Cys Loop Receptors PDF

Ligand-Gated Ion Channels- the Cys-Loop Receptors Dr Stephen Kelley Senior Lecturer in Pharmacology [email protected] [Park...

Ligand-Gated Ion Channels- the Cys-Loop Receptors Dr Stephen Kelley Senior Lecturer in Pharmacology [email protected] [Parker et al. (2022), Acta Cryst. F78, 313–323]. 1  Learning outcomes Be able to discuss the structure and cellular and physiological function of receptors within the Cys-loop receptor family Be able to discuss drug action at various receptors within the Cys-loop receptor family Recommended reading: See noted sections in the slides  Some relevant terms Receptor- a term used in pharmacology to denote a class of cellular macromolecules that are concerned specifically and directly with chemical signalling Ligand – A substance that is bound to a protein From the Latin word ‘ligare’ meaning ‘to bind’ Affinity- the tendency of a ligand to bind to its receptor Agonist - A ligand that binds to a receptor and alters the receptor state resulting in a biological response. Antagonist - A drug that reduces the action of another drug, generally an agonist. Many antagonists act at the same receptor macromolecule as the agonist. Channel blocker- a molecule or ion that binds to the pore of an ion channel and prevents flow of ions through that channel Protein subunit- a single protein molecule that assembles with other protein molecules to form an oligomer Oligomer- protein macromolecule formed from two or 3 more protein subunits  The Ligand-Gated Ion Channel Receptors Cys-loop receptors- nicotinic acetylcholine receptors, 5- HT3 receptors, GABA-A receptors, the strychnine sensitive glycine receptor and the zinc-activated channel (ZAC) Ionotropic glutamate receptors- NMDA receptors, AMPA and kainate receptors P2X receptors See Table 6-2 in Boron & Boulpaep, Medical Physiology pp 234-235 (eBook)) 4  The Cys-loop receptors: Basic Structure Pentamers- that means composed five protein subunits Cys-loop receptors can be either homo- oligomers or hetero-oligomers, that means the subunits can be all the same type or composed of different types of subunits Very important- subunit composition will affect the pharmacology 5  TM2 TM2 Kelley et al., (2003) Each receptor subunit is composed of extracellular N-terminus large intracellular loop small intracellular loop four hydrophobic transmembrane domains small extracellular loop extracellular C-terminus Stereo view of the Torpedo nACh receptor showing the arrangement the transmembrane domains surrounding the channel pore (Miyazawa et al., 2003) M2 M3 pore M4 M1 The Cys-loop receptors: Basic Structure 1 1 A B C  F E D F E D C  B A 1 Corringer et al.(2000) TM2 TM2 For the nicotinic acetylcholine receptor, Kelley et al., (2003) the ligand binding site is located between the subunit interfaces 8  Brejc et al. (2001) Equivalent location for the ligand-binding domain found between the subunit interfaces of the ACh binding protein of TM2 TM2 the mollusc Lymnaea stagnalis Kelley et al., (2003) The Cys-loop receptors: Basic Structure Unwin (1995) TM2 TM2 The five subunits Kelley et al., (2003) surround a centrally located ion channel 10 The Cys-loop receptors: Basic Structure Torpedo marmorata Tubular formation Containing nAChRs Cryo-electron microscopy 11  The agonist-induced conformational change has been best described in detail for the Torpedo nACh receptor TM2 TM2 Miyazawa et al., (2003) 12  Nicotinic acetylcholine receptors N a + Permeable to Na+, K+ and Ca2+ Nonspecific cation channels Modulate fast synaptic TM 2 TM 2 excitation 13 Detailed overview of the nicotinic acetylcholine receptor in Boron & Boulpaep, Medical Physiology pp 331-334 (eBook  Nicotinic Acetylcholine Receptors 1905- Cambridge physiologist, John Langley examines the effects of nicotine on denervated skeletal muscle. Application of nicotine continued to produce its characteristic contractile response. Application of the John Newport