the main component of the secretin GPCR family of receptors
o Hormone receptor motif domain
What technological developments were vital in determining how GPCRs activate G proteins and what did each involve/demonstrate?
• Advent of cryo-electron microscopy and X-ray crystallography were vital in determining how GPCRs activate G proteins
o X-ray crystallography involved getting extremely high-affinity antagonist states of the receptor crystallised
μ-opioid receptor antagonist bound crystal
o Cryo-electron microscopy
μ-opoid receptor
Antagonist bound crystal
Agonist and Gi bound (cryo-EM)
Shows orientation of helices
Describe the conformational changes of GPCRs from an inactive state to an active state?
• Conformational switch from inactive to active state
o Small transformation/reorientation in transmembrane 5 and 6 (barrels in the membrane) and α helix 8 that sits inside the C terminal region
o Transduction process is a reorientation of the C terminal and intracellular helices of the protein to reduce the activation of the GTP binding protein
Describe the G-protein receptor activation sequence (how GPCRs work)
o The portion of receptor on the inside of the cell can bind to the G protein which has a guanosine diphosphate attached to an alpha subunit
o When the ligand binds to the receptor site, the G protein changes conformation and guanosine triphosphate replaces the guanosine diphosphate (GDP) that was previously on the alpha subunit of the G protein
o The activated alpha subunit separates from the beta and gamma subunits- this step can be repeated as long as the ligand remains bound to the receptor
When the ligand separates from the receptor site, additional G proteins are no longer activated
• The GPCR cycle amplifies the receptor signal whilst the agonist is resident on the receptor
This separation produces a set of intracellular signals
Beta and gamma subunits (which usually has a lipid attached to it and is hence trapped to the cell membrane) also produces signals
• Lateral diffusion of the subunits
• Signal mostly to ion channels, PI3Kγ and PLCβ
Alpha subunit diffuses to intracellular sites (even the nucleus) to cause their effects
o Inactivation of the alpha subunit occurs when its own hydrolytic activity removes a phosphate from the GTP, leaving GDP
GTP is hydrolysed to GDP
How quickly this hydrolysis occurs depends on the G protein
o The alpha subunit regains affinity for the receptor and the G protein subunits then recombine and attach to the receptor in the cell membrane
In GPCRs, what determines the type of signal sent during the G-protein receptor activation sequence?
o The Gα subunit type largely determines type of signal that α subunits send during the G-protein receptor activation sequence
Alpha family type determines type of signal
Does the G-protein cycle of GPCRs amplify or dampen signals?
o The G-protein cycle amplifies signals
What are the main different Galpha subunit types in GPCRs?
- Gαs
- Gαi/αo
- Gαq
- Gα12 etc.
What is the role of Gas subunits in GPCRs and what molecules do they act on?
o Activates cAMP cascade
o Gs stimulates cAMP formation, etc. (e.g. D1R, βAR)
o Stimulates activity of Adenylyl cyclase, Axin, cAMP and PKA
What is the role of Gai/Gao subunits in GPCRs and what molecules do they act on?
o Gαi coupled receptors are generally inhibitory: the major transducers in neurons are GIRK and VGCCs
o Acts on adenylyl cyclase, phosphodiesterases, phospholipases, cAMP
o Gi/0 inhibits cAMP, activates or inhibits ion channels (e.g. D2R, α2AR, 5HT1, GABAB, u-opioid)
Describe how the Gai/Gao subunits in GPCRs work to produce an inhibitory effect on the neuron and why this works
Gβγ closes N-type calcium channels which inhibits neurotransmitter release and opens GIRK potassium channels
• GIRK- inhibits action potentials via dissociated βγ- subunit action in opening GIRK channels
• N-, P/Q-type voltage-gated calcium channel- inhibits neurotransmitter release via βγ subunit action on presynaptic Ca-channels
o Negative feedback mechanism to control level of neurotransmitter release (homosynaptic or heterosynaptic self-auto feedback)
• This mechanism operates quickly in cell membrane due to proximity of these channels to βγ subunits
Gαi inhibits formation of cAMP (particularly if cAMP formation is activated by another mechanism)
What is the role of Gaq subunits in GPCRs and what molecules do they act on? How do they work?
