Receptors Flashcards
What response do ligand-gated ion channels cause?
Hyperpolarisation or Depolarisation
What is meant by the Cys Loop Superfamily?
Refers to a characteristic loop formed by 13 highly conserved amino acids between two cysteine (Cys) residues, which form a disulfide bond near the N-terminal extracellular domain
What is the common structure of the Cys Loop Superfamily?
Composed of five protein subunits which form a pentameric arrangement around a central pore
What is the structure of the nicotinic acetylcholine receptor?
Consists of a pentameric assembly of different subunits, of which there are four types, termed α, β, γ and δ, each 40-58 kDa
Subunits show marked sequence homology, and each contains four membrane-spanning α helices.
Extracellular N and C terminal tails
ACh binding site is on the α - subunit and either γ- or δ subunit
Explain the binding and activation of nAChR
When acetylcholine molecules bind, a conformation change occurs in the extracellular part of the receptor, which twists the alpha subunits, causing the kinked M2 (one of the transmembrane helices) segments to swivel out of the way and open the channel.
The channel lining contains a series of anionic residues (Glu, Gln and Asp), making the channel selective permeable to cations
Explain the differences of γ and ε subunits in muscle nAChRs
In foetal muscles, the stoichiometry of nAChR is α(2)βγδ, but in adult muscle, the γ subunit is replaced with an ε subunit to give a stochiometry of α(2)βεδ
Adult nAChRs have higher single channel conductances but shorter open times compared to the foetal form of the receptor
What are positive allosteric modulators?
enhance the effects of a ligand bound to the orthosteric site
They do not cause any response when bound by themselves
What is a silent allosteric modulator?
They bind to the receptor but have no effect on orthosteric agonist affinity or efficacy.
However, SAMs can act as competitive antagonists at the same allosteric site, blocking PAM or NAM activity.
What is a negative allosteric modulator?
inhibits the effects of a ligand bound to the orthosteric site
They do not cause any response when bound by themselves
What is the structure of the GABAA receptors?
GABAA receptors are generally pentameric proteins composed of different subunits, similar structures shared by an entire superfamily of Cys loop-type ligand-gated ion channels
Selectively transport Chloride ions
Explain the diversity of GABAA R subunits
At least 16 human GABAA receptor proteins have been described, and these have been classified under five distinct subfamilies of protein subunits termed α, β, γ, δ and ε.
There are six α subunits, four β subunits with two splice variants, three γ subunits with two splice variants, one δ subunit, and one ε subunit
Describe the GABA binding site
For α1β2 GABAA receptos, important binding residues are found upon 3 sections (Loop A-C) of the β-subunit (Principle face) and participating residues are found upon 3 sections of the adjacent subunit (Complementarity Subunits) - α subunit in the GABAA receptor
What is the action of benzodiazepines (e.g. diazepam) on GABAA receptors?
On their own, they generally have no effects, but they potentiate the action of GABA
They cause an increased affinity and/or efficacy of GABA
Thus they are a positive modulator
What is the basic structure of GPCRs?
Consists of a single polypeptide chain, usually of 350 -400 amino acid residues
Their characteristic structure comprises seven transmembrane α helices with an extracellular N-terminal domain of varying length, and an intracellular C-terminal domain
G-proteins bound to cell membrane via a fatty acid chain (prenylation)
What are the three main classes of GPCRs?
Classes A, B and C
Describe Class A GPCRs
Rhodopsin-like Receptor Family
Short extracellular N-terminal tail. Small ligands (amines) bind between TM Helices and Larger ligands (peptides) bind to extracellular loops
Describe class B GPCRs
Secretin/Glucagon Receptor-like Family
Intermediate extracellular N-terminal tail which contributed to binding of ligands (peptide hormones)
Describe class C GPCRs
Metabotropic Glutamate Receptor-like Family
Long extracellular N-terminal tail, fully responsible for ligand binding (encloses on the ligand)
Use β2-AR to give an example of ligand binding
Site-directed mutagenesis revealed that β-agonists bind to residues on the hydrophobic region within the cell membrane between the third and sixth transmembrane domain
How can GPCRs be activated by proteinases?
