神经生物学：Excitatory Amino Acids

1、Excitatory Amino Acids,20 Amino Acids Used for Protein Synthesis,Non-essential (Our bodies can make them) Alanine Arginine Asparagine Aspartate Cysteine Glutamate Glycine Glutamine Proline Serine Tyrosine,Essential (body cannot make them must get from diet) Histidine Isoleucine Leucine Lysine Methio

2、nine Phenylalanine Threonine Tryptophan Valine,Life Cycle of neurotransmitters,Biosynthesis A family of transmitter transporters,Cytosolic transport,NATURE REVIEWS | NEUROSCIENCE 2011,Y,Processes involved in the inactivation of synaptic transmission,Y,Y,Y,Y,Y,Y,Y,Y,Y,Y,Inactivation ,Glutamate uptake

3、,Neuronal/glial (Retinal),Neuronal (cerebellum),Neuronal,Glial,Glial,Cellular Distribution,EAAT 5,EAAT 4,EAAC 1,EAAT 3,GLT-1,EAAT 2,GLAST,EAAT 1,Alternative Names,Name,Types of EAATs,Probable, primary function:Presynaptic and/or glial neurotransmitter transporters control synaptic levels of neurotra

4、nsmitter,NT,Glial cell,Presynaptic terminal,EAAT4 and EAAT5 mediate presynaptic inhibition in cerebellum and retina,Pre-synaptic Neurone,Shaping synaptic responses ?pH regulation ?Reverse transport ?,But, at synaptic level, glutamate transporters may have other secondary (but potentially important)

5、roles.,Presynaptic,Postsynaptic,G,G,Glutamate-mediated synaptic transmission,SUMMARY,G,Glutamate is a neurotransmitter,ASP,T,ASP,ASP,G,Is aspartate a neurotransmitter?,Sialin is a vesicular aspartate/glutamate transporter,PNAS 2008 vol. 105 11721,Proteoliposomes 脂蛋白体,ATP,Postsynaptic Receptors,Pharm

6、acology of ionotropic glutamate receptors,AMPA receptors and synaptic signalling,Molecular structure of AMPA receptors AMPA receptor-mediated excitatory post synaptic potential (EPSPs),major excitatory neurotransmitter in CNS around 70% of all CNS synapses are glutamatergic,GluA1-4 subunit structure

7、,Long extracellular amino terminus Shorter intracellular C terminus Three transmembrane spanning domains (TM1, TM3 and TM4) TM2 forms a re-entry loop which lines the channel pore AMPA receptors are tetrameric (ie 4 GluR subunits combine to form receptor),GluA,AMPA receptor subunit isoforms,GluA1-4,I

8、MPERMEABILITY OF AMPA RECEPTORS TO CALCIUM GENERATED BY RNA EDITING（编辑）,谷胺酰胺 CAG (Q) 精氨酸 CGG (R),Expression of AMPA receptor subunits in the rat brain,In situ hybridization in horizontal brain sections of adult rats,Distribution of AMPA receptor subunits in the mouse brain,Immunostaining in mouse sa

9、gittal brain sections,Glutamate Receptors,Kainate and AMPA receptors are very similar EPSPs are generated by Na+ influx,海人藻酸/红藻氨酸,Synaptic responses to glutamate usually have both AMPA- and NMDA-receptor mediated components,4 outside cell Glutamate Glycine Obligatory co-agonist Zinc (inverse agonist

10、) Polyamine (indirect agonist) 2 inside cell Magnesium (inverse agonist) PCP (inverse agonist) 苯环己哌啶,N-methyl-D-aspartate (NMDA) receptors,- Unique features,As all ligand-operated ion channels, the NMDA-receptor complex has an agonist binding site.,OUT,IN,Gate,Agonist binding site,But, occupation of

11、 the agonist site by NMDA or glutamate is NOT sufficient to open the gate.,Ionophore (channel),Gate-opening, requires a second amino acid, glycine, to be present and to bind to an allosteric site on the receptor complex.,OUT,IN,Glutamate NMDA,Gate-opening, requires a second amino acid, glycine, to b

12、e present and to bind to an allosteric site on the receptor complex.,OUT,IN,Glycine Co-agonist (activator),Glutamate NMDA,Allosteric modulation of NMDA receptors by D-serine,Why ?,OUT,IN,Glycine Co-agonist (activator),Glutamate NMDA,OUT,IN,Glycine Co-agonist (activator),Allosteric site,Glutamate NMD

13、A,Ca2+,Na+,Ca2+,The membrane must be independently depolarised to relieve the Mg2+-block and allow ions to flow through.,Mg2+,Independent membrane depolarisation,OUT,IN,Glycine Co-agonist (activator),Allosteric site,Glutamate NMDA,Mg2+,Multiple regulatory sites,Voltage-dependent Mg2+-block,OUT,IN,Gl

