Receptors And Ion Channels Flashcards
What is the rINN name for the following drugs
a) butoxamine
b) clorgyline
c) amphetamine
a) butaxamine
b) clorgiline
c) amfetamine
What is 5-HT
5-hydroxytryptamine (serotonin)
Are steroid receptors membrane bound or cytosolic
They are cytosolic proteins
Give the five forces involved in the structure of a receptor in diminishing order of strength
Covalent bonds
Ionic bonds
Hydrogen bonds
Van der Waals Forces
Hydrophobic forces
How are large ligands believed to bind to receptors
Through a zipper interaction
Define agonist in terms of a receptor
An agonist binds to receptors and produces a response
Describe the approximate relationship between the size of the response and the concentration of agonist
What about when the response is plotted against the logarithm of the agonist concentration
Hyperbolic
Sigmoidal
What is the amount of a receptor that is bound by a given concentration of a drug governed by?
The affinity of the drug for the binding site
What is efficacy
The relationship between the amount of receptor occupied by a drug and the size of the response produced
True or false
An agonist has both affinity and efficacy
True
as they both binds to and activates a receptor to produce a response
True or false
Both antagonists and agonists display efficacy
False
both display affinity as antagonists bind but elicit no response
How is efficacy usually measured
What does this allow calculations of
On a log graph
EC50 - A measure of potency for agonists
What is EC 50
The concentration of the agonist that causes 50% of the maximum response
The response does NOT indicate the concentration of agonist occupying half of the receptors
What is potency
The concentration causing a particular magnitude of response (e.g. EC 50) - not to the size of the maximal response
What happens to the log concentration/response curve for an agonist in the presence of a competitive antagonist
It is shifted to the right in a parallel fashion
Can a maximal response be obtained if the agonist is in the presence of a competitive antagonist
Therefore what is another name of competitive antagonism
Yes, if enough agonist is added then the same maximal response is obtained
Surmountable antagonism
Does the addition of a competitive antagonist increase or decrease the EC 50
Increase
Maximal response remains the same
What is a structure activity series
A sequence of experiments to determine the relative potency of a group of structurally related compounds
What can be used to aid receptor classification
Give an example
In the case of ACh, two structure activity series can be seen and these help demonstrate the existence of two types of receptor, nicotinic and muscarinic receptors
Why can drugs with the same chemical formula offer different potencies
What does this demonstrate
Stereoisomers act differently
The complementarity of the interaction between drug and receptor
What is acetyl-β- methylcholine
What does its stereoisomer do
What does this mean for the racemate of acetyl-β-methylcholine?
Cholinergic agonist
Isomer has no affinity
Effectively reduces the concentration of the active isomer by half
What does isoprenaline do
What is the effect of its stereoisomer
What happens if you increase concentrations of the racemate
β- adrenoceptor agonist
Inactive isomer has affinity but no efficacy so acts as an antagonist
Simultaneously increases [agonist] and [antagonist]
What is the forward and backward reactions of the following know as:
D+R↔️DR
What does the rate of each equal?
Forward: K+1; rate =K+1[D][R]
Backwards: K-1; rate = K-1[DR]
What is the notation for the affinity constant of drug D
Ka (=k+1/k-1)
The higher the Ka, the ____ the affinity of D for R
Higher
What is Kd
How to work out Kd
The dissociation constant
1/Kd
What are the units of Ka
What about for Kd
M-1 or L/mol
M or mol/L
Describe D+R↔️DR in terms of α, where α is a fraction of receptors that combine with drug D
Therefore, what does Ka= in terms of α
Thus, what does it mean if α= 0.5
Describe α in terms of Ka and [D]
α=?
[D] Ka
————-
1+[D]Ka
When α=0.5, [D]=1/Ka=Kd. What does this mean in reality?
The affinity constant is the reciprocal of the drug constant that produces 50% occupancy of the receptors
The affinity constant is the reciprocal of the drug constant that produces 50% occupancy of the receptors.
This is usually the concentration of an agonist that causes the half maximal response - true or false?