Proteinases activate protease-activated receptors (PARs) by snipping off the end of the extracellular N-terminal tail of the receptor to expose five or six N-terminal residues that bind to the receptor domains in the EC loop
Function as a ‘tethered agonist’
Describe the light-mediated activation of rhodopsin
Rhodopsin consists of a complex of a rod-type opsin (rhodopsin) with the chromophore ligand 11-cis-retinal acting as an inverse agonist
Photoisomerisation of retinal converts the ligand into its all-trans form now acting as the receptor agonist
Describe the conformational changes in agonist-activated β2-AR
Agonist ligand (NA) interacts and induces separation of the inner ends of TM5 and TM6 to allow G protein access
What are the 4 main families of G proteins and their function?
- Gs –> activates adenylyl cyclase to produce cAMP
- Gi/o –> inhibits adenylyl cyclase
- Gq/11 –> activate phospholipase C to produce IP3 and DAG
- G12/13 –> activate small G protein Rho to cause cytoskeletal changes
How does phosphorylation desensitise GPCRs?
On receptor activation, GRK3 and GRK2 are recruited to the plasma membrane by binding to Beta/gamma complex.
They are able to phosphorylate the C-terminal cytoplasmic tail which allows for arrestins to bind
What are the effects of arrestins on GPCRs?
Phospo-GPCR invades polar core of arrestin; exposed regions can bind to intracellular loop of GPCR and block G protein interaction –> homologous desensitization.
also targets the receptor for endocytosis in clathrin-coated pits.
can now be dephosphorylated and re-inserted into the plasma membrane (resensitisation) or trafficked into lysosomes for degradation (inactivation)
Explain a general scheme for GPCR signalling
Under resting conditions, the inactive alpha subunit is bound to one molecule of GDP and Beta/gamma complex
Agonist binds and receptor undergoes a conformational change that increase affinity for g protein.
G protein interacts with active receptors stimulates release of GDP from alpha subunit
High [GTP] means GTP replaces GDP
Active alpha subunit dissociates from receptor and Beta/gamma complex
Signal terminated by intrinsic GTPase activity of alpha subunit followed by reassociation
What is the random collision theory?
experimental observations and computational studies have revealed that agonists alone often do not stabilize the active conformation of the GPCR, hindering the subsequent recruitment of GP.
Thus, complex formation between GPCRs and their cognate GPs greatly relies on random collisions between the pair
Explain the adenylyl cyclase pathway of Gs and Gi proteins
Gs stimulates and Gi inhibits this pathway
cAMP is synthesised from ATP by the action of adenylyl cyclase.
Regulated and inactivated by hydrolysis to 5’-AMP by phosphodiesterase
The varied effects of cAMP are brought about through the activation of protein kinases by cAMP
Give examples of regulation by protein kinase
PKA phosphorylates voltage-gated calcium channels in heart muscle cells
Phosphorylation increases the amount of Ca2+ entering the cell during the action potential
Increase force of contraction
Give an example of a phosphodiesterase inhibitor
PDE4 (inflammatory cells) - Rolipram (asthma)
Explain the phospholipase C pathway of Gq proteins
PIP2 is the substrate for a membrane-bound enzyme, phospholipase Cβ which splits into DAG and Inositol (1,4,5) triphosphate (IP3) both of which function as a second messenger
After cleavage of PIP2, DAG being phosphorylated to form phosphatidic acid (PA) and the IP3 is dephosphorylated and then recoupled with PA to form PIP2 once again
What is the function of IP3 and DAG?
IP3: Water soluble mediator that is released into the cytosol and acts on a LGIC to control the release of Ca2+ from intracellular stores
DAG: activates protein kinase C which catalyses the phosphorylation of several intracellular proteins. It is highly lipophilic so remains within the membrane and will bind to a specific site on PKC molecule cause the enzyme to migrate from the cytosol to the cell membrane and thereby becoming activated.
How may ACh cause blood vessels to relax?
- ACh binds to M3 receptor (linked to Gq protein) on endothelium instead
- ACh stimulates an influx of Calcium ions
- Calcium binds to Ca Modulin which causes activation of eNOS
- eNOS catalyses the production of NO through the conversion of L-Arginine into L-Citrulline
- NO activates soluble Guanylate cyclase
- Guanylate cyclase catalyses GTP to cGMP which activates PKG
- Smooth Muscle Relaxation
NOTE: ACh typically stimulates contraction if directly binds to smooth muscle AChR
Why are ion channels important?