• Gαq
o Gαq coupled GPCRs are generally excitatory
o Gq stimulates phospholipase-C then protein kinase-C and mobilises calcium (e.g. M1R, α1AR)
o Acts on PLCβ, Lbc, Ca2+, PKC, Rho
What molecules do Ga12 subunits in GPCRs act on?
• Gα12 etc. o Acts P115-RhoGEF o LARG o PDZ-RhoGEF o AKAP-Lbc o Rho
Are GPCRs selective or non-selective for specific alpha subunit types? Why/why not?
Each GPCR, because of its unique C-terminal, has selectivity for a specific α subunit type (Gs….)
• Although some can sometimes act non-specifically
What signals do Gbetagamma subunits (Gby subunits) modulate/result in in GPCRs? What is the role of these signals?
o Gβγ subunit signals are complex and experience some selectivity (but not as distinct as alpha subunits)
Modulate adenylyl cyclase (cAMP formation)
• Depends on subtype of adenylyl cyclase- complicated signalling
Inhibit N-type calcium channels (inhibit neurotransmitter release)
• N-type calcium channel in CNS is the major type of calcium channel that mediates neurotransmitter release
Activate phospholipase C (many cellular processes)
Activate GIRK potassium channels (G-protein Inwardly Rectifying K-channels) (inhibits action potential activity)
• Stimulated GIRK channels hyperpolarise cell membranes and inhibit action potential activity
Activate PI3 kinase (many cellular processes)
Where are u-opioid receptors mostly distributed and why?
• μ-opioid receptor distribution in spinal cord where pain transmission neurons are located (on surface of dorsal horn)
o Morphine etc… act on μ-opioid receptors
• But not where non-noxious transmission neurons are deep in the dorsal horn of the spinal cord, as μ-opioid receptors do not dampen down non-noxious stimuli
What do GPCR actions depend on?
• GPCR actions depend on cellular and circuit location of types of receptor
What does GPCR signal strength depend on?
• GPCR signal strength varies according several variables
o Receptor number
o Intrinsic efficacy of agonist
o Downstream amplification
o GPCR signaling is strongly regulated by phosphorylation and endocytosis
What structures amplify GPCR signals?
• Ligand signals-> GPCR causes G-signal to amplify: as long as the receptor is bound, the GPCR cycle can continue amplification -> G-effectors amplify by allowing influx of ions or through other mechanisms
What two factors are affected by ligand-receptor affinity and why?
• Increased ligand-receptor affinity improves signal detection but affects time of action
o If affinity is high, GPCR is more sensitive to signals and vice-versa
What does the affinity of a receptor depend on? What is the formula for it?
o Affinity of a receptor depends on the rates of association on the receptor vs rates of dissociation from receptor
k-1/k1 gives information about affinity (rate function)
What determines the strength of the GPCR response to a stimulus?
[A]+[R]↔[A.R]↔[A.R]* →response
kon is when equilibrium shifts to the right on first step, and k1 is when equilibrium shifts to the right on second step
koff is when equilibrium shifts to the left on the first step, and k-1 is when equilibrium shifts to the left on the second step
Where A stands for agonist
[A] is the concentration of the agonist
Where R stands for receptor
[R] is the concentration of the receptor
What is the dissociation ratio?
o KA(d)= koff/kon
How can the GPCR response to a stimulus be increased?
• To increase response, can either increase the number of active receptors (up to saturation) or increase concentration of the drug (up to saturation)
Do hormones acting on GPCR have very high affinity or very low affinity? Why? What is the consequence of this?