Langley toxin curare abolished this (1852-1925) response. Langley concluded that both nicotine and curare act at some 14 ‘receptive substance’ on the  Neuromuscular Junction Detailed overview of the neuromuscular junction in Boron & Boulpaep, Medical Physiology pp 322-330 (eBook)) 15 Sir Bernard Katz 16 Nicotinic acetylcholine receptor- Myasthenia gravis Skeletal muscle autoimmune disorder associated by loss of nAChRs at the neuromuscular junction Symptoms include weakness fatigue in skeletal muscle with the muscles of the face and eyes particularly affected Can occur in all ethnic groups and ages, although more common in women under 40 years and men over 60 years Diagnosis can be initially difficult, as muscle fatigue is associated with other disorders Blood test for antibodies for nAChRs Edrophonium test Single fibre electromyography Dr Kelley 17 Myasthenia gravis - Pharmacotherapy Neostigmine (Prostigmin®) – reversible competitive antagonist of AChE- medium duration of action (1-2 hours). Pyridostigmine – reversible competitive antagonist of AChE – longer duration of action (30 minutes – 5 hours). Better absorbed than neostigmine. Edrophonium – reversible competitive antagonist of AChE, short duration of action (10 minutes), used for diagnosis of myasthenia gravis. Corticosteroids- immunosuppressants – work by inhibiting inhibiting genes that code for the cytokines and TNFγ 18 Congenital myasthenic syndrome (CMS) Mutations that affect nACh receptor expression Defects in nACh receptor kinetics Mutations affecting agonist binding to nACh receptors Mutations that affect channel gating May be presynaptic (↓ACh) or postsynaptic (↓AChRs, fast channel CMS, slow channel CMS) CMS can be down to a variety of mutations affecting presynaptic ACh vesicular transport, acetylcholinesterase activity, the expression of post-synaptic nicotinic acetylcholine receptors. For further reading see the following review paper by Beeson (2024) and there is a good description of both myasthenia gravis and congenital myasthenic syndrome in Boron and Boulpaep Medical Physiology pp 223 (print), pp 345-346 (eBook). 19 Channel open single channel recording of a recombinant wildtype nicotinic acetylcholine receptor Channel closed activated by acetylcholine (ACh) ACh activation of a recombinant nicotinic acetylcholine receptor expressing a mutation identified in a patient with CMS Note the decreased open-channel adapted from Shen et al., 2012 frequency 20  Drugs that act at as agonists at nicotinic acetylcholine receptors (nAChRs)  Acetylcholine Full agonist at both nAChRs and mAChRs Indicated for cataract surgery  Nicotine Full agonist at nicotinic acetylcholine receptors Delivery of nicotine via controlled release is indicated for smoking cessation  Varenicline Inhibits the binding of nicotine to the α4β2 nicotinic acetylcholine receptor (predominant brain nAChR), and exerts partial agonist activity at the receptor, eases nicotine withdrawal symptoms. Affinity for the α4 subunit, pKi = 10. 21 For further reading see pp 192-193 in Rang & Dale’s Pharmacology, 10th Edition  Varenicline is a b2 a4 partial agonist here 42  nAChR β2 a4 The main nAChR in the brain a7 a7 7 a7 nAChR a7 a7 Lesser importance 22 But Varenicline is a full agonist here! Mihalak et al., 2005 in the brain  N a + 5-HT3 receptors Permeable to Na+, K+ and Ca2+ Nonspecific cation channels Modulate fast synaptic excitation TM 2 TM 2 Adapted from Kelley et al., (2003) Adapted from 23 Derkach, Surprenant & North (1989) X-ray crystal structure of the mouse 5-HT3 receptor (adapted from Hassaine et al., 2014, Nature. 2014 Aug 21;512(7514):276-81 Architecture of the 5-HT3 receptor in complex with VHH15. X-ray crystal structure of the mouse 5-HT3 receptor (adapted from Hassaine et al., 2014, Nature. 