Biochemistry - Cholinergic Synapses

A cholinergic synapse Choline Na+, ClAction potential Acetyl-CoA Acetyl-Choline Ca + + Ca + + Acetyl-Choline A cholinergic synapse (2): Rapid transmitter inactivation by cholinesterase Choline Action potential Acetyl-CoA Acetyl-Choline Acetate Ca + + Choline esterase Types of cholinergic receptors The „nicotinergic‟ acetylcholine receptor: • Activated by nicotine • A pentameric protein transmembrane channel • Permeability for small cations • 16 different alleles; the gene products can combine into heteromers (221 and so on), giving rise to an even greater number of variants • Related to GABAA, glycine, and 5-HT3 receptors The „muscarinergic‟ acetylcholine receptor: • Activated by muscarine • A single chain transmembrane protein, not a channel • Relays signals through G-proteins (various types) The nicotinic acetylcholine receptor: A ligand-gated channel There are two important functional classes of ion channels: 1. Voltage-gated channels. With these, a change in the transmembrane potential (=voltage) effects a transient opening. Channels of this type are the basis of action potential propagation along excitable membranes. 2. Ligand-gated channels. Here, the transient opening is effected by the binding of specific ligand molecules – neurotransmitters, or, with intracellular channels, second messengers (IP3, cAMP). Ligand-gated channels are important for rapid intercellular transmission of action potentials. NAR in / isolated from electric ray cell membranes How fish electric organs work a) NAR +++ - - Nerve endings b) +++ - - - - - +++ +++ - - - + + - + + - + + - +++ - - - + + - + + - + + - - - +++ - - +++ - - +++ Contour maps of the NAR receptor Top view Side views NAR structure (electron density map) ACh Leucine residues ACh The „bottleneck‟ of NAR in the open state The intracellular „vestibule‟ and its role in conductance and ion selectivity Kelley et al., Nature 424:321-324 (2003) Characterization of the NAR acetylcholine binding site by photoaffinity labelling acetylcholine O O + CF3 N N (C H 3 ) 3 N 4-[(3-Trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine Biochemistry, 42 (2), 271 -283, 2003 Narrowing down the site of labelling with proteolytic fragmentation Biochemistry, 42 (2), 271 -283, 2003 Excision of V8 fragment from gel HPLC purification of radioactive fragment Protein sequencing, identification of residue Biochemistry, 42 (2), 271 -283, 2003 What forces are responsible for interaction of acetylcholine with the NAR? • • • Acetylcholine and all (?) related agonists and antagonists have a positive charge Ion bond? Binding pocket has no complementary negative charges Cation-pi interaction? Binding pocket has aromatic (tryptophan) residues O O + C H3 (C H 3 ) 3 N H2 C N H Protein Experimental proof of a cation-pi interaction between acetylcholine and the NAR (1): Fluorination of Trp should weaken the interaction O O + O C H3 + O C H3 (C H 3 ) 3 N H2 C N H (C H 3 ) 3 N H2 C Protein F F N H P rotein Experimental proof of a cation-pi interaction between acetylcholine and the NAR (2): Construction of NAR with fluorinated trp wild type mRNA UGG (Trp) UAA (Stop) AUU mutant mRNA suppressor tRNA, coupled in vitro to a synthetic tryptophan derivative Trp derivative injection into single cells Fluorination of trp149 in the -chain reduces the agonist sensitivity of NAR Trp F-Trp F2-Trp F3-Trp F4-Trp EC50 from PNAS 95:12088-93, 1998 Cation-pi interaction between acetylcholine and trp149 Acetylcholine O O + C H3 (C H 3 ) 3 N H2 C N H Protein Trp 149 from PNAS 