• For hormones acting on GPCR, very high affinity (KA about 1 pM) is needed so that only very low concentrations are used to signal (as it takes a lot of energy expenditure to produce the hormones)
o But kon is limited by maximal rate of diffusion to a point (surface)- it takes a while for activation
Maximum possible kon= 2.5x109 sec mol-1
o If KA = 1pM then koff= 2.5x10-3 sec-1 that is τ= 400 sec (more than 6 minutes)
Appropriate for hormones, as their signals are not too time-sensitive, but not appropriate for signals that need to travel fast
• Signals that need to travel fast tend to have very low affinity
What determines the strength of a stimulus?
o Strength of the stimulus (S) = ε. [A.R]
ε is efficacy of transduction
• Range:
o Starts from 0- antagonist (binds to receptor and produces no signal)
o Up to agonists that produce an extremely strong signal
Increasing effective receptor number (R(n)) improves signal strength
Increasing ε improves signal strength: partial agonists, receptor reserve
How is the affinity trade off issue solved in a GPCR system?
• Increasing gain of amplification is a manner in which the system solves the limitation of the affinity trade-off issue
Does GPCR signal response increase linearly towards infinity?
Eventually, signal response reaches a threshold as signalling system cannot produce a bigger response
• However, if there are too few receptors or efficacy is too low, signalling system may not reach this maximum response
Is GPCR signalling speed fast or slow compared with ionotropic receptors? Why?
• GPCR actions are slow compared with ionotropic receptors
o This is because ionotropic receptors have low affinity for their agonists and require a large amount of them to turn them on
Are modulatory signals from GPCRs slow or fast? Describe
• Modulatory signals from GPCRs are slow:
o Fastest GPCR action is Gβγ modulation of channels with lag of about 50 ms and usual action in range of 100s of ms to seconds
Compare with fast synaptic transmission (about 1 ms)
o Slower actions more common: seconds to minutes
o Very slow actions on nucleus (hours to days)
What modifies the rate of signalling?
• Number of receptors also modifies rate of signalling
o The more receptors, the faster the response
GPCR response to light is extremely fast
How does intrinsic efficacy influence GPCRs?
• Intrinsic efficacy influences response at GPCRs
What is the efficacy of methadone (opioid)
Methadone: high intrinsic efficacy
• When one molecule reacts with a GPCR, then a strong signal is produced
What is the efficacy of morphine (opioid)
Morphine: moderate intrinsic efficacy
• When one molecule reacts with a GPCR, then a moderate signal is produced
What is the efficacy of buprenorphine (opioid)
Buprenorphine: low intrinsic efficacy
• When one molecule reacts with a GPCR, then a small signal is produced
• It cannot ever fully stimulate the receptors
Describe the process of GPCR regulation
o Phosphorylation
When GPCRs are stimulated and alpha/betagamma subunits are released to produce a response, if the alpha and betagamma subunits are not sitting on intracellular C terminal region and intracellular loops of the receptor, then receptor becomes a substrate for kinase proteins and it gets phosphorylated
o GRK and arrestin binding: reduces coupling
When GPCRs are phosphorylated, they become a substrate for proteins called arrestins (scaffolding proteins)
Binding drags the receptor into clathrin- the receptor pits into the membrane
o Endocytosis reduces effective [R]
Pits containing receptors get pinched off as vesicles, and the receptor disappears from the membrane into an endosyme
o Both processes reduce ε. [R]
From the endosyme, the receptor may get back into the membrane, signal or get degraded
What is the role of GPCR regulation?
• GPCR regulation reduces effective receptor number or signalling efficacy (same net effect).
• GPCR regulation produces desensitization and tolerance
o Overall strength of signal declines in long-term exposure to high concentrations of agonists
o Auto-regulatory mechanism
Where in the cell can GPCRs signal? Describe the commonality of the locations
• GPCRs can signal both at surface and in endosomes
o In most GPCRs, surface signals dominates
o For some GPCRs, endosomal signal dominates
Surface signal not as important because GPCR spends most of the time in the endosome
Possible alpha/beta/gamma signalling from endosome
What can GPCR selectivity be developed by? Rate by potential.
