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Presynaptic terminal Glial Postsynaptic neuron 1 GABA and GABA receptors Lecture 1. GABAA receptors Lecture 2. GABAB receptors Lecture 3. GABA homeostasis Lecture 4. Modulation of GABAergic synaptic transmission 2 1 What is GABA? γ-aminobutyric acid • Inhibitory neurotransmitter. • ~1/3 of synaptic transmission in the brain is mediated by GABA. • Neurons that synthesize and release GABA is called GABAergic neurons. 3 GABA receptors • GABAA receptors – Ligand-gated ion channels – Fast synaptic inhibition • GABAB receptors – GTP-binding protein coupled receptors – Slow synaptic inhibition • GABAC receptors 4 2 Lecture 1. GABAA receptors • • • • • molecular structures location and function single channel recording pharmacology two types of inhibition 5 Benzodiazepines Barbiturates Neurosteroids Anesthetics 6 3 GABAAR is ligand-gated ion channel Receptor Transmitter Pore Channel Extracellular side Gate Cytoplasmic side 7 Molecular structure of GABAA receptors ρ1-ρ3 Moss & Smart 2001 8 4 ρ subunits are part of GABAA receptor family ρ1-ρ3 Moss & Smart 2001 •structure •function •pharmacology Dendrogram of the deduced amino acid sequences of GABAAR subunits. from Cherubini and Conti 2001 9 Distribution of GABAAR α-subunit mRNA in rat brain 10 5 Molecular structure of GABAA receptors • Multiple subunits: α1−6, β1−3, γ1−3, δ, ε, π, θ, ρ1−3 • Each subunit contains 4 putative transmembrane domains, TM2 is believed to form the lining of the channel. • Hetero- or homo-oligomeric proteins. • Pentamer with α:β:γ at a ratio of 2:2:1 • subunit composition determines functional properties and pharmacology. 11 Location Presynaptic terminal Glial Synaptic cleft GABAAR Cl - Postsynaptic neuron IPSP Cl- and HCO312 6 Nernst equation - Cl 10 mM Cl125 mM RT ln [Cl]o zF [Cl]i [Cl] o ECl = -60 log [Cl] i ECl = -66 mV ECl = z = charge of diffusible ion (Cl- = -1) R = universal gas constant T = absolute temperature F = Faraday's Constant 13 Current passing recording Current passing recording Inhibitory interneuron Motor neuron IPSP AP 14 7 Reversal potential of IPSP Postsynaptic potential - Cl flux Postsynaptic current -35 -55 ECl- -74 -99 (mV) Current clamp Voltage clamp 15 IPSP reduces cell excitability • Membrane hyperpolarization – drive membrane potential away from the threshold potential. • Reduction in membrane resistance – reduce the excitatory input. This is known as “shunting inhibition”. Threshold potential ECl- Threshold potential ECl16 8 Single channel recording Open GABA Closed Bound Closed Open Bound Open Bound Closed 17 ligand binding sites on GABAA receptors benzodiazepine picrotoxin 18 9 • Agonists – GABA, muscimol Response (%) GABAA receptor pharmacology • Antagonists Response (%) Zn2+ Neurosteroids Benzodiazepines Anesthetics Barbituates Alcohol 1 10 KD 1000 Dose 100 affinity 50 0 Response (%) • Modulators – – – – – – 50 0 – Bicuculline, picrotoxin, gabazine Maximum Response 100 affinity 1 10 100 Dose efficacy 100 50 0 1000 efficacy 1 10 100 1000 Dose 19 Phasic and tonic inhibitions 50 pA Bicuculline 20 pA 40 ms – high agonist dose ~1 mM – quantal release • Action potentialdependent IPSPs • Action potentialindependent mIPSPs – synaptic receptors – sensitive to gabazine 1 min – low agonist dose ~1µM – unknown mechanisms • • • • reverse uptake spill over extracellular matrix channel spontaneous open – extrasynaptic receptors – insensitive to gabazine 20 10 GABAAR and disease • GABAAR is a major target for developing therapeutics. – – – – – pain epilepsy anxiety depression sleeping disorders • Mutations in GABAARs are found to be linked to epilepsy. 21 GABAAR Summary • GABA-gated anion channels. • The primary inhibitory receptors in the mammalian brain. • Pre-, post-synaptic and extrasynaptic area, mediating inhibitory postsynaptic potentials (IPSPs) and tonic inhibition. • Important targets for therapeutic agents. • Mutations in the genes encoding GABAA receptor subunit correlate with certain type of epilepsy. 22 11 Questions 23 Lecture 2. GABAB receptors • molecular structures • location and function • modulation 24 12 GABABRs are G-protein coupled receptors Transmitter Receptor Channel G protein βγ Gate α GTP NH2 P P P P Extracellular side Cytoplasmic side COOH 25 Molecular structure of GABAB receptors • Heterodimer linked by coiled-coil domain • GABAB1a-f • GABAB2, 35% homology with GABAB1 • coupled to Gi/Go Marshall, FH et al, 1999 26 13 Function of GABABRs 2+ + Ca K GABABR βγ βγ βγ α GTP PP P Adenylyl cyclase 27 GABABRs - postsynaptic GABABR fast IPSP ECl - slow IPSP EK + 28 14 GABABRs - presynaptic Glutamatergic GABAergic GABAARs GABABR GluRs GABABR IPSP Threshold EPSP 29 GABABR function • opening K+ channels in the postsynaptic membrane. • closing Ca2+ channels in the presynaptic terminal. – GABAergic: autoreceptor – glutamatergic: heteroreceptor 30 15 GABABR pharmacology • Agonists – GABA, (-)baclofen, APPA • Antagonists – saclofen, phaclofen, CGP35348, CGP55845A • Modulators – CGP7930 31 GABABR and disease • GABABR agonist – antispasticity – antinociceptive – suppression of drug craving • GABABR antagonist – suppress absence seizure in animal models 32 16 GABABR Summary • G-protein coupled receptors. • Heterodimer with GABAB1 and GABAB2. • Mediate slow IPSP via opening K+ channel at postsynaptic membrane. • Decrease synaptic release via inhibit Ca2+ channels in the presynaptic terminal. • targets for therapeutic agents. 33 Function of GABAARs and GABABRs • Under several conditions GABARmediated response can be excitatory. – GABAARs • EIPSC higher than the threshold (Cl- or HCO3-). • Disinhibition – GABABRs • Presynaptic inhibition on inhibitory neurons • Activating K+ channel may recruit T-type Ca2+ channel to induce oscillation in thalamus. • Disinhibition 34 17 Reference books • Principles of neuroscience 4th Edition Eric R. Kandel Jame H. Schwartz Thomas M. Jessell • From neuron to brain 4th Edition John G. Nicholls Robert Martin Bruce G. Wallace Paul A. Fuchs New York: Elsevier Sunderland: Sinauer Associates 35 18