False
How can you measure the binding of a drug to its receptor directly
Using radioligand binding
What does the binding of a radio ligand consist of
Saturable component (specific binding to receptors) and amazing effectively linear non-saturable component (non - specific binding to non receptor material)
How is specific and non specific binding of radio ligands affected in the presence of a large excess of unlabelled ligand
Specific- almost completely abolished
Non- specific- almost unaffected
How can specific binding of radioligands be calculated
The difference between total binding and non specific binding in the presence of a large excess of non labelled ligands
Two of those
Specific binding sites always represent functional receptors
False
Conclusions from binding experiments must be supported by data about the functional behaviour of the putative receptor
How does the graph of specific binding and non specific binding differ on a chart of [radioligand] vs bound radioligand
Specific and total: rectangular hyperbola
Non specific: linear
Describe α in terms of B (specific binding at the radiolabelled concentration [D]) and Bmax (maximal specific binding)
α= B/Bmax
Describe the equation for the double reciprocal graph of the following equation
α= B/Bmax= [D]Ka/1+[D]Ka
How to calculate Ka and Bmax
1/B=1/[D] x 1/BmaxKa + 1/Bmax
1/Bmax= y intercept -Ka= x intercept
What are the 2 axes for the Scatchard plot
How do you find out Ka and Bmax?
What does [D] refer to
B(x) vs B/[D] (y)
Gradient = -Ka
X intercept= Bmax
Free drug (NOT total concentration added)
Why is the Scatchard plot useful
Helps demonstrate situations where there maybe more than one binding site on a receptor
It can show the different affinities of each site
Can the different binding sites on a receptor affect the affinities of different binding sites
Yes - oxygen binding to haemoglobin - positive cooperativity
What graph is cooperativity seen in
A plot of frequency of channel opening vs agonist concentration
It is sigmoidal and NOT hyperbolic
What is the Hill plot
The equation relating binding to drug concentration can be modified to generate the Hill plot which reveals cooperativity
What are the axes of the Hill plot
x= log[D] y= Log B/(Bmax-B)
What indicated positive and negative cooperativity on a Hill plot
nH >1 is positive
nH <1 is negative
What does the nH =1 mean
In this condition, what does X=, if y=0
1:1 binding between drug and active site
No interaction between binding sites
LogKd
Describe a Hill plot line where there are multiple non interacting binding sites
nH <1 but line will not be straighten
When a fixed concentration of radioligand is used to describe the shape of the graph of ligand binding against log concentration of competing unlabelled drug
What do we need to know for this
Sigmoid
The concentration of unlabelled drug that displaces 50% of the specifically bound radio ligand
What is IC50?
The concentration of unlabelled drug that displaces 50% of the specifically bound radio ligand
What is the equation for the occupancy of receptors by unlabelled ligand α
When [U]=IC50, What does α=?
[U] x KU
———————————-
1+([U]xKU)+([L] x KL)
Where KU and KL are affinity constants for the binding of labelled and unlabelled drugs
α=0.5
Give an equation for KU, when [U]=IC50
KU=…
1+[L]KL
————
IC50
Is there a direct linear relationship between affinity and efficacy
This was thought to be true up until the 1960’s when it was believed that Kd=EC50
This was proved to be incorrect through experiments with Benzilylcholine mustard
What did experiments with BzCh reveal
Describe associated efficacy/ affinity graphs
The concept of ‘spare’ receptors
Affinity does not equal efficacy
Efficacy results from significant amplification of the response so is always left shifted from affinity
Both are sigmoidal
What does BzCh mustard (an antagonist) do to its receptor
What would be expected if the agonist response generated were directly proportional to receptor occupancy? What actually happens?
Irreversibly alkylates the muscarinic receptor
As progressive alkylation occurs thete should be a decrease in maximum response from the agonist with no change in apparent affinity of remaining receptors
With ACh, on the guinea pig ileum, only a fraction of the receptors need to be bound to to produce a maximal response
What happens as the amount of BzCh increases with the amount of ACh?
How can we estimate which percentage of receptors are spare?
