Neurotransmitter Receptors I: Ionotropic Receptors PDF

2024-10-09 Neurotransmitter Receptors I: Ionotropic Receptors NROC36H3F © Arruda Carvalho UTS...

2024-10-09 Neurotransmitter Receptors I: Ionotropic Receptors NROC36H3F © Arruda Carvalho UTSC The Life Cycle of Neurotransmission I. Synthesis (1-2) II. Storage (3) Lecture 3 III.Release Lecture 4 From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc IV. Receptor Binding (4-5) V. Transmitter Inactivation (6-9) Lecture 3 © Arruda Carvalho UTSC 1  2024-10-09 Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term What is the Outcome of Neurotransmitter Release? ? Type of Response – Postsynaptic Receptor Magnitude of Response – Receptor number, "state" of the receptors, and amount of neurotransmitter released © Arruda Carvalho UTSC 2  2024-10-09 Ionotropic and Metabotropic Receptors Fast synaptic actions lasting Slower synaptic actions lasting seconds to minutes only milliseconds Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 3  2024-10-09 Ionotropic Receptor Families composed of 3 to five subunits Each subunit has a membrane domain with four membrane-spanning -helices (M1– M4) and a short extracellular c-terminus. The M2 helix lines the channel pore Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 4  2024-10-09 nACh Receptor: an Iconic Ionotropic Receptor Torpedo Ray Panoramic view of the postsynaptic membrane of an electrocyte in the Torpedo electric organ. The vase-like structure in the center of the field is the external surface of the postsynaptic membrane. Clusters and linear arrays of 8 to 9 nm protrusions represent the AChR oligomers. © Arruda Carvalho UTSC nACh Receptor: an Iconic Ionotropic Receptor Two extracellular binding sites for Ach: In the clefts between each α-subunit and its neighboring γ- or δ-subunit The plant alkaloid nicotine can bind to the ACh binding site to activate the receptor Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 5  2024-10-09 nACh Receptor: an Iconic Ionotropic Receptor Each subunit contributes one cylindrical component to form the ion channel through the middle of the complex From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC nACh Receptor: an Iconic Ionotropic Receptor Each subunit contains four hydrophobic regions of approximately 20 amino acids called M1 to M4, each of which is thought to form an α-helix that spans the membrane Negatively charged amino acids on each subunit repel anions and establish cation selectivity Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 6  2024-10-09 nACh Receptor: an Iconic Ionotropic Receptor The membrane-spanning segments that line the pore are the five M2 regions, one contributed by each subunit. The amino acids that compose the M2 segment are arranged in such a way that three rings of negatively charged amino acids are oriented toward the central pore of the channel – providing cation selectivity Modified from Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC nACh Receptor: an Iconic Ionotropic Receptor or AcH Some neuronal nicotinic ACh receptors are composed of five identical subunits and are thought to bind five molecules of Ach. Each subunit binds one molecule of Ach at the ACh binding site, which is located at the interface between neighboring subunits Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 7  2024-10-09 nACh Receptor: an Iconic Ionotropic Receptor ACh gains access to its binding sites by entering the central pore of the receptor where it then enters small channels that provide access to the binding sites. Once the binding sites are occupied, the receptor rapidly opens to allow ion flow. From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC nACh Receptor: an Iconic Ionotropic Receptor The closed-to-open transition of the receptor is When the M2 segments associated with a subtle rotation of its M2 segments. rotate because of ACh binding, the kinks also rotate, The M2 segments are helical and exhibit a mild kink in relaxing the constriction their structure that forces a leucine (L) residue from each formed by the leucine ring, segment into a tight ring that effectively blocks the flow and ions can then flow of ions through the central pore of the receptor. From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 8  2024-10-09 nACh Receptor: Posttranslational Modification nACh Receptor is phosphorylated by three kinases: PKA phosphorylates the γ and δ subunits PKC phosphorylates the δ subunit Unidentified tyrosine kinase phosphorylates the β, γ, and δ subunits Phosphorylation sites are within the intracellular loop between M3 and M4 Phosphorylation increases the rapid phase of desensitization of the receptor, thereby limiting ion flux through transitions into a closed state TM = M From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC nACh Receptor: Structural Variability Functional neuronal nAChRs can be assembled from a single subunit (as in α7, α8, and α9), and a single type of α subunit can be assembled with multiple types of β subunits (e.g., α3 with β2 or β4 or both) and vice versa, producing an array of receptors with distinct single channel kinetics