- Neuronal excitability e.g membrane potential, action potential, the release of neurotransmitters
- Excitation-contraction coupling in muscles
- Volume control (counteracts swelling by maintaining OsM)
- Maintenance of high external [K+] by marginal cells of the cochlea
- Regulate the cell cycle and cell division
- Mutations in voltage-gated channels lead to forms of epilepsy, heart disease and deafness
How does the animal kingdom emphasise the importance of ion channels?
Potent neurotoxin in the venom of Black Mamba snake (dendrotoxin I and - K, calciseptin) act by selectively blocking different types of voltage-gated ion channels
How does TTX block voltage-gated sodium channels?
It contains a guanidium moiety that enters the mouth of the Na+ channel
Other parts of TTX cannot and will interact with acidic amino acid residues around the entrance to generate a high affinity and highly selective block of the channel
Why doesn’t TTX block puffer fish and mammalian heart muscle Na+ channels?
The P-loop that links membrane-spanning regions 5 and 6 dips into the membrane to for the mouth of the Na+ channel
Amino acids from each of the four P-loops also form selectivity filters
Residue changes have been shown to remove selectivity:
Position 387 –> Glutamate to Glutamine
Position 385 –> Cysteine to Phenylalanine
Position 388 –> Arginine to Asparagine
Latter are ones found in mammalian heart
What makes voltage-gated K+ channels specific?
K+ channel signature sequence:
- Thr - (Val or Iso) - Gly - Tyr - Glu -
creates the selectivity filter
Amino acids provide a carbonyl in a position that specifically interacts with dehydrated K+ ions but are too far apart to do the same for Na+
P loops point their negative charge towards the cavity to attract cations
What is the proposed mechanism of K+ channel activation?
- The S4-S5 linker interacts through non-covalent forces with the inner parts of S6
- Voltage-sensor movement exerts movement in the S4-S5
- Due to its interaction with S6, S4-S5 movement causes a conformational change in the inner part of S6 around the PVP hinge in the S6 alpha helix
- Pore opened
How is the voltage-gated Na+ channel inactivated?
- Inactivation modulated by antibodies, enzymes
- Three key amino acids in linker, I1488, F1489 and M1490 form the IFM motif
- The intracellular III-IV linker (containing IFM) acts as a hinged lid
- Opening exposes residues that IFM interact with
- Inactivation restored by pentapeptide containing IFM motif
What is the general structure of a local anaesthetic molecule?
Local anaesthetic molecules consist of an aromatic part linked by an ester or amide bond to a basic side chain
What are the pharmacokinetic properties of local anaesthetic molecules?
They are weak bases, with pKa values mainly in the range 8-9, so that they are mainly, but not completely, ionized at physiological pH. This is important in relation to their ability to penetrate nerve sheath and axon membranes
What are the pharmacokinetic properties of local anaesthetic molecules?
They are weak bases, with pKa values mainly in the range 8-9, so that they are mainly, but not completely, ionized at physiological pH. This is important in relation to their ability to penetrate nerve sheath and axon membranes
What are Local Anaesthetics?
A class of drugs that have their effects by preventing nerve conduction through blocking voltage-gated Na+ channels
What is the Hydrophilic Pathway of LA action?
The charged form of LA enters through the aqueous environment of the voltage-gated Na+ channel
Once inside, the charged form blocks Na+ channel from an intracellular site
What is the hydrophobic pathway?
Entry of the uncharged molecule into the channel directly from the membrane phase
X-crystallography suggest presence of portals in the side of the channel protein allows access to central cavity in the ion permeation pathway –> possible route for uncharged molecule
What does use-dependence mean?
The more the channels are opened, the greater the block becomes
Why is pain blocked more effectively then other sensory modalities?
The passage of a train of action potentials, for example, in response to a painful stimulus, causes the channels to cycle through the open and inactivated states, both of which are more likely to bind local anaesthetic molecules than the resting state
Thus both mechanisms contribute to use dependence
What is the mechanism of action of LA?
A major determinant of LA binding in hNav1.5 is a phenylalanine residue (F1759) located on TM IV of domain 4.
Allows LA to bind at site in the channel pore, on the cytoplasmic side of the selectivity filter
Could lead to physical block or introduce a positive charge that repels positive Na+
How doe LA’s structure contribute to its duration?
The presence of the ester or amide bond determines its susceptibility to metabolic hydrolysis
ester-containing compounds are fairly rapidly inactivated in plasma and tissues by non-specific esterases.
amides are more stable and these anaesthetics generally have longer plasm half-lives