At first maximal response is still attainable as BzCh is only affecting spare receptors
Once BzCh fills all spare receptors, maximal response is still just possible
Any extra BzCh results in a fall off of the maximal response
By measuring dose ratio at the point where maximal response is just attainable
What is the dose ratio for ACh in the guinea pig ileum
What does this mean
100
99% of the receptors are spare
Why does it make sense for a receptor to have a low affinity for an agonist
What does this mean for the number of spare receptors
So the offset rate is high and the response is terminated quickly
The existence of spare receptors here increases the sensitivity of the tissue to the agonist
How can you determine the affinity constant of an agonist from a log conc/ response curve
It is not possible because response is not directly proportional to receptor occupancy
What does the dose ratio determining method assume
Why do we know this
Only have equal responses are produced by equal agonist
Antagonist affinities determined in this way are the same as those determined by direct binding
What is the effect of adding a partial agonist alongside a full agonist
Why does this happen
Partial agonist acts like a competitive antagonist
By occupying receptors relatively ineffectually and preventing the full agonist from binding
What does α= for drug D and antagonist A
α=
[D]K1
————————-
1+[D]K1 +[A]K2
Give an example of drugs that don’t need to bind to receptors to have an effect
Ant acids - only change pH
Some diuretics only exert osmotic pressure
What is the shape of a graph of bound receptors vs [D]
Hyperbolic
What kind of bond is formed by an irreversible inhibitor
Covalent
Which kind of inhibitor reduces Vmax
Irreversible
Why can irreversible inhibitors still be surmountable
They may only block spare receptors so enough receptors are still present for a full response to occur
Why do partial agonists act as inhibitors
Partial agonists require all receptors so its weaker effect blocks the more powerful effect that could be elicited by the full agonist
What does the drug ratio (R)-1 of an antagonist (B) equal
What kind of antagonist is this for
R-1=[B] x Kb
Reversible antagonist
What are the agonist, partial agonist and antagonist for μ-opioid receptors
Morphine = full agonist Buprenorphine = partial agonist Naloxone= competitive antagonist
Define agonist
A substance that initiates a physiological response when combined with a receptor
Define antagonist
A substance which interferes with/ inhibits the physiological action of another
Define competitive antagonist
Bonds to the same site as the agonist but does not activate it, thus blocking the agonist’s action
Define partial agonist
A substance that binds to and activates a given receptor, but only has partial efficacy at the receptor relative to a full agonist
Define inverse agonist
A drug that binds to the same receptor as an agonist but induces a pharmacological response opposite to that of the agonist
Define biased agonism
AKA functional selectivity
Ligand dependant selectivity for certain signal transduction pathways relative to a reference ligand at the same receptor
V1=
C2 x V2
—————-
C1
Should there be air in the intra-abdominal cavity?
What should you be thinking
No
This suggests a perforation
PEPTIC ULCERS
What is the dose ratio
[D2]
—— = 1+[A]K2
[D1]
How does K1 affect the dose ratio
Dose ratio is independent of K1
What is the problem with dose ratio
We only have a single concentration of the antagonist to calculate affinity which is unsatisfactory experimentally
Normally multiple doses are used to compete with the agonist allowing a Schild regression to be generated
What are the axes of the Schild regression
What can this be used to find
Log[antagonist]
y= Log[dose ratio-1]
X intercept = affinity of the competitive antagonist (if the slope is of unity)
What are the 5 drug receptor families
Give an example for each
- Intracellular (NO or steroid receptors)
- Directly operated ion channels (nicotinic ACh receptor)
- Intrinsic enzyme activity receptors (eg RTK)
- Those linked to enzymes (eg EPO)
- GPCRs (eg β adrenoceptor or muscarinic ACh receptor)
Which 2 groups can intracellular receptors be divided into
Those with enzymatic activity (eg soluble guanylyl cyclase
Those that bind to lipid soluble agonists eg steroids
Describe the activity of soluble guanylyl cyclase
NO is released from endothelial cells to cross the PM and interact with soluble guanylyl cyclase, which converts GTP to cGMP
cGMP activates PKG, promoting smooth muscle relaxation
How is cGMP broken down
How can this be inhibited