95:12088-93, 1998 The NAR at work Presynaptic action potentials (extraneously triggered) Postsynaptic action potentials NAR desensitization constant flow of acetylcholine 60 sec NAR functional cycle Ligand binds Resting-L • • Three interconvertible conformations Ligand binding favors the open and inactivated states over the resting state Resting Open-L Inactivated Inactivated-L • Ligand leaves Open state is favored kinetically, but inactivated state is favored thermodynamically The „bottleneck‟ of NAR in open state ? The $0.50 (Canadian Tire) question: If desensitization occurs fast, how can we even observe, let alone „crystallize‟ the open state? An ingenious apparatus for trapping NAR in the open state NAR in lipid membranes, letting go forceps triggers gun crystalline but „alive‟ gun blows acetylcholine Cholinergic agonists 1. Direct agonists: Bind to the receptor and stimulate it Acetylcholine C H3 + Carbamoylcholine C H3 + H3 C C O O C H2 C H2 N C H3 C H3 H2 N C O O C H2 C H2 N C H3 C H3 C H3 H3 C C O O H C C H3 + C H3 C H3 H2 N C O O H C C H3 + C H2 N C H3 C H2 N C H3 C H3 Metacholine Betanechol Cholinergic agonists 1. Direct agonists bind to the receptor and stimulate it NAR MAR CE C H3 + H3 C C O O C H2 C H2 N C H3 C H3 Acetylcholine +++ +++ +++ C H3 + H2 N C O O C H2 C H2 N C H3 C H3 Carbamoylcholine ++ +++ (-) C H3 H3 C C O O H C C H3 + C H2 N C H3 C H3 Metacholine - +++ + Muscarinic agonists HO C H3 H3C + O N C H3 C H3 Muscarine (Amanita muscaria = toadstool) H3C H2 C C H2 N C H3 O O N Pilocarpine (Pilocarpus – some South American shrub) Effects of muscarinic agonists • • • Slowed heartbeat Stimulation of intestinal and urinary bladder motility Secretion of exocrine glands (saliva, intestinal, sweat) • Miosis; eye accommodation, eased flow of humor Nicotinic agonists Nicotine (Nicotiana tabacum) N N C H3 H + N N C H3 O C C H2 H + N C H3 C H2 OH C Lobeline (Lobelia inflata = „American tobacco‟) C H3 N + N C H3 Dimethylpiperazinium (synthetic) Effects of nicotine (and nicotinic agonists) 1. „Preganglionic‟ stimulation of both sympathetic and parasympathetic effectors in the autonomic nervous system: • • Increased heart rate / blood pressure Increased intestinal motility (boy runs for the bathroom after purloining one of grandpa‟s cigars) Effects on motor endplate: negligible in normal dose range („depolarizing blockade‟ can be experimentally observed at high dosages) • 2. Stimulation of nicotinic synapses in the brain: Increased vigilance or whatever, ask smoker Vomiting, tremor, … Muscarinic antagonists H + N C H2O H C O C H3 H3C + C H3 Atropine O N C H2O H C O O C H3 Ipratropium H + N C H3 Benztropine C O Nicotinic antagonists (I): „Ganglion blockers‟ C H3 C H3 + Hexamethonium + H3 C N C H3 C H2 C H2 C H2 C H2 C H2 C H2 N C H3 C H3 C H3 C H3 C H3 C H3 + Mecamylamine N H C H3 C H3 C H3 H+ N H2 C H3 Trimethaphan has a sulfonium ion instead of an amino group O C H2 N N C H2 + S Nicotinic antagonists (II): Motor end-plate blockers C H3 + HO O O C H3 NH d-Tubocurarine H3 C O O H H + HO H3 C N C H3 O H3 C C O H + + C H3 N C H3 H C H3 C H3 H H Pancuronium H3 C C O N H O Depolarizing motor endplate „blockers‟ Succinyl-bis-choline C H3 + C H3 + H3C N C H3 C H2 C H2 O C O C H2 C H2 C O O C H2 C H2 N C H3 C H3 C H3 H3C N + C H3 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 C H2 N + C H3 C H3 C H3 Decamethonium Do d-tubocurarine and pancuronium actually occupy both binding sites on the NAR? Clinical use of neuromuscular blockade („muscle relaxation‟) 1. Supplementary to systemic narcosis • Prevents reflex