2014 Aug 21;512(7514):276-81 The ion permeation pathway. The 5-HT3 receptor as a drug target 5-HT3 receptors are greatly expressed in the gut and modulate peristalsis. The anti-emetic drugs ondansetron (Zophran®), granisetron (Navoban®), tropisetron (Kytril®) dolasetron (Anzemet®) and palonosetron (Aloxi®) are 5-HT3 receptor antagonists. Alosetron (Lotronex®) which is used to treat severe diarrhoea-predominant irritable bowel syndrome in women is also a 5-HT3 antagonist. For further reading see pages 422 & 424 in Rang & Dale’s Pharmacology, 10th Edition 26  The GABAA Receptor is a target for positive allosteric modulators Etomidate (anaesthetic) Pentobarbitone Propofol (barbiturate) (anaesthetic) Halothane (Volatile anaesthetic) Diazepam Ethanol (alcohol) (benzodiazepine) Allopregnanolone (Steroid) Detailed overview of GABAA receptor structure and function in Boron & Boulpaep, Medical Physiology pp 471-474 ( eBook)  Benzodiazepines are positive allosteric modulators of the (synaptic) GABAA receptors Diazepam – status epilepticus, severe acute anxiety, panic attacks, acute alcohol withdrawal Alprazolam – short term use in anxiety Clobazam – adjunct in epilepsy Clonazepam – all forms of epilepsy, anxiety/panic disorder Lorazepam – status epilepticus Midazolam – status epilepticus Flurazepam – short term use in anxiety Loprazolam – short term use in anxiety Lormetazepam- – short term use in anxiety Nitrazepam – short term use in anxiety Oxazepam – short term use in anxiety Temazepam – short term use in anxiety 28 For further reading see pp 518-520, & 606-610 in Rang & Dale’s Pharmacology, 10th Edition  Key anaesthetics are positive allosteric modulators of GABAA receptors Propofol – intravenous anaesthetic, binds to 132GABAA receptors (the main synaptic GABAA receptors)(Yip et al., 2013) Thiopental – short acting barbiturate, intravenous anaesthetic, binds to GABAA receptors Etomidate –intravenous anaesthetic, binds to GABAA receptors 29 For further reading see page 676 in Rang & Dale’s Pharmacology, 10th Edition  Non-benzodiazepine hypnotics are positive allosteric modulators of GABAA receptors- Bind to the benzodiazepine binding site on the synaptic GABAA receptor Zolpidem, Zopiclone and Zaleplon – indicated for the treatment of insomnia 30 For further reading see page 611 in Rang & Dale’s Pharmacology, 10th Edition  Pregnane Steroids Pregnane neurosteroids contains methyl groups at C18 and C19, and an ethyl side chain at C17. O 20 CH3 CH3 CH3 21 CH3 12 18 H 17 11 13 CH3 H 16 CH3 H 1 19 9 14 2 10 8 15 H H H H 3 5 7 4 6 HO Pregnane Pregnenolone For further reading see pp 519-520 Rang & Dale’s Pharmacology, 10th Edition  N a- + Cl O CH3 CH3 CH3 H HO NH HO NH Neurosteroids can O H O H HO H H directly activate the H GABAA receptor at high enough concentrations and Allosteric under certain binding of conditions TM 2 TM 2 neurosteroids to the GABAA receptor increases the duration that the ion channel remains open Evidence from Single Channel Recordings: Neurosteroids Increase the ‘Open-Time’ of the Channel closed open Twyman and MacDonald, 1992 Evidence from Single Channel Recordings: Neurosteroids Increase the ‘Open-Time’ of the Channel N closed open H H H HH closed HO H H open ACN (synthetic neurosteroid) Akk et al., 2005  Neurosteroids and GABAA function Neurosteroid levels in the brain change during pregnancy, stress and ageing Neurosteroid levels can also be altered by alcohol and some SSRI antidepressants Postnatal depression, premenstrual tension, and some epilepsies might be due to alterations in pregnane steroid levels in the brain Belelli & Lambert, 2005 Phasic and Tonic Conductance Phasic conductance is mediated by post-synaptic receptors activated by synaptic GABA release Tonic conductance is mediated by activation of extra-synaptic receptors by ambient concentrations of GABA. This tonic current may significantly influence neuronal excitability. The tonic current has also been shown to be potentiated by comparatively low neurosteroid concentrations b2/3 a1 b2/3 a4/6 g δ β2/3 β2/3 a4/6 a1 Post-Synaptic Extra-Synaptic Receptor Receptor