• GPCR selectivity can be developed by:
o Structural knowledge of orthosteric site (certainly)
o Signalling bias for different effectors (maybe)
o Allosteric modulation (in its infancy but there is evidence for)
o Heterodimerization (perhaps)
o Better structural knowledge (certainly)
Why is it hard to develop structurally selective drugs for closely related GPCRs?
• Closely related GPCRs have very similar binding pockets: hard to develop drug selectivity due to this structure
o Very closely related GPCRs are hard to discriminate pharmacologically although they are enormously physiologically different
o More so for agonists in the orthosteric binding sites because of strong structural constraints on agonist binding
Why is important for drugs to be selective to specific receptors? Give an example
o D2/D3 dopamine receptor antagonists are antipsychotics
o D2 receptors appear to mediate worst side effects
o D2 and D3 dopamine receptors are very similar
o No highly selective antagonists
o Structure of the D3R bound to eticlopride suggests selectivity can be developed
o Drug selectivity still very difficult because orthosteric sites do not differ greatly
How would GPCR drug selectivity from signal bias work? Give examples. WHat is required for GPCR drug selectivity from signal bias to work?
• GPCR drug selectivity from signal bias
o Could potentially explore the fact that different GPCRs signal differently
Could potentially exploit the ability that some agonists will produce a strong G protein signal and strong arrestin signal whilst others will produce different signalling patterns
• E.g. Opioids
o Methadone has strong G-protein signalling and strong arrestin signalling
o Morphine oxycodone has moderate G-protein signalling and little arrestin signalling
o Endomorphin-2 has moderate G-protein signalling and strong arrestin signalling
o Structural basis needs to be understood
o Established at many GPCRs
What is orthosteric agonism and what kind of signalling does it lead to?
Orthosteric agonism= normal signalling
• Agonist binds to usual binding site that turns on G proteins and leads to a signal
What kind of signalling do positive allosteric modulators lead to?
Positive allosteric modulator= increases signalling
What kind of signalling do negative allosteric modulators lead to?
Negative allosteric modulator= decreases signalling
What kind of signalling do neutral allosteric ligand lead to?
Neutral allosteric ligand= unaltered signalling
Where is the binding site of M2 muscarinic receptors and what increases its binding capabilities?
o M2 muscarinic receptor
Binding residues available near extracellular vestibule
Alcuronium enhances N-methyl scopolamine binding
How is GPCR drug selectivity from allosteric actions proposed to work? Give an example
o Physiological GPCR selectivity from allosteric actions
Allosteric modulators can enhance (or dampen) physiological signal
Selectivity is very high because binding is remote from orthosteric site
• Regions of receptors that are away from orthosteric site tend to be more divergent-> increases selectivity
Many examples emerging: positive allosteric enhancer (cinacalcet) of a calcium sensing receptor is in clinical use
Why would GPCR drug selectivity from dimerization be able to work? What isthe evidence behind it?
• GPCR drug selectivity from dimerization
o Most GPCRs exist as dimers and can be promiscuous
o Evidence is often indirect
o If different monomers can assemble as dimers then selective drugs may be found
o Still not clearly established whether or not such heterodimers exist widely but there is in vitro evidence for many
o Some GPCRs only function as heterodimers
In what kind of dimers can GPCRs exist as? When can GPCRs form dimers?
o Most GPCRs exist as dimers and can be promiscuous
Different subtypes of receptors can form dimers if they’re related closely enough
Many GPCRs are obligate dimers (they covalently bond)
Some are transient dimers (stick together for several seconds/minutes, then fall apart and drift off into the membrane)
o Some GPCRs only function as heterodimers
What kind of dimer is the GABAB receptor? Explain
GABAB receptor is an obligate dimer
• Receptor won’t function if it hasn’t got a GABAB1 AND GABAB2 subunit that assembles covalently in the membrane
o One member (B1) binds GABA
o Other member (B2) contributes to transduce signal
Ligand doesn’t bind to this subunit
o G protein signal comes from transduction into the other subunit
What is the main difference between GABAA receptors and GABAB receptors?
