Receptors

Question 1

What response do ligand-gated ion channels cause?

Answer 1

Hyperpolarisation or Depolarisation

Question 2

What is meant by the Cys Loop Superfamily?

Answer 2

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

Question 3

What is the common structure of the Cys Loop Superfamily?

Answer 3

Composed of five protein subunits which form a pentameric arrangement around a central pore

Question 4

What is the structure of the nicotinic acetylcholine receptor?

Answer 4

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

Question 5

Explain the binding and activation of nAChR

Answer 5

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

Question 6

Explain the differences of γ and ε subunits in muscle nAChRs

Answer 6

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

Question 7

What are positive allosteric modulators?

Answer 7

enhance the effects of a ligand bound to the orthosteric site

They do not cause any response when bound by themselves

Question 8

What is a silent allosteric modulator?

Answer 8

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.

Question 9

What is a negative allosteric modulator?

Answer 9

inhibits the effects of a ligand bound to the orthosteric site

They do not cause any response when bound by themselves

Question 10

What is the structure of the GABAA receptors?

Answer 10

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

Question 11

Explain the diversity of GABAA R subunits

Answer 11

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

Question 12

Describe the GABA binding site

Answer 12

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

Question 13

What is the action of benzodiazepines (e.g. diazepam) on GABAA receptors?

Answer 13

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

Question 14

What is the basic structure of GPCRs?

Answer 14

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)

Question 15

What are the three main classes of GPCRs?

Answer 15

Classes A, B and C

Question 16

Describe Class A GPCRs

Answer 16

Rhodopsin-like Receptor Family

Short extracellular N-terminal tail. Small ligands (amines) bind between TM Helices and Larger ligands (peptides) bind to extracellular loops

Question 17

Describe class B GPCRs

Answer 17

Secretin/Glucagon Receptor-like Family

Intermediate extracellular N-terminal tail which contributed to binding of ligands (peptide hormones)

Question 18

Describe class C GPCRs

Answer 18

Metabotropic Glutamate Receptor-like Family

Long extracellular N-terminal tail, fully responsible for ligand binding (encloses on the ligand)

Question 19

Use β2-AR to give an example of ligand binding

Answer 19

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

Question 20

How can GPCRs be activated by proteinases?

Answer 20

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’

Question 21

Describe the light-mediated activation of rhodopsin

Answer 21

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

Question 22

Describe the conformational changes in agonist-activated β2-AR

Answer 22

Agonist ligand (NA) interacts and induces separation of the inner ends of TM5 and TM6 to allow G protein access

Question 23

What are the 4 main families of G proteins and their function?

Answer 23

1. Gs –> activates adenylyl cyclase to produce cAMP
2. Gi/o –> inhibits adenylyl cyclase
3. Gq/11 –> activate phospholipase C to produce IP3 and DAG
4. G12/13 –> activate small G protein Rho to cause cytoskeletal changes

Question 24

How does phosphorylation desensitise GPCRs?

Answer 24

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

Question 25

What are the effects of arrestins on GPCRs?

Answer 25

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)

Question 26

Explain a general scheme for GPCR signalling

Answer 26

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

Question 27

What is the random collision theory?

Answer 27

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

Question 28

Explain the adenylyl cyclase pathway of Gs and Gi proteins

Answer 28

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

Question 29

Give examples of regulation by protein kinase

Answer 29

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

Question 30

Give an example of a phosphodiesterase inhibitor

Answer 30

PDE4 (inflammatory cells) - Rolipram (asthma)

Question 31

Explain the phospholipase C pathway of Gq proteins

Answer 31

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

Question 32

What is the function of IP3 and DAG?

Answer 32

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.

Question 33

How may ACh cause blood vessels to relax?

Answer 33

1. ACh binds to M3 receptor (linked to Gq protein) on endothelium instead
2. ACh stimulates an influx of Calcium ions
3. Calcium binds to Ca Modulin which causes activation of eNOS
4. eNOS catalyses the production of NO through the conversion of L-Arginine into L-Citrulline
5. NO activates soluble Guanylate cyclase
6. Guanylate cyclase catalyses GTP to cGMP which activates PKG
7. Smooth Muscle Relaxation

NOTE: ACh typically stimulates contraction if directly binds to smooth muscle AChR

Question 34

Why are ion channels important?

Answer 34

• 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

Question 35

How does the animal kingdom emphasise the importance of ion channels?

Answer 35

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

Question 36

How does TTX block voltage-gated sodium channels?

Answer 36

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

Question 37

Why doesn’t TTX block puffer fish and mammalian heart muscle Na+ channels?

Answer 37

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

Question 38

What makes voltage-gated K+ channels specific?

Answer 38

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

Question 39

What is the proposed mechanism of K+ channel activation?

Answer 39

1. The S4-S5 linker interacts through non-covalent forces with the inner parts of S6
2. Voltage-sensor movement exerts movement in the S4-S5
3. 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
4. Pore opened

Question 40

How is the voltage-gated Na+ channel inactivated?

Answer 40

1. Inactivation modulated by antibodies, enzymes
2. Three key amino acids in linker, I1488, F1489 and M1490 form the IFM motif
3. The intracellular III-IV linker (containing IFM) acts as a hinged lid
4. Opening exposes residues that IFM interact with
5. Inactivation restored by pentapeptide containing IFM motif

Question 41

What is the general structure of a local anaesthetic molecule?

Answer 41

Local anaesthetic molecules consist of an aromatic part linked by an ester or amide bond to a basic side chain

Question 42

What are the pharmacokinetic properties of local anaesthetic molecules?

Answer 42

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

Question 42

What are the pharmacokinetic properties of local anaesthetic molecules?

Answer 42

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

Question 43

What are Local Anaesthetics?

Answer 43

A class of drugs that have their effects by preventing nerve conduction through blocking voltage-gated Na+ channels

Question 44

What is the Hydrophilic Pathway of LA action?

Answer 44

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

Question 45

What is the hydrophobic pathway?

Answer 45

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

Question 46

What does use-dependence mean?

Answer 46

The more the channels are opened, the greater the block becomes

Question 47

Why is pain blocked more effectively then other sensory modalities?

Answer 47

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

Question 48

What is the mechanism of action of LA?

Answer 48

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+

Question 49

How doe LA’s structure contribute to its duration?

Answer 49

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