Receptor theory Flashcards
By whom and when were ligand-gated ion channels first described?
J. N. Langley in 1905 first wrote that since muscle conctraction inhibitors do not prevent contraction via direct muscle stimulation, there must be an accessory substance they act through.
What happens at neuromuscular junctions?
Acetylcholine binds to nicotinic receptors, allowing Na+ to flow through and K+ to flow out. Na+ ions dominate, leading to depolarisation of the muscle fibre.
This triggers an AP which travels along the t-tubules ultimately leading to the release of calcium ions from the sarcoplasmic reticulum (SR).
The released calcium ions bind to troponin, causing a conformational change and allowing the myosin and actin filaments to slide past each other and cause muscle contraction.
When was the activity of nicotonic receptors first recorded and how?
Hamill et al (1981) recorded the activity of the receptors by fillimg a patch pipette with ACh and sealing it on the membrane
Were able to record a movement of ions into the cell
What is the other name for ligand gated ion channels and what sort of transmission do they mediate?
Ionotropic receptors
Mediate fast synaptic transmission in the CNS and PNS
What are the different families of ligand gated ion channels, what is there neurotransmitter, where are they located and how many subunits does each receptor have?
Pentameric, tetrameric and and trimeric
What is the resting membrane potential of across the cell membrane?
~ 70 mV
What is the balance of Na, Cl- and K+ ions in and out of the cell?
K+ = 140mM inside 3mM outside
Reversed for Na+
High levels of Cl- outside cell and lower inside
What are inhibitory and excitatory ligand-gated ion channel examples?
Excitatory= Nicotinic (Na+, K+), 5-HT3 (Na+, K+), AMPA, (Na+, K+) and NMDA (Na+, K+, Ca2+)
Inhibitory= GABAa (Cl-) and glycine (Cl-)
What is the structure of the nicotinic acetylcholine receptor?
It is pentameric with 4 transmembrane domains
M2 (green)= is the transmembrane section
Extracelluarly located NH3+ and COO-
What are the two main types of ACh nicotinic receptor?
The muscle type at the NMJ
The neuronal type in the autonomic ganglia
They have completely seperate subunits
What is homology like between the subunits of nicotinic ACh receptors?
- Homology between the same subunit across different species is very high (e.g. rat and human there is 90% homology)
- Homology between different subunits in the same species is lower (30-60%).
Where is the ACh binding site on nictonic ACh receptors located?
ACh binding site is on the α-subunit + either γ- or δ-subunit; not exclusively contained in 1 subunit but at the interface.
What type of charge lines the channel pore of nicotinic ACh receptors?
Rings of negative charge- attracts cations through the receptor
What happens when ACh binds to nicotinic receptors?
There is a confirmational change and twisting of the TM2 to open the channel
What are the subunits of nicotinic ACh receptors (muscular) and how does this change throughout development?
The subunits are designated as α (alpha), β (beta), γ (gamma), δ (delta), and ε (epsilon).
Whereas expression of the γ-subunit predominates in embryonic muscle, expression of the ε-subunit occurs during early neonatal development and persists in adults
How does the depolrisation due to nicotinic ACh receptors differ between foetal and adult muscle?
Adult nAChRs have higher single channel conductances but shorter open times compared to the foetal form of the receptor
May need prolonged depolarisation in younger tissue to trigger an action potential
What is an example of a drug that can be given for NMJ dysfunction?
Cholinesterase inhibitors- inhibits the enzyme that breaks down ACh and is currently being used in AD and myasthenia gravis (auto-immune disease)
What protein has been used as a functional mimic for the ligand binding domain of nicotinic ACh receptors?
Acetylcholine binding protein (AChBP)
Found in certain molluscs such as snails and shares structural similarities with the ligand binding domain of nAChRs in vertebrates
It has been widely used as a surrogate for studying ligand-receptor interactions
It also serves as a model for screening and designing drugs that modulate the activity of these receptors
What are the different subunits in neuronal and muscle nicotinic ACh receptors?
- Neuronal subunits= alpha 2-10 and beta 2-4
- Muscle subunits alpha-1, beta-1, gamma (foetal), epsilon, and omicron (adult)
How do neuronal nictonic ACh receptor have different number of binding sites?