14、ycine Co-agonist (activator),Allosteric site,Glutamate NMDA,Mg2+,Polyamine,Multiple regulatory sites,Voltage-dependent Mg2+-block,Glutamate NMDA,Multiple regulatory sites,OUT,IN,Glycine Co-agonist (activator),Allosteric site,Mg2+,Polyamine,Voltage-dependent Mg2+-block,Phosphorylation site,NMDA recep

15、tors are large hetero-oligomeric complexes including at least two copies of an NR1 subunit and two copies of an NR2 subunit.,Agonist Sensitivity and Deactivation Rates Among NMDA Receptor Subtypes,Immature Neurons-higher agonist sensitivity, slower off-rate (NR1/2B, NR1/2D) Mature Neurons-lower agon

16、ist sensitivity, faster off-rate (NR1/2A),VOLUME 15 | NUMBER 10 | 2012,The NMDA Glutamate Receptor,Requires both NT and depolarization to open Blocked at RMP by Mg2+ Depolarization displaces Mg2+ ions “Ligand and Voltage-dependent” Channel is permeable to Na+, K+ and Ca2+,NMDA and AMPA Receptors are

17、 Colocalized at Excitatory Synapses,But, the number of AMPA receptors is tightly regulated by activity, so amplitude of fast component of the EPSP/Cs can be rapidly altered!,Metabotropic glutamate receptors,mGluRs fall into one of three categories based on sequence homology, effector mechanisms and,

18、 to some extent, their pharmacolgy.,Brain distribution of mGluR receptors,Metabotropic glutamte receptors (Eight different subtypes) When localized to the presynaptic termianl, inhibit neurotransmtter release When localized to the postsynaptic membrane, exert complex modulatory effects through speci

19、fic signal transduction cascades.,FUNCTINAL ROLES EPILEPSY PAIN DEVELOPMENT MEMORY EXCITOTOXICITY,AMPA Receptor Function Garden variety excitatory signals Learning Sensory processing Pain regulation Induction of seizures,Glutamate Receptors,Nature neuroscience 5 (9) 2002.,Spatial memory dissociation

20、s In mice lacking GluR1,Functional Consequences of Glutamate Receptor/Transporter Knockouts,AMPA receptors -GluR1-impaired LTP -GluR2-reduced exploratory behavior, normal or enhanced LTP -GluR2-no Q/R editing-severe seizures, death at PN20,NMDA Receptor Function Developmental Plasticity/Learning Inc

21、reased synaptic density increased dendritic branching increased density of spines increased neurite sprouting Learning: Long-term Depression (LTD) Long-term Potentiation (LTP),Glutamate Receptors,Functional Consequences of Glutamate Receptor/Transporter Knockouts,NMDA receptors -NR1, NR2B-early post

22、-natal death (1 day)-respiratory failure, feeding impairment, reduced LTP in hippocampal specific NR1 knockout -NR2A, C, D-normal development, reduced LTP, impaired motor coordination,Long Term Potentiationlasting, activity-dependent increase in synaptic efficacy,1st observed by Bliss Corpus callosu

23、m：胼胝体,Deficiency in GAD67: death soon after birth.,GAD65,Caudate nucleus：尾状核;,Deficiency in GAD65: spontaneous seizures.,A transgenic mouse that expresses the green fluorescent protein (GFP) in GAD65 positive neurons.,bis-benzimide苯甲亚胺,Storage of GABA VGAT/VIAAT: 520aa, 10 transmembrane domains; Con

24、ferring vesicular GABA and glycine transport;, ,Chaudhry et al. 2002 JCB.,Vesicular accumulation of amino acids results from both a gradient of membrane potential and pH.,GABA Reuptake,GATs Belong to a Family of Na+/Cl-dependent Neurotransmitter Transporters (SLC6 family) Plasma

25、 Membrane GABA Transporters Glycine Transporters DAT, NET, SERT Orphan Transporters,GABA Reuptake in the CNS,GAT1: mainly in the neurons, some in glial cells. GAT2: in cells of ependyma and arachnoid membrane (室管膜和 蛛网膜) in the brain and in liver. GAT3: principally in glia, also in some neurons. BGT-

26、1: in the brain and kidney, transporting the osmolyte (渗透 物) betaine as well as GABA.,Distribution of GAT-1 (A), GAT-2 (B), GAT-3 (C), VGAT (D), GABA(E) and thionine(亚氨嗪)-stained section (F) in three adjacent sections of developing SI cortex(感觉皮层)(P5 rat). Bar: 100 m for AE.,A,B,Distribution of GAT-

27、1 (A), GAT-2 (B), GAT-3 (C) , GAD67(E) , and the thionine-stained section (D) in three adjacent sections of SI cortex in an adult rat.,cRNA; mRNA,Translation Modification Sorting,Function analysis,Reuptake (Functions of GATs) A fraction of GATs is strategically placed to mediate GABA uptake at fast

28、inhibitory synapses, terminating GABAs action and shaping inhibitory postsynaptic responses; GATs may contribute to the dysregulation of neuronal excitability that accompanies major human diseases: epilepsy, ischemia, etc.,Tiagabine/Gabitril:硫加宾,Obvious phenotype: appearance of spontaneous strong tr