By cGMP phosphodiesterase
Sildenafil citrate
What are the 3 domains of the steroid receptors
Lipid binding domain
DNA binding domain
Transcription activating domain
Where do steroid receptors usually reside
Name an exception
In the cytoplasm but trans locates to the nucleus upon activation by ligand
Oestrogen receptor - May be located in the nucleus even in the absence of a ligand
Describe the time period of steroid activity
Which other group of hormones work in this way
Effects typically appear after a lag and may persist for hours or days
Thyroid hormones
What kind of communication do ligand gated ion channels allow
Fast communication between cells
What is the best characterised ligand gated ion channel
How has it been studied
the nicotinic ACh receptor
Using the patch clamp technique
Describe quantitively the behaviour of a nicotinic ACh receptor (nAChR)
In response to ACh, the channel opens for about 1ms, allowing passage of ~10,000 Na+ ions
Frequency of channel opening increases with increasing [agonist]
What is the Hill coefficient for nAChR
2
Describe the structure of the nAChR
Pentameter
2α, β, γ, and δ
α and its neighbour bind ACh and Both must be occupied to elicit a response
Each subunit has 4 hydrophobic stretches (M1-4)
M2 May form an α helix within the membrane and the 5 M2 regions apparently line the pore of the channel
Is GC soluble or membrane bound
Both
Membrane bound GC is similar to soluble guanylyl cyclase function except the receptor is bound to the membrane
Describe the structure of a membrane bound GC
Receptor is a dimer and binds molecules such as ANP
How does ANP bind to membrane GC
Binds to the extracellular face of the receptor dimer which activates the guanylyl cyclase domain on the intracellular face
What are RSKs
Receptor serine/ threonine kinases
Transmembrane proteins of the plasma membrane and Are characterised by extracellular ligand binding domains and cytoplasmic kinase domains
What superfamily do RSKs belong to
Thus what roles are they involved in
Transforming growth factor β (TGF-β)
Physiological and pathological processes of the metres are in life including early embryogenesis, atherosclerosis and cancer
What are the 2 sy types of RSKs
Type 1 and 2
Based on primary amino acid sequence - upon ligand binding a heteromeric complex of both types is formed and Type 2 phosphorylates and activates Type 1
Type 1 then plays a role in downstream signalling
What do activated RSKs recruit
SMADs - when SMAD4 is recruited, a complex of activated SMADs and SMAD4 translocate to the nucleus and influence gene expression for cell proliferation
What does every RTK have
Name 2 molecules that bind to them
What do all RTK do at some point
An intracellular catalytic domain and an extracellular regulatory domain
Insulin
EGF
PDGF
Activate the G protein Ras
How many polypeptide chains do the receptors for the following contain
Insulin
EGF
Insulin: Contains two different peptide chains linked as a dimer (βα-αβ)
EGF: a single polypeptide chain
Does dimerisation come before or after autophosphorylation in EGF RTK?
Dimerisation precedes auto phosphorylation
Are RTK responses rapid
They can be fast or delayed
Why are RTKs of interest to virologists
Several. Viral oncogenes encode mutated forms of either receptors or ligands
Eg v-erb encodes a truncated, constitutively active EGF receptor
V-sis encodes a PDGF chain
How do RTKs and RSKs differ from receptors linked to soluble kinases
In receptors linked to soluble kinases, the kinase domain is encoded on separate genes from the transmembrane receptor but then associate together
Give an example of receptors linked to soluble kinases
The EPO receptor
Signalling is facilitated by the JAK-STAT pathways - inducers of TFs
Disrupted JAK-STAT signalling May lead to many different disorders
How many subunits do receptors of GPCRs have
1 of a size or 40-50kDa
What do all GPCRs have
7 transmembrane α helices (N extracellular, c intracellular)
Where is the GPCR’s ligand binding site
How does the receptor interact with the G protein
Usually buried within the transmembrane helices
Via the 3rd intracellular loop and C terminal tail
How does cAMP control cell function
Through PKA
How may GPCRs affect AC
Activate (β1-AR) - Gs
or
inhibit (α2 -AR) - Gi
Describe the structure of G proteins
Where can the difference between Gi and Gs be found
Heterotrimers, consisting of an α subunit, a β subunit and a γ subunit
In the α subunit which binds and hydrolyses GTP
What happens to a G protein when a GPCR is occupied by an agonist
The receptor forms a transient complex with Gs, which is occupied by GDP