Made up of different combinations of alpha (2-10) and beta (2-4) subunits
5 alpha-7 subunits can form a receptor, meaning there is 5 binding sites.
There is different combinations in the CNS, PNS and immune system
What conditions have agonists of nicotinic ACh receptors been trialled for?
Schizophrenia, AD, ADHD (attention deficit hyperactivity disorder), nicotinic addiction, pain and Parkinson’s disease
Autism, depression, cognitive deficits and psychosis
How successful have nicotinic ACh receptor agonists been for treatment of neurology conditions?
Largerly failures of nAChR ligands in AD and schizophrenia in clinical trials have reduced enthusiasm for this therapeutic strategy and many pharmaceutical companies have now abandoned this field of research.
However, there is still nAChR modulating drugs in clinical trials
What are examples of successful nictonic ACh modulating drugs?
Galantamine, a acetylcholinesterase inhibitor and positive allosteric modulator indicated for AD. It only reduces symptoms however, it does not cure it.
What are the 4 receptor superfamilies and how long do they take to exert changes?
- LGICs/ ionotrophic receptors e.g. nicotinc AChR. Very fast reaction- ms
- GPCRs e.g. muscarinic AChR. Fast reaction- seconds
- Kinase linked receptors e.g. insulin or cytokines. Hours to activate
- Nuclear receptors e.g. oestrogen or cortisol. Activation takes hours
Where are nuclear receptors located?
Intracellularly (although are some caveats)
What activates nuclear receptors?
Activated by steroid hormones: oestrogen (ER), testosterone (AR), cortisol (GR), aldosterone (MR), progesterone (PR) as well as thyroid hormone (TR), vitamin D (VDR)
How does a nuclear receptor exert its changes?
They are ligand-activated transcription factors
Once a ligand binds, the receptor translocates to the nucleus where RNA polymerase acts.
Cause gene expression modulation
What are the main domains of a nuclear receptor?
What domain has a high level of homology between different receptors?
DNA binding domain
What is the role of the regulatory domain in nuclear receptors?
- Regulatory domain= very variable between receptors, both in sequence and length. It contains an important transcriptional activation function AF-1.
- AF-1 binds elements (co-activator or co-repressor proteins) that modify how the receptor interacts with transcription machinery- to silence or start transcription
What is the role of the DNA binding domain
Highly conserved between receptors, it localises on DNA and is involved in dimerisation
What does the DNA binding domain contain and how does this help its function?
It lcontains 2 Zn2+ fingers that wrap around the DNA helix
The C1 figer is involved in recognising specific DNA sequences to bind to
For example, the GR homodimer contains 4 zinc fingers which holds the protein in the correct shape to interact with the DNA
What is dimerisation?
Two receptors binding togther
Can be a heterodimer or a homodimer
How do nuclear receptors dimerise to each other?
Two receptors bind in mirror to each other
Some receptors (class 2 receptors) bind not in mirror but as ‘spooning’ each other. This includes the retinoid X receptor, thyroid hormone receptor and Vit D receptor
What is the function of the hinge domain in nuclear receptors?
A flexible hinge that links the ligand binding domain to the DNA binding domain and gives the receptor conformational flexibility
Has a role in dimerisation and allowing the receptor to find the nucleus
What is the role of the ligand binding domain in nuclear receptors?
The ligand typically binds within the ligand-binding pocket of the LBD, inducing structural alterations that expose the AF-2 domain.
The exposed AF-2 domain serves as a docking site for coactivator proteins which helps to enhance gene transcription
Where and what is the AF-2 region in nuclear receptors?
Sits in helix 12 of the ligand binding domain
Agonist ligands stabilise the receptor conformation that is optimal for efficient interaction with co-activators, with AF-2 domain exposed. Causes increased gene transcription
Antagonists have bulky side chains that hinder H12 from aligning in the agonist conformation.
What are the two classes of nuclear receptors?