29、emor in GAT1-/- mice,A. GAT1(-/-) mice show strong spontaneous tremor , ataxia, and nervousness,GABA Receptors in the CNS,Benzodiazepine sensitive,GABAA GABAB GABAC,Neuronal excitability (seizures) Memory ? Rapid changes in mood (anxiety) Mood (depression) Sleep Analgesia,痛觉丧失,GABAA receptors Anion

30、permeable ion channel (Cl-) Multi-Subunit Complex (pentameric) Major inhibitory transmitter in adult brain Target for many drugs and compounds,GABAA Binding Sites,GABA Muscimol (蝇蕈醇direct agonist); bicuculine (荷包牡丹碱 direct antagonist) Benzodiazepine (indirect agonist) Natural inverse agonist binds h

31、ere (fear, tension, anxiety) Tranquilizing drugs (anxiolytics): valium, librium (安定) (利眠宁) Likely site for alcohol Barbiturate (indirect agonist) Phenobarbital; pentobarbital Steroid (indirect agonist) Picrotoxin (Antagonist) :印防己毒素 causes convulsions,GABAA receptor structure,nature reviews | neuros

32、cience 2008,Table 1. GABAA receptor subtypesa,aThe term benzodiazepine refers to diazepam and structurally related agents in clinical use.,Subcellular Distribution of GABAA Receptors,Double label cell for different GABAA subunits 2/3-10 nm gold particles -synaptic localization- -20 nm gold particles

33、 -extrasynaptic localization-,mouse cerebellum,Modes of GABAA Receptor Activation,mIPSC,eIPSC,Synaptic GABA Receptors (EC50 10-20 M) (1,2,3,6)(2,3)2,Subunit Composition of Synaptic and Extrasynaptic GABAA Receptors,Extrasynaptic GABA Receptors (EC50 0.5 M) Cerebellar granule neurons 6(2,3) Thalamus,

34、 dentate gyrus Hippocampal pyramidal 532,Excitatory Effects of GABA in Immature Neurons,Hippocampal slices/pyramidal neurons,Stimulus elicits 4 post-synaptic responses Two of these are blocked by AMPA and NMDA antagonists The remaining two are blocked by GABAA antagonist bicuculline So GABAA recepto

35、rs can excite neurons also-how?,GABAB Receptors,First suspected in 1981; l-baclofen(-对氯 苯基-氨基丁酸肌松药) induced responses not blocked by bicucculine, the GABAA antagonist Expression cloning in oocytes not successful cDNA isolated in 1997,Neuropharmacology 60 (2011),GABAB Actions,GABAB,G,K+,Postsynaptic,

36、Presynaptic,GABAB,G,Ca+,GABA,GABA,GABAC Receptor Ligand-gated Cl- channel (similar to GABA-A) Not a target of benzodiazepines, barbiturates, ethanol Physiologic role less clear (at present),Pharmacology and Structure of GABAc Receptors,Cis-4-transamino crotonic acid:反-丁烯酸 TPMPA：1,2,5,6 - 四氢吡啶-4 - 基-

37、甲基次膦酸,昏迷,癫痫,GABA and,PND6-7 7 days later,GABAA receptor agonist,British Journal of Pharmacology (2010) 161 271287,Motor Control,Trends in Neurosciences 2011,Reward System,Science. 2003 May 2;300(5620):812-5. GABA and its agonists improved visual cortical function in senescent monkeys Leventhal AG, W

38、ang Y, Pu M, Zhou Y, Ma Y,Formation of Serine,Glucose,Glycolysis,3-Phospho- glycerate,3-Phospho- hydroxypyruvate,3-Phosphoserine,Serine (Ser),Pyruvate,Dehydrogenase,NAD+ NADH + H+,Glutamate,a-Ketoglutarate,Transaminase,Phosphatase,3 Steps,Inhibits,磷酸甘油酸,羟基丙酮酸,Conversion of Serine to Glycine,Folate,T

39、etrahydrofolate (FH4),Dihydrofolate reductase,N5, N10-Methylene FH4,Serine,Glycine,Serine hydroxymethyl transferase (PLP-dep.),Key intermediate in biosynthesis of purines and formation of thymine,Important in biosynthesis of heme and porphyrins,Glycine,Located by radioligand binding occurs mainly in

40、 spinal cord & brain stem Overview only 1 receptor type ionotropic opens channel permeable to Cl- reverses -75mV therefore generates fast IPSP in spinal cord & brain stem,Release inhibitor: Tetanus toxin 破伤风毒素 Pharmacology agonists = glycine & alanine and taurine antagonist = strychnine, picrotoxin (Cl- channel blocker) Molecular biology of glycie receptor nicotinic like form pentamers Physiological/pathological roles Renshaw cells (脊髓前柱内侧短轴突神经元)are glycinergic spastic patients show loss of strychnine binding genetically spastic(痉挛的