Note: GABAA are the IONOTROPIC receptors, whilst GABAB are the METABOTROPIC (GPCR) receptors
What is DREADDS? Give an example of how it works
• Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
o Muscarinic receptor with TM3 and TM5 point mutations
Receptors stops responding to muscarinic receptor agonists
Did respond to novel agonist CNO (clozapine N oxide)
• CNO does not act on any other GPCRs
o Can test drugs by applying them to a single population of genetically modified neurons
What is optogenetics? Describe the channels that underlie it and how it works
• Optogenetics is based on channelrhodopsin: a GPCR o Channelrhodopsin (ChR2) is an algal motility photoreceptor cation conducting GPCR Transmembrane barrel in the GPCR allows ions to go through o Halorhodopsin (ChR2) is a chloride conducting homologue. More recently ACR (anion conducting rhodopsin has been engineered) o Introduction of a ChR2 channel in the brain allows that channel to turn on neurons/inhibit neurons when activated by light (especially blue light)- good experimental tool
What is the difference between receptors and transporters? Give examples
o Receptors-
Passive transport
Ligand receptor channel and ion channel receptor
• Activated by ligands- ligands bind to the receptor through extracellular space and produces conformational change-> then unbinds through extracellular space
o Ligands don’t move through membrane
o E.g. mGluR and AMPA
o Transporters
Passive OR active transport
Transporters move a substrate across the cell membrane
E.g. EAAT
What is the general role of membrane transporters?
Membrane proteins control movement of everything across the cell membrane: nutrient uptake, waste disposal and cell to cell communication (neurotransmitters)
What is passive transport and an example of a passive transporter?
• Passive transport- movement of molecules or ions from an area of higher to lower concentration
o Does not required additional energy
o Passive transporters:
Facilitators
• E.g. Na+ independent glucose transporter
What is active transport?
• Active transport- movement of molecules or ions against a concentration gradient (from an area of lower to higher concentration)
o Enzymes and energy are required
What are two main types of active transporters?
o Active transporters (pumps)
Primary active transporters
Secondary active transporters
What are primary active transporters? Give an example and how it works
Primary active transporters
• Derive energy required for transport process from either light or ATP hydrolysis
o E.g. Na+/K+ ATPase
Pumps 2 potassium into the cell for every 3 sodium out of the cell using ATP hydrolysis
What are secondary active transporters? Give an example and how it works
Secondary active transporters
• Derive energy from pre-existing ion gradients (mostly set up by primary active transporter Na+/K+ ATPase)
o E.g. plasma membrane glutamate transporter (and all neurotransmitter transporters)
What are the two main families of neurotransmitter transporters fond on post- and pre-synaptic neurones and glial cells?
- Two main families of neurotransmitter transporters found on post- and pre-synaptic neurones and glial cells
- Glutamate transporter family
- Neurotransmitter sodium symporter (NSS) family
What is the general role of neurotransmitter transporters and why is it important?
• Role is to clear neurotransmitter from the synaptic cleft
o Important as otherwise, neurotransmitters would continually activate their targets
What are the two main types of glutamate transporters?
• Glutamate transporter family
o VGLUT
o EAATs
What are VGLUTs and what is their purpose/how do they work
Vesicular Glutamate Transporter
• Package glutamate into somatic vesicles
• Somatic vesicles transferred into presynaptic neuron
• Once cell is depolarised, packages will be released in synaptic cleft
What are EAATs and what do they transport?
Human plasma membrane glutamate transporters
EAATs- Excitatory Amino Acid Transporters
Can transport glutamate and aspartate with similar affinities (2-20 uM)
How many subtypes of EAAT are there and how similar are they?