- Class I
Endocrine ligands
High affinity
Mainly homodimers (although ERa/ERb; oestrogen receptor has two forms)
Cytosolic location (float around in the cytosol)
e.g. Steroid receptors: MR, GR, ER, AR, PR - Class 2
Lipid ligands
Low affinity
Mainly heterodimer with RXR (except RXR)
Nuclear location- bound to DNA in the nucleus in an inactivated state (until ligand binds)
e.g. PPAR, LXR, RAR - Also get hybrids
These have endocrine ligands, that heterodimerise with RXR. Include TR (thyroid) and VDR (Vit D)
What happens when a ligand binds to a nuclear receptor
In the AF-2 region, when not activated, the receptor is bound to co-repressor proteins.
Once ligand binds, co activator proteins bind and there is an opening up (acetylation) of the chromatin structure (or closing)
What are the different co-activator and co-repressor proteins that influence nuclear receptors?
- Co-activators: recruited by DNA bound, ligand bound receptor. Agonist ligands stabilise the receptor in a conformation that is optimal for efficient interaction with co-activators and facilitates transcriptional activation.
- Co-repressor: recruited by DNA bound unliganded receptor or antagonist bound receptor help repress transcription
- Different cohorts of co-activator and co-repressor proteins are expressed in a tissue specific manner
What is an example of a drug that acts as a ligand for nuclear receptors and acts in more than one way?
Glucocorticoids are used in asthma inhalers and for arthritis. They bind to the glucocorticoid receptor.
- Classical regulation pathway= GRs bind to the regulatory region of the gene encoding the anti-inflammatory protein annexin 1 (lipocortin) and increases its synthesis
- Alternative pathway= GRs repress COX and iNOS formation by binding to the pro-inflammatory transcription factors AP-1 and NFkB (which normally drive inflammation, and inhibiting their ability to promote gene transcription.
What is an example of a selective nuclear receptor modulator drug?
SERMS- selective oestrogen receptor modulators e.g. raloxifene
Mimic oestrogen in some tissues but antagonises it in others.
It is an agonist in bone and lipids (decreases LDL) but an antagonist on breast and endometrium
Can also get SARMS (selective androgen receptor modulators) that mimic testosterone in selected tissues only.
Useful as increases bone integrity and muscle strength without driving prostate cancers.
What evidence is there for non-genomic signalling from steroid receptors?
- Classical nuclear receptor signalling takes hours to transduce signal
- But also get very rapid effects of some steroids e.g. effect of oestrogen on blood vessels
- This only takes minutes and thus is not due to protein transcription.
What is the model of oestrogen action in endothelial cells?
The ER can be coupled with scaffolding to hold it to the cell membrane.
The receptor can then act clasically, or like a g protein coupled receptor
What do class II nuclear receptors dimerise with?
RXR (Retinoid receptor) which strengths DNA binding and transcriptional activity
What are the two types of class II nuclear receptor dimerisation?
Can be permissive (the complex can be activated by either RXR or the partner ligand) or non-permissive (the complex can only be activated by the partner ligand)
What year was the first nuclear receptor cloned?
1985
What are orphan nuclear receptors?
An orphan receptor whos endogenous ligand has not yet been identified
Orphan nuclear receptors are largely receptors for lipids: cholesterol (and metabolites), fatty acids and bile acids.
If the ligand is identified, the receptor becomes an adopted orphan
What are class II nuclear receptors mainly involved in?
Mainly involved in metabolism e.g. lipid profile in blood
Abnormal function leads to different blood lipid profile, insulin sensitivity, glucose tolerance, inflammation and adiposity
What are PPARs?
Peroxisome proliferator activated receptor
Activation reduces triglyceride levels
All PPARs herterodimerise with RXR
Endogenous ligands include free fatty acids and Vit B3- no domiant ligand however
What drugs target PPARs and what could be improved by them?
Clofibrate= ligand. Promotes fat efflux from tissue to liver for excretion. Lipid lowering agent
Glitazones= partial ligand of the PPAR-gamma receptor. Oral anti-biotic drugs designed to treat patients with type 2 diabetes
There is side effects of activating PPAR-gamma such as increased Na+ and water retention
How is pharma cuurently working to develop therapeutics targeting nuclear receptors?
- Pharma currently working to develop a series of ligands that result in different conformational transitions. Different ligands can differentially regulate gene expression by the same receptor by inducing selective co-activator/repressor binding
- This can be used to exploit the good effects of nuclear receptor signalling whilst not causing the bad.
