BS3054 TOPIC ONE: Receptor Diversity

Define the term receptor.

A molecule (usually a protein) inside, or on the surface of a cell that binds to a specific substance (ligand) and causes a consistent physiological effect in the cell

What are the four things a molecule must demonstrate to be classed as a receptor?

Saturability, specificity, reversibility and reconstruction of function.

Define saturability.

A finite number of receptors means that at a certain concentration of the ligand no further increase in the magnitude of response is observed.

Define specificity.

Only specific ligands will bind to the receptor: classically this is shown by stereospecificity

Define reversibility.

The ligand binds and unbinds the receptor and is chemically unaltered by the interaction.

Outline how enantiomers are an example of receptor specificity.

Ligands must match up every area of the receptor, in fact only one enantiomer will be able to bind to a receptor. - add image

Define reconstruction of function.

The active cellular component can be isolated and reconstituted in an artificial (membrane) system.

Briefly define the term novel molecular entity.

Although a receptor responds only to one or a very limited number of endogenous ligands, it is possible to synthesize chemicals (also known as novel molecular entities (NMEs)), which interact with the receptor.

Note: This also occurs in nature where one biological organism generates a chemical, which can be delivered (e.g. through a bite / sting / etc.) to another species and, via receptor interactions, cause a change in the targeted animal's physiology (e.g. incapacitation / paralysis).

Outline how snake venom e.g. eastern green mamba venom, is an example of the importance of receptors.

Snake venom can be used for its properties:

Venom contains:

Ca2+-channel blockers (calcicludines) - cardiotoxins

K+-channel blockers (dendrotoxins) - neurotoxins

over 10 muscarinic acetylcholine receptor toxins,

e.g. MT7 is a highly M1-selective antagonist

(strictly speaking, it acts as a negative allosteric modulator, rather than a true antagonist ). Add definition of negative allosteric modulator

Define the following terms: agonist, antagonist and allosteric modulator.

Mimic the action of endogenous ligand - AGONIST

Block the action of endogenous ligand - ANTAGONIST

Act at a site distinct from the ligand binding ("orthosteric") site - ALLOSTERIC MODULATOR

Outline allosteric ligand binding to non-orthosteric sites on receptors.

Muscarinic acetylcholine receptors are G protein-coupled receptors. mAChRs are activated by the binding of acetylcholine to the orthosteric binding site. MT7 binds to distinct surfaces of the mAChR which allow this peptide to bind allosterically and non-competitively decrease the ability of acetylcholine to activate the receptor i.e. MT7 is an negative allosteric modulator (NAM). - add image

Name the receptor superfamilies.

GPCRs (mebatropic receptors),ligand-gated (receptor-linked) ion channels, receptor tyrosine kinases (RTKs) and nuclear receptors).

Briefly summarise the features of GPCRs.

With over 800 GPCRs identified in the human genome they form 1-2% of identified genes, and all share a common basic structure. Their structure consists of a single polypeptide chain ranging from 300 to 1200 amino acids in length, an extracellular N-terminal , an intracellular C-terminal and 7-Transmembrane spanning domains (TMD).

Outline ligand gated ion channels.

Outline Receptor Tyrosine Kinases (RTKs).

RTKs are often single, transmembrane-spanning polypeptides which dimerize on ligand binding (N.B. the insulin receptor is an exception to this). Following dimerization, the C-terminal domain of each monomer phosphorylates the other monomer of the dimer pair (autophosphorylation) on specific tyrosine residues to form phosphotyrosine (Y-P) docking-sites for proteins possessing SH2 (Src-homology-2) domains. This allows the RTK to act as a scaffold for intracellular signalling pathways to be assembled.

Note: Insulin receptor is an exception to this and slightly different

Outline the therapeutic use of RTKs (Receptor Tyrosine Kinases)

HER2 (ERBB2) is an RTK closely related to EGFR. No specific ligand has been identified for HER2, so it is believed that it must heterodimerise with EGFR (or ERBB3, ERBB4) to become active. HER2 is overexpressed in 30% of breast cancers, thus various approaches to blocking HER2 signalling have been devised as anticancer agents, e.g. lapatinib a HER2-selective tyrosine kinase inhibitor.

Name the two classes of nuclear receptors.

The two classes are class one and class two receptors.

Class one examples: Glucocorticoid receptor (GR) Androgen receptor (AR) Progesterone receptor (PR) Estrogen receptor (ER)

Class two examples:

Thyroid hormone receptors Retinoic acid receptors Retinoid X receptor (RXR) Peroxisome-proliferator-activated receptors (PPARs)

What are the nuclear receptor ligands?

They have endogenous ligands (e.g. steroid hormones, thyroid hormones, vitamin A derivatives aka retinoids and exogenous liagnds such as tamoxifen, which binds to the oestrogen receptor in the breasts as an antagonists, while it is an agonist in the bones and uterus . Thus it is used in the treatment of hormone (oestrogen) positive breast cancer.

Outline the mechanism of action of nuclear receptors.