5 subtypes (EAAT1-5)
5 subtypes share around 50-60% amino acid identity
In what cells are EAAT1 and 2 usually found and what is their expression level throughout the CNS?
• EAAT 1 and 2: found mainly on astrocytes and glial cells surrounding synapses
o Very highly expressed
o Clear glutamate through astrocytes
What percentage of total brain membrane protein is EAAT2?
o EAAT2 is around 1% of total brain membrane protein
In what cells are EAAT3 and 4 found and what is their role?
• EAAT 3 and 4: found on post-synaptic neuron extrasynaptically
o Catch glutamate that is spilling over from other synapses
Where is EAAT3 expressed in the CNS?
Widely expressed
Where is EAAT4 expressed in the CNS?
Cerebellum
In what cell type is EAAT5 expressed and where in the CNS is it expressed?
Neuron
-Can sometimes be pre-synaptic neuron
Retina
What is the role of EAATs?
Take up glutamate/aspartate from synaptic cleft (reuptake)
Keeps resting glutamate level low (10 nM)
Can maintain a 10^6 fold gradient across the membrane
How do glutamate transporters work?
Glutamate and sodium bind-> using ATP, glutamate and sodium are transported to the inside
Potassium binds and is transported to the outside so as to prepare the channel for future glutamate transport
For every glutamate transport, there is a net transfer of two positive charges
• 3 sodium comes in, 1 potassium comes out
What neurotransmitters can the neurotransmitter sodium symporter family transport and what ions do they use to do so?
o Can transport GABA, glycine, dopamine, noradrenaline, serotonin
o All human subtypes are coupled to the co-transport of sodium and chloride ions
What symporter is used as a model structure of all transporters in this family?
o Bacterial homologue from Aquifax aeolicus (LeuTAa) has been crystallised and is a model of the structure of all transporters in this family
Describe the coupling of GlyT1 receptors
o Is coupled to 2 sodium ions
Describe the coupling of GlyT2 recptors
o Is coupled to 3 sodium ions
Where are GlyT1 receptors localised and why?
o GlyT1 are highly localised in astrocytic processes
Allows glycine availability for excitatory neurons
What are examples of GlyT2 inhibitors, where would they be used and why?
o GlyT1 inhibitors:
In schizophrenia there is reduced NMDAR activity (NMDAR hypofunction hypothesis)
Inhibition of GlyT1 will elevate [Gly] at excitatory synapses and stimulate NMDAR
Examples: Sarcosine, NFPS
Where are GlyT2 receptors localised and why?
o GlyT2 is highly localised in inhibitory neurons
Tightly controls glycine availability
What is the use of GlyT2 inhibitors and how does that purpose work?
o GlyT2 inhibitors
Enhancement of glycinergic inhibition in the spinal cord
Reduces excitatory transmission of pain signals from the spinal cord up to the brain
Reduces pain perception
In spasticity there is impaired glycinergic neurotransmission- GlyT2 inhibitors may be beneficial
What are GATs and how many subtypes of them are they?
o Plasma membrane GABA transporters (GATs)
o There are 4 subtypes of GABA transporters (GAT1-4)
Where are GATs transporters expressed in the CNS?
o Expressed in neurons and glial cells throughout the CNS
What ions are GATs transporters coupled to?
o Coupled to 2 sodium ions and one chloride ion
What examples of drugs that modulate GATs and what do they do?
o Drug modulation of GABA transporters Inhibition of transporters will elevate GABA and stimulate GABA neurotransmission • Nipecotic acid- selective for GAT1 • Tiagabine- selective for GAT1 o Anticonvulsant
What is the ion coupling for DAT transporters?
DAT
• 2 sodium and 1 chloride ion
What is the ion coupling for NET transporters?
NET
• 1 sodium and 1 chloride ion
What is the ion coupling for SERT transporters?
SERT
• 1 sodium and 1 chloride ion
What is the history of amphetamine and methamphetamine?