What are liver X receptors and what are the potential therapeutic benefits?
LXRs are cholesterol sensors and regulate cholesterol homeostasis
They are activated by sterols such as oxysterol which are intermediates of cholesterol synthesis
Drug development pathway for cholesterol lowering, Parkinson’s, cancer and inflammation
What are the subunits of ligand gated ion channels made up of?
4 membrane spanning helices
A pentameric receptor will contain 20 membrane spanning helices
How are the M2 subunits in ligand gated channels at rest and once opened?
Sharply kinked inwards at rest, forming a constriction
Once the channel is activated, a confirmational change occurs in the extracellular part of the receptor, causing the kinked M2 segments to swivel out of the way
How many molecules of ACh are needed to open the nAChR?
2- both binding sites need to be occupied for the receptor to open
What may the function of orphan receptors be?
One possible explaination may be their ‘promiscuous’ ability to bind to many related compounds with low affinity
What is the structure of GPCRs?
7 transmembrane alpha-helices
What happens when a signalling molecule binds at GPCRs?
The alpha subunit exchanges GDP for GTP and dissociates
The alpha subunit and the beta-gamma dimer target anothe protein in the membrane and alter/regulate protein function
Target proteins can be channels or enzymes
What ligands are GPCR activated by?
Photons, hormones, N terminal protelytic cleavage
Not always ligands
What are the different types of allosteric ligands acting on GPCRs?
- Allosteric antagonists= Ligands that bind to the allosteric site but do not have efficacy, yet their binding disrupts or inhibits the binding of other allosteric ligands.
- Neutral allosteric ligands= (Previously referred to as silent allosteric modulators). Allosteric ligands that bind to a G protein-coupled receptor but do not alter the affinity or efficacy of the orthosteric ligand.
- Negative allosteric modulators= Allosteric modulators that decrease the affinity and/or responsiveness of the orthosteric ligand.
What is biased signalling?
There can be two signalling pathways within one receptor and an agonist can act quicker in one pathway- An agonist can have a higher efficacy in one pathway, but lower efficacy than a different agonist in a different pathway
What are the different ways that GPCRs can cause signalling?
What does G alpha s and i do?
s= Stimulates adenylyl cyclase, causing increased cAMP formation
i= inhibits adenylyl cylase, decreasing cAMP
How are arrestins involved in GPCR recycling?
Arrestin binds with AP-2 and clarthin which are found in clarthrin coated pits (destined to become endoscopes)
The endosome is either recycled or degraded
What are the ionotropic glutamate receptors?
When do NMDARs open?
NMDA receptors open following depolrisation of the postsynaptic (dendritic) spine by AMPA receptor activation
The influx of sodium causes an action potential. NMDAR usually blocked by Mg2+ sitting in the pore, however depolarisation causes the Mg2+ to leave.
- Ca2+, N+ and K+ flows through the NMDA receptor
How do the AMPAR subunits change with age and how does this affect current?
GluA2-lacking AMPARs are predominantly expressed in early development and GluA2 AMPARs increase with age
GluA2 lacking allow Ca2+ to move through
Therefore, there is a greater variability of current in young ages
What is a blocker of AMPARs that lack GluA2?
NASPM
1-naphthyl acetyl spermine (NASPM) blocks AMPAR-currents at Week 2 but not at Week 5
What subunit do all NMDARs contain?
All NMDARs contain one or more of the obligatory GluN1 subunits
These are required for he binding of glycine, which acts as a co-agonist
How are GluN2 NMDA subunits spatially expressed?
An in situ study of mRNAs encoding NMDA receptor subunits in the developing rat CNS revealed that, at all stages, the N1 gene is expressed in virtually all neurons, whereas the four N2 transcripts display distinct expression patterns.
What are the different domains of an NMDAR?
Amino-terminal domain
Ligand-binding domain
Mmebrane domains
Carboxy-terminal domain
What pharmacological and biophysical properties do the N2 subunits of NMDARs determine?
2A receptors have low GluN1 and GluN2 agonist potency as well as fast deactivation rate. Also has higher Mg2+ sensitivity, higher Ca2+ permeability and higher conductance.
Opposite for 2D