GDP-GTP exchange promotes Gs release and causes a reduction in binding affinity of agonist to receptor
βγ is also released, while αs forms an active enzyme complex with AC
How does G protein activity end
Agonist activates GTPase activity terminates the active state of αs and βγ recouples
How does Cholera toxin act
Causes ADP-ribosylation of αs
This inhibits GTPase activity and causes sustained activity of AC
How can the βγ complex be inhibitory
Through the mopping up of free αs
Can also have effects on AC (independent of αs)
How does pertussis toxin work
ADP-ribosylation of αi and prevents activation of Gi in response to receptor stimulation
Name a group of organists that stimulate the metabolism of inositol phospholipids in the plasma membrane
ACh (at mAChR)
Noradrenaline ( at α1 ARs)
Substance P
Which G proteins activate phospholipase C
G11 and Gq
What are the cleavage products of PIP2 and what do they Do
DAG - Stimulate membrane-bound PKC, which modifies properties of various proteins such as ion channels by phosphorylation
IP3- Causes the release of calcium from the endoplasmic reticulum
How is IP3 metabolised
By phosphorylation to IP4 or by dephosphorylation successively to inositol
Why is lithium used to treat bipolar disorder
That’s the uncompetitively inhibits the phosphatase that is responsible for converting IP1 to inositol thereby blocking recycling of inositol
Overall what does it mean if a receptor is desensitised
Transduction mechanism is uncoupled
What are the three processes involved in desensitisation
Uncoupling
Sequestration
Down-regulation
Describe the uncoupling part of desensitisation
This is uncoupling of the receptor from its G-protein
Takes place over the course of seconds two minutes and is due to phosphorylation events at the receptor
Describe the sequestration part of desensitisation
Receptors are removed from the plasma membrane by endocytosis and transferred to vesicles in the cytoplasm
This takes place over minutes
The receptors can be shuffled back to the plasma membrane but can also be destroyed in lysosomes this destruction can be elevated in the down regulation stage
Describe the time regulation stage of desensitisation
Takes place over minutes two hours
The number of receptors available to be shuttled to and from the plasma membrane is reduced this is possibly due to reduced receptor synthesis by effect of PKA on mRNA stability
Can sequestration or down-regulation in desensitisation take place without uncoupling
No
What are the different types of desensitisation
Homologous and Heterologous
Described a homologous desensitisation using the example of the β2adrenoreceptor
The adrenoreceptor can upon ligand
binding be phosphorylated by GRK (usually GRK2/3)
GRK phosphorylates sites at the end of the C terminal membrane which increases the infinity of the beta 2 adrenoreceptor for another protein called β arrestin
The binding of β arrestin to receptor uncouples the receptor from the α-subunit of Gs thus producing desensitisation
How many GRKs are there
GPCR kinases - 7 types with 2,3,5 being the most important
Why is it called homologous desensitisation
GRKS only work on agonist occupied receptors so does not affect any other receptors that might be present
How does heterologous desensitisation work
Stimulation of β2 ARs increases cAMP, stimulating PKA
If the receptor is disproportionately stimulated, there will be a large rise in PKA activity, which is able to phosphorylate site on the β2- receptor itself
This phosphorylation uncouples the receptor from the α-subunit of Gs, thus rendering it ineffective
Where does high levels of PKA phosphorylate the β2 receptor
At site on the third cytoplasmic loop and the first part of the C terminal domain
Why is heterologous desensitisation of adrenoreceptors considered heterologous
PKA can also phosphorylate receptors with similar amino acid sequences so other receptors than in the β2 ARs can be desensitised without having first been stimulated by a ligand
Can one GPC are couple to more than one G protein
Yes and can also be activated by multiple agonists
What is the bias of an agonist
The degree by which the agonist activates different pathways
What can β arrestin do for GPCRs
Recruitment of β arrestin blocks the G protein interaction but can now enable additional G protein independent signalling events
Give an example of a drug that uses GPCR bias
Oliceridine
TRV130
μ-opioid receptor biased agonist that elicits robust G protein signalling, with potency and efficacy to morphine but with less β arrestin 2 recruitment and receptor internalisation thus having reduced adverse effects compared to morphine