- First synthesis
- Uses throughout history
- Street names
• First synthesised from ephedrine in 1890s
• Used in WW2 by German pilots and Tank Crews (used by Adolf Hitler)
• 1950s- prescribed for narcolepsy, Parkinson’s disease, alcoholism, depression and obesity
• 1960s- used as a recreational drug
• 1980s- made illegal
o Use escalated after it was made illegal, common among truck drivers
• Since 2000, ice (crystal methamphetamine) has become the more common street form and is more associated as a party drug
• Common names:
o Methamphetamine- ice, meth, crystal, crystal meth, shaboo
o Amphetamine- speed
Does amphetamine or methamphetamine produce more exaggerated effects?
Comparison between amphetamine and methamphetamine
• Generally, methamphetamine produces more exaggerated effects, related to the higher effective doses used and greater propensity for addition
What are the effects of amphetamines?
Effects- • Arousal • Euphoria • Alertness • Capable of conducting over-learnt tasks”
What is the mechanism of action of amphetamine on DAT?
o AMP is a false substrate
It is a transportable substrate- moves into the cell
o AMP reduces dopamine uptake and causes reverse transport of dopamine
How do amphetamines act?
o Competes with DAT/NET
AMP accumulates in cytoplasm
DA is elevated in synapse
o Depletes cytosolic DA- reverse transport
o AMP inhibits MAO
o Competes with VMAT- depletes storage of DA in vesicles
What are the effects of methamphetamine associated with short-term use?
Effects-
• Euphoria
• Increased energy and attentiveness
• Diarrhea, nausea
• Excessive sweating
• Loss of appetite, insomnia, tremor, jaw-clenching
• Agitation, compulsive fascination with repetitive tasks
• Talkativeness, irritability, panic attacks
• Increased libido
What are the effects of methamphetamine associated with chronic use?
Chronic use • Drug craving • Withdrawal-related depression • Rapid tooth decay • Degeneration of the dopaminergic neurons • Weight loss • Psychosis
What are the effects of methamphetamine associated with overdose?
- Brain damage
- Sensation of flesh crawling with bugs with associated compulsive picking and infecting sores
- Paranoia, delusions, hallucinations
- Muscle breakdown which leads to kidney failure
- Death from overdose is usually due to stroke, heart failure, but can also be caused by cardiac arrest (sudden death) or hyperthermia
From what is cocaine isolated?
Isolated from leaves of the cocoa plant
What are the uses of cocaine?
Stimulant, appetite suppressant
Topical anaesthetic, used in eye, nose and throat surgery
How does cocaine work?
Na+ channel blocker
DAT, NET and SERT inhibitor
Mechanism of action
• Competitive blocker of dopamine, noradrenaline and serotonin transport
• Non-transportable inhibitor- does not interfere with dopamine loading
o Due to its larger structure
Why is cocaine addictive?
Actions on the mesolimbic system cause it to be addictive
What is the history of cocaine?
- Uses over the years
- Purification
- Illegality
- Used for thousands of years by South American Indians
- Cocaine first purified in 1855
- 1880s used as an anaesthetic- inspiration for a number of anaesthetics (novocaine, lignocaine)
- Cocaine was added to Coca Cola between 1886 and 1906
- 1914- illegal to sell or distribute cocaine, but rarely prosecuted
- 1970- the controlled substances act in USA declared its use illegal
- 1980s and 1990s trade and use escalated especially with the introduction of crack cocaine
What is the transporter for glycine, and what is the use of therapeutic drugs that act on these transporters?
Transporter: GlyT1, GlyT2
Therapeutic drugs: Pain and antipsychotics
What is the transporter for GABA, and what is the use of therapeutic drugs (give examples) that act on these transporters?