Lipophilic ligand permeates the plasma membrane and binds receptor → Receptor undergoes ligand-dependent release from cytoplasmic sequestration → Liganded receptor dimerises and translocates to the nucleus → Receptor binds to DNA and recruits coactivators to enhance transcription of specific target genes

Define the term druggability.

Our concept of what is 'druggable' and 'non-druggable' changes over time 'Undruggable' definition originally based on whether a small molecule might be designed to alter directly the function of the target protein.

Outline therapeutic approaches in pharmacology.

• Biopharmaceuticals (e.g. mAbs, nanobodies, etc.)• Alter de novo synthesis of target ('knockdown' strategies)• Increase/decrease rate of degradation (alter turnover)• Alter ability of target to localize (e.g. to membrane)• Alter ability of target to scaffold to other protein

Ion channels are...

integral membrane proteins, which form pores that allow ions to flow according to an electrochemical gradient. They can be ligand-gated, voltage-gated or other e.g. inward rectifiers

What electrophysiological technique measures changes in membrane potential (voltage) over time?

Current-clamp.

What electrophysiological technique is used to measure whole-cell ion channel currents by controlling the voltage?

Voltage-clamp.

Which patch-clamp recording configuration is achieved by applying a pulse of suction to rupture the membrane patch under the pipette?

Whole-cell recording.

Which patch-clamp configuration involves pulling a small piece of membrane away from the cell so that the extracellular side faces the bath solution?

Outside-out patch.

Which patch-clamp configuration involves pulling a membrane patch away so the intracellular side is exposed to the bath solution?

Inside-out patch.

What is the relationship between single-channel open events and whole-cell current?

Single-channel open events over time add up to the total whole-cell current.

On what factor does the probability of a voltage-gated ion channel opening primarily depend?

The membrane potential.

What are the two primary classifications of ion channels based on their gating mechanism?

Ligand-gated and voltage-gated channels.

Which superfamily of ligand-gated ion channels, including nicotinic ACh receptors, forms pentamers?

The Cys-loop receptor superfamily.

To which structural class (trimer, tetramer, or pentamer) do P2X receptors belong?

Trimers.

Which receptor family commonly forms tetramers?

The glutamate receptor family which includes , NMDA and AMPA receptors

What receptor antagonist is to treat Alzheimer's disease?

Memantine (Ebixa) a non-competitive, low-affinity NMDAR antagonist is used.

What is the pathological rationale for using $NMDA$ antagonists in Alzheimer's disease?

To combat neurotoxicity caused by excessive glutamate activity.

What is the common name for the Kv11.1 channel encoded by the KCNH2 gene?

The ether-a-go-go related gene (ERG) channel.

Why must all new drugs be screened for Kv11.1 (hERG) block before clinical trials?

To prevent acquired Long QT syndrome, which can lead to fatal arrhythmias.

What specific cardiac arrhythmia is characterised by the QRS complex twisting around the isoelectric point on an ECG?

Torsades de pointes.

Identify three structural reasons why many diverse drugs bind to the Kv11.1 channel.

A large inner cavity, aromatic residues, and polar residues.

In voltage-gated sodium and calcium channels, which transmembrane spanning domain acts as the voltage sensor?

The S4 domain.

What must happen to an inactivated sodium and calcium channel before it can open again?

It must cycle back through the closed (resting) state.

What does 'use-dependence' mean regarding ion channel drugs?

Drugs block more effectively in cells where channels are frequently active (open or inactivated).

Which class of calcium antagonists is exemplified by Nifedipine and is most effective in vascular smooth muscle?

Dihydropyridines.

Which class of calcium antagonists is exemplified by Verapamil and is primarily used for cardiac dysrhythmias?

Phenylalkylamines.

How does the binding of Verapamil differ from Nifedipine in terms of channel state?

Verapamil is strictly use-dependent, binding only when the channel is in the open state.

What is the primary clinical use for Nifedipine?

Treatment of hypertension by relaxing arteriole smooth muscle.

Name the analgesic drug derived from $\omega$-conotoxins found in fish-hunting cone snails.

Ziconotide (Prialt).

What is the molecular target and location of Ziconotide action?

Presynaptic N-type calcium channels ($C_{a,v}2.2$) in the dorsal horn of the spinal cord.

By what route must Ziconotide be administered for patients with morphine-unresponsive chronic pain?

Intrathecal injection.

Which auxiliary calcium channel subunit is the target for Gabapentin and Pregabalin?

The $\alpha_{2}\delta$ subunit.

What is the primary effect of Gabapentin binding to the alpha-2-delta subunit at synapses?

Modulation of calcium influx and subsequent reduction of neurotransmitter release.

Where does Lidocaine bind to the voltage-gated sodium channel?

The intracellular side of the channel pore.

In which states of the sodium channel does Lidocaine primarily produce a block?

The open or inactivated states.

How does action potential frequency affect the depth of block by local anaesthetics?

The depth of block increases with higher action potential frequency (use-dependence).

What are the three structural components typically found in a local anaesthetic drug?

An aromatic region, an ester or amide bond, and a basic amine side-chain.

What is the therapeutic use of Lidocaine when administered via intravenous injection?