movements in e.g. abdominal surgery • Permits narcosis to be less severe: Just knock out consciousness and arousal by pain, not the brain stem / spinal chord 2. Treatment of tetanus • Tetanus: Toxin-mediated permanent and maximal activity of skeletal muscle • Life-threatening by interfering with respiration • Treatment: Muscle relaxation, artificial respiration until toxin effect has abated (usually weeks) Cleavage and regeneration of acetylcholine H3 C C O S CoA Choline acetyltransferase CoA-SH C H3 + C H3 + H3 C C O O C H2 C H2 N C H3 C H3 HO C H2 C H2 N C H3 C H3 H2O H3 C C O OH Choline esterase Acetylcholine esterase Acetylcholinesterase has a catalytic triad in the active site H Ser C 3 O H Ser H C 3 O H N N O O His C H Glu 3 C H3 HN N O O HO HN N O HN N + H O O C H3 + C H3 H C H3 C H3 C H3 C H3 „short, strong hydrogen bond‟ The catalytic mechanism of acetylcholinesterase (I) Enzyme H C 3 Enzyme O C H3 H3C C O O + H3C O H3C O O + H C H3 + C C N C H3 H2 H2 C H3 C C N C H3 H2 H2 C H3 Acetylcholine Tetrahedral transition state Enzyme H C 3 O O C H3 HO C H3 + C C N C H3 H2 H2 C H3 Acetylated enzyme intermediate Choline The catalytic mechanism of acetylcholinesterase (II) Enzyme H3 C O H Enzyme H3 C O O C H3 O HO C H3 H+ HO- H O H H N N O O His C H Glu 3 C H3 Carbamoylation of acetylcholinesterase is slowly reversible Enzyme H C 3 Enzyme H C 3 O C H3 + O C H3 + H3 C C O O C H2 C H2 N C H3 C H3 H2 N C O O N C H3 C H3 fast O H3 C O C H3 fast Enzyme H2 N O O C H3 Enzyme fast O O Hydrolysis C H3 slow O O C H3 H3 C OH H H2 N OH H Carbamoylation of acetylcholinesterase by carbamoylcholine Enzyme H C 3 O C H3 + H2 N C O O C H2 C H2 N C H3 C H3 O H2 N O C H3 Enzyme Hydrolysis O slow C H3 O H2 N OH H Covalent acetylcholinesterase blocking agents C H3 C H3 O H SerC 3 C H3 F O O P O O C H3 SerC H 3 F O P O O C H3 C H3 C H3 C H3 Diisopropylfluorophosphate (DFP) „Nerve gases‟ such as soman and sarin are cholinesterase blockers C H3 C H3 O F P O C H3 C H3 O C H3 F P O C H3 H3 C C H3 C H3 N C C H3 O C H2 O P N C H3 O C H3 Diisopropylfluorophosphate (DFP) Soman Tabun Cholinesterase blockers are widely used as insecticides C H3 C H2 O N O2 O P O C H2 C H3 O NO2 O C H3 C H2 O P O C H2 C H3 S C H3 O H3 C H3 C C H2 C H2 O O C C O H C C H2 S O P O C H3 S Paraoxon Parathion Malathion Reactivation of alkylphosphorylated acetylcholinesterase C H3 C H3 + C H3 SerC H 3 OH O C H3 H C Ser 3 H O O P O O H2 N O P O O H2 N O C H3 H C H3 C H3 C H3 Hydroxylamine + N C H N OH + N C H3 C H N OH C H2 O C H2 + Obidoxime C H N OH Pralidoxime N Indirect-acting „cholinomimetics‟: cholinesterase-blocking agents C H3 (Acetylcholine) O + H3 C C O O C H2 C H2 N C H3 C H3 C H3 O C H3 N H3 C N H C O H3 C N H C O N Carbaryl H3 C N H3 C O C O C H3 + Physostigmine C H3 + C H3 N C H3 C H3 HO N C H3 C H2 C H3 Neostigmine Edrophonium Medical applications of cholinesteraseblocking agents • Act on both muscarinic and nicotinic synapses • Nicotinic: „Mysasthenia gravis pseudoparalytica‟ • Autoimmune disease: Antibodies against NAR diminish number of functional receptors in neuromuscular junction • Compensate by increasing the lifetime of endogenously released acetylcholine by inhibition of cholinesterase Muscarinic: • Activate ciliary muscle to lower intra-ocular pressure • Stimulate intestinal activity (sluggish e.g. post-surgery) • The „Ordeal Bean‟ “The Calabar negroes call the seed eséré, and use it as an ordeal for the purpose of deciding the guilt or innocence of persons accused of crimes.” http://www.ibiblio.org/herbmed/eclectic/kings/physostigma.html