Transporter: GAT1-4
Therapeutic drugs: Anticonvulsants (tiagabine)
What is the transporter for dopamine, what is the use of therapeutic drugs (give examples) that act on these transporters and what drugs of abuse act on this transporter(s)
Transporter: DAT Therapeutic drugs: Antidepressants (TCAs) Drugs of abuse: Amphetamine Meth Cocaine MTPT (MPP+)
What is the transporter for noradrenaline, what is the use of therapeutic drugs (give examples) that act on these transporters and what drugs of abuse act on this transporter(s)
Transporter: NET Therapeutic drugs: Antidepressants (TCA's, SNRI's) Drugs of abuse: Amphetamine Meth Cocaine
What is the transporter for serotonin, what is the use of therapeutic drugs (give examples) that act on these transporters and what drugs of abuse act on this transporter(s)
Transporter: SERT
Therapeutic drugs: Antidepressants (SSRIs, TCAs)
Drugs of abuse: MDMA, Cocaine
What type of active transporters are all neurotransmitter transporters?
• All neurotransmitter transporters are secondary active transporters that are coupled to pre-existing ion gradients
What is the process of excitotoxicity? How does it happen and in what conditions can it occur in?
• Process:
1. Ischaemia (lack of oxygen supply to the brain)
a. Depletion of glucose
b. Reduction in ATP
2. Failure of the Na+/K+- ATPase
3. Rundown of membrane potential and uncoordinated action potential generation
4. Excessive glutamate release and glutamate transporter failure
a. Failure as glutamate transporters need sodium/potassium balance to work
5. Excessive stimulation of glutamate receptors
6. Excessive calcium influx
7. Activation of: proteases, lipases, NO synthase, endonucleases
8. Cell death by necrosis or apoptosis
• This process can occur in neurodegenerative conditions
o Alzheimer’s disease
o Parkinson’s disease
o Motor neuron disease
• Episodic ataxia
o Recurrent/periodic periods of paralysis
o EAATs could be a good drug target
What are the substrates, substrate inhibitors and blockers of EAAT1?
Substrates: L-Glutamate,
L-Aspartate
Substrate inhibitors: D-Aspartate, 4-MG
Blockers: TBOA
What are the substrates, substrate inhibitors and blockers of EAAT2?
Substrates: L-Glutamate,
L-Aspartate
Substrate inhibitors: D-Aspartate
Blockers: DHK, TBOA, 4-MG
What are the substrates, substrate inhibitors and blockers of EAAT3?
Substrates: L-Glutamate,
L-Aspartate, L-Cysteine
Substrate inhibitors: D-Aspartate, 4-MG
Blockers: TBOA
What are the substrates, substrate inhibitors and blockers of EAAT4?
Substrates: L-Glutamate,
L-Aspartate
Substrate inhibitors: D- Aspartate
Blockers: TBOA, 4-MG
What are the substrates, substrate inhibitors and blockers of EAAT5?
Substrates: L-Glutamate,
L-Aspartate
Substrate inhibitors: D-Aspartate
Blockers: TBOA
What is the structure of TBOA and what was learnt about EAAT due to its structure?
o Aspartate backbone
o Benzyl ring
o While it binds in same place as aspartate, benzyl ring props open HP2
Does not allow HP2 to close down-> HP2 closure is vital for transport cycle to continue
What is glutamate transporter structure and what transporter specifically was used to demonstrate this/where did it come from? How close to human EAAT2 is this transporter? Describe its use.
• GltPh- structure of the transporter homologue from Pyrococcus horikoshii (archea- thermophile)
o Trimer- 3 identical subunits
Long alpha-helical transmembrane domains
2 hairpins (HP1 and HP2)
o Each subunit is capable of transport
o 37% identity to human EAAT2
o Is an Na+-dependent aspartate transporter
What is the structure of neurotransmitter sodium symporters and what transporter specifically was used to demonstrate this? Where does the substrate bind, how is it regulated and how does it work?
• Drosophila Dopamine Transporter
o 12 transmembrane domains
o Shot glass shape
Tight structure and exists as a monomer
Open to outside and inside of cell, but closed in the middle
o Substrate buried
o Co-transport 2 sodium ions and 1 chloride ion
o Regulated by cholesterol