Treatment of tachycardia as a Class 1 anti-dysrhythmic drug.

Why is there a time-dependence for Lidocaine to reach its maximum anti-dysrhythmic effect?

It takes time for enough channels to cycle through the open state to be blocked.

What is the generic term for drugs that block more effectively in cells firing many action potentials because they only bind the open state?

Strictly use-dependent drugs.

Which channel type allows calcium entry and is a target for pain treatment via the pharmacology of chillies?

TRPV1 (Transient Receptor Potential Vanilloid 1).

Which ligand-gated ion channel is a target for antiemetic drugs?

The 5-HT3 receptor.

What component of the ion channel protein pore discriminates between different ion species?

The selectivity filter.

How many genes approximately encode ligand-gated ion channels in humans?

84 genes.

Name a drug that acts as a partial agonist at nicotinic receptors.

Varenicline.

Which class of GABA drugs acts as agonists at GABAA receptors?

Benzodiazepines.

What is the primary role of Kv11.1 in the cardiac action potential?

Repolarisation of the cardiac action potential.

Which antihistamine was withdrawn from the market due to its dangerous block of Kv11.1?

Terfenadine.

How many domains are found in the alpha1 pore-forming subunit of calcium and sodium channels?

Four domains (I, II, III, and IV).

What type of calcium channels are also referred to as 'dihydropyridine receptors'?

L-type calcium channels e.g. Cav1.1, Cav1.4

Which neuropeptide's release is inhibited by the action of Ziconotide in the dorsal horn?

Substance P.

Which specific local anaesthetic contains an ester bond instead of an amide bond?

Procaine.

What ion is Kv11.1 primarily selective for?

Potassium ions

In the context of local anaesthetics, what role does the aromatic region play?

It facilitates diffusion through the nerve sheath and lipid membranes.

Describe the state of a channel when no current is recorded in a voltage-clamp experiment.

The channel is closed.

Which ligand-gated channel family is non-selective for cations and allows calcium ion entry?

The NMDA receptor family

What does the term 'ionotropic receptor' refer to?

The ion channel nature of a receptor that opens upon ligand binding.

Outline the crystal structure of rhodopsin.

It has an

Which GPCR taxonomy group corresponds to the 'Rhodopsin-like' family?

Family A

List the five families of GPCRs represented by the acronym GRAFS.

Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin

What specific membrane structures within retinal rod outer segments contain Rhodopsin?

Intercalating discs

What structural feature often anchors the C-terminal of a GPCR to the plasma membrane, as seen in Rhodopsin?

An 8th cytoplasmic helix (H8) and palmitoylation (cysteine bridges)

Which GPCR family contains the largest number of members, including most sensory receptors?

Family A (Rhodopsin-like)

In the crystal structure of Rhodopsin, what molecule is covalently bound as the inverse agonist in the dark state?

11-cis-retinal

Which GPCR taxonomy group corresponds to the 'Secretin' family in the GRAFS classification?

Family B

Which GPCR taxonomy group corresponds to the 'Metabotropic glutamate-like' family?

Family C is the Metabotropic glutamate-like'

In Family B GPCRs, which two structural regions contribute to ligand binding?

The extracellular N-terminal and the transmembrane domains

Define the term RAMP.

Receptor activity-modifying proteins (RAMP) that interact with certain Family B GPCRs to alter their pharmacological properties.

Which GPCR family is characterised by a very large extracellular N-terminal domain often described as a 'Venus flytrap' mechanism?

Which GPCR family is characterised by a very large extracellular N-terminal domain often described as a 'Venus flytrap' mechanism?

Family C

In the GRAFS system, what does the 'A' represent?

Adhesion

In the GRAFS system, what does the 'F' represent?

Frizzled

What is GRAFS classification?

A system used to subdivide the GPCR superfamily into five main families based on phylogenetic criteria.

Approximately what percentage of currently licensed drugs target G protein-coupled receptors (GPCRs)?

35%

What is the approximate number of GPCRs identified within the human genome?

Over 800

How many transmembrane-spanning domains (TMDs) do all members of the GPCR superfamily have?

7 hence why they are also known as 7 transmembrane spanning domain receptors (7 TMDs)

In the basic structural motif of a GPCR, where is the N-terminal located extracellularly or intracellularly?

The N-terminal domain is located in the extracellular space

In the basic structural motif of a GPCR, where is the C-terminal located?

In the Intracellular space (cytosol)

What was the first mammalian GPCR to be successfully cloned (in 1986)?

The beta-2-adrenoceptor

In Family A GPCRs, where is the orthosteric ligand-binding site primarily located?

Within the transmembrane (TM) regions

The Calcitonin receptor-like receptor (CRL) requires co-expression with _____ to function as a high-affinity CGRP receptor.

It requires co-expression with RAMP1

When the Calcitonin receptor-like receptor (CRL) is co-expressed with RAMP2, for which ligand does it show high affinity?

Adrenomedullin

Migraines are primarily treated using?

CGRP (Calcitonin gene-related peptide = CGRP) receptor antagonists or monoclonal antibodies against CGRP