and rates of desensitization Hendrickson et al., Frontiers in Psychiatry, 2013 © Arruda Carvalho UTSC 9  2024-10-09 nACh Receptor and the Rest of the Family From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 10  2024-10-09 5-HT3 Receptor Homomeric complex composed of five copies of the same subunit (most related to α7 subtype of neuronal nAChRs), Permeable to Na+ and K+ but not Ca2+ Sparsely distributed on peripheral primary sensory nerve endings and in the mammalian CNS Antagonists used to treat vomiting, nausea, anxiety, psychosis © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 11  2024-10-09 GABAA and Glycine Receptors GABAA Jacob et al., Nat Rev Neuro, 2008 GABAA receptors are usually composed of two α-, two β-, and one γ- or δ-subunit. The receptors are activated by the binding of two molecules of GABA in clefts formed between the two α- and β-subunits, allowing the opening of the channel and influx of Cl- Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC GABAA and Glycine Receptors Glycine Glycine receptors are composed of three α- and two β-subunits and require the binding of up to three molecules of ligand to open Du, Lu et al., Nature, 2015 Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC 12  2024-10-09 GABAA and Glycine Receptors GABAA TM = M Jacob et al., Nat Rev Neuro, 2008 https://aopwiki.org/events/667 GABA and glycine receptor-channels contain either neutral or positively charged basic residues flanking the M2 domain, which contributes to the selectivity of these channels for anions © Arruda Carvalho UTSC GABAA and Glycine Receptors GABAA In Which Ways Could Modifications of GABAARs or Glycine Receptors Interfere with their Function? What could be the Consequences for Behaviour? © Arruda Carvalho UTSC 13  2024-10-09 GABAA Receptors and Disease Underlying repetitive, non-abating seizures is a decrease in the phosphorylation of GABAAR β3 subunits by PKC. This leads to an increased association with the clathrin-adaptor protein 2 (AP2) complex, followed by increased internalization through clathrin-mediated endocytosis. Decreased numbers of synaptic GABAARs lead to reduced synaptic inhibition (= increased excitatory drive and lower seizure threshold) Jacob et al., Nat Rev Neuro, 2008 © Arruda Carvalho UTSC Glycine Receptors and Disease Strychnine, a GlyR antagonist Pesticide, commonly used in rodents and small birds Muscle spasms, followed by respiratory failure and brain death Agatha Christie, in 1925. Wikimedia Commons © Arruda Carvalho UTSC 14  2024-10-09 Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term Purinergic Receptors Ionotropic receptors for adenosine triphosphate (ATP), which serves as an excitatory transmitter Kaebisch et al., Comp Struct Biotech J, 2015 Smooth muscle cells innervated by sympathetic neurons of the autonomic ganglia as well as on certain central and peripheral neurons © Arruda Carvalho UTSC 15  2024-10-09 Purinergic Receptors Comprised of only two transmembrane domains, with some homology in its pore- forming region with K+ channels. Each subunit contributes two transmembrane (TM or M) domains and three subunits congregate together to form the native receptor Permeable to both monovalent cations and Ca2+ Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC Purinergic Receptors ATP binding causes the expansion of the pore region in an iris-like manner by twisting of the transmembrane (TM) helices to allow permeation of cations Browne et al., PNAS, 2014 © Arruda Carvalho UTSC 16  2024-10-09 Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term Glutamate Receptors Development of agonists that could pharmacologically distinguish between different glutamate receptor subtypes N-methyl-D-aspartate (NMDA), amino- 3-hydroxy-5- methylisoxazoleproprionic acid (AMPA), Kainate © Arruda Carvalho UTSC 17  2024-10-09 Glutamate Receptors (Non-NMDA) Ionotropic (NMDA or non-NMDA) Metabotropic Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC Ionotropic Glutamate Receptors (Non-NMDA) CNQX APV (2-amino-5-phosphonovaleric acid) (6-cyano-7-nitroquinoxaline-2,3-dione) Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC 18  2024-10-09 Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term AMPA Receptors Receptor assembly and trafficking Three transmembrane α-helixes (M1, M3, and M4) and a loop (M2) between the M1 and M3 helixes that dips into Glutamate binding and out of the cytoplasmic side of the membrane. This M2 loop is thought to form the selectivity filter of the channel Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC 19  2024-10-09 AMPA Receptors: subunit composition Sobolevsky et al., Nature, 2009 GluA4 Mod from Henley and Wilkinson, Dial Clin Neurosci, 2013 AMPARs are tetrameric structures, comprised of different combinations of the four types of subunits, GluA1-4. Most AMPARs in the brain contain GluA2, a subunit that renders them impermeable to Ca2+. Increasing amounts of evidence point towards an important role of GluA2-lacking AMPARs, which are calcium permeable (cp-AMPARs), in synaptic plasticity and learning © Arruda Carvalho UTSC AMPA Receptors: subunit composition AMPARs containing the GluA2 subunit (light blue) in its unedited (Q; pink) form also gate calcium. However, the presence of a GluA2 subunit that has been RNA edited to replace Q607 with an arginine (R; dark blue) renders the AMPAR impermeable to calcium Henley and Wilkinson, Nat Rev Neuro 2016 © Arruda Carvalho UTSC 20  2024-10-09 AMPA Receptors: subunit composition GluA2 After transcription the codon for glutamine in the M2 loop of the GluA2 mRNA is replaced with one for arginine (a positively charged basic amino acid) because of a chemical modification of a single nucleotide base through an enzymatic process termed RNA editing. Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC AMPA Receptors: subunit composition AMPA receptor-channels with this edited GluA2 have a very low permeability to Ca2+, most likely as a result of strong electrostatic repulsion by the arginine From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 21  2024-10-09 AMPA Receptors: subunit composition RNA editing switches single amino acid in GluA2 reducing Ca2+ permeability GluA2 From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC AMPA Receptors: subunit composition Deletion of the editing site complementary sequence (ECS) in the intron impairs GluA2 Q/R RNA editing. This resulted in approximately 25% unedited GluA2 mRNA at the Q/R site compared to age-matched wild-type littermates © Arruda Carvalho UTSC 22  2024-10-09 AMPA Receptors: Desensitization Extended exposure to glutamate can lead to a change in the dimer bond at the ligand binding domain which leads to closure of the channel. The rate and speed of desensitization will be affected by composition of receptor subunits From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 23  2024-10-09 NMDA Receptors Critical role in development, learning and memory and brain injury Unique properties: Permeable to Ca2+ Glycine as a co-factor Voltage-dependent Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC NMDA Receptors Membrane Depolarization Expels Mg2+ to Allow Ion Flux Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC 24  2024-10-09 NMDA Receptors: Structure Paoletti et al., Nat Rev Neuro 2013 Karakas and Furukawa, Science 2014 Similar to AMPARs, NMDARs are tetrameric structures with four distinct domains: the N-terminal domain (NTD), the agonist-binding domain (ABD), the transmembrane domain (TMD) containing the ion channel, and an intracellular C-terminal domain (CTD) © Arruda Carvalho UTSC NMDA Receptors: Structure Paoletti et al., Nat Rev Neuro 2013 Seven NMDAR subunits: GluN1, GluN2A– GluN2D and GluN3A and GluN3B. NMDAR is composed of two GluN1 subunits and either two GluN2 subunits or a combination of GluN2 and GluN3 subunits. © Arruda Carvalho UTSC 25  2024-10-09 NMDA Receptors: Structure Glutamate binding site Glycine binding site Paoletti et al., Nat Rev Neuro 2013 GluN1 is essential for the formation of a functional pore to permit the flow of ions. The GluN1 or GluN3 subunits provide the glycine-binding site for the receptor, while the GluN2 subunits contain the glutamate binding sites. © Arruda Carvalho UTSC NMDA Receptor Subunit Diversity Subunit composition determines receptor properties Paoletti et al., Nat Rev Neuro 2013 © Arruda Carvalho UTSC 26  2024-10-09 NMDA Receptors: Function The individual contributions of NMDA and AMPA receptors to the total excitatory postsynaptic current (EPSC) can be dissected using pharmacological antagonists in voltage-clamp The difference between the traces (blue region) represents the contribution of the NMDARs to the EPSC The current that remains in the presence of APV is the contribution of the AMPARs Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC NMDA Receptors: Function What Happens if NMDAR Function is Disturbed? © Arruda Carvalho UTSC 27  2024-10-09 NMDA Receptors, Hallucinations and Schizophrenia Hallucinogenic compounds Phencyclidine (PCP), Ketamine, and dizocilpine (MK-801) are NMDAR pore-blockers From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC NMDA Receptors, Hallucinations and Schizophrenia Glutamate hypothesis of Schizophrenia Glutamatergic projection neuron Ketamine Interneuron Mod from Stone et al., Ther Adv Psychopharm, 2011 © Arruda Carvalho UTSC 28  2024-10-09 NMDA Receptors and Glutamate Excitotoxicity Kritis et al., Front Cell Neurosci 2015 © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 29  2024-10-09 Distribution of Ionotropic Glutamate Receptors: Postsynaptic Density (PSD) View of PSD from outside of the cell View of PSD from inside of the cell A typical PSD is around 350nm long, with approximately 20 NMDARs (located by the center of the PSD), and 10 to 50 AMPARs (less centrally localized). mGluRs are located on the periphery, outside the main PSD Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC Distribution of Ionotropic Glutamate Receptors: Postsynaptic Density (PSD) PSD-95: Binds to the NMDAR, AMPAR and other proteins, localizing and concentrating them at postsynaptic sites Transmembrane AMPA receptor regulatory proteins (TARPs): Strongly regulates the trafficking, synaptic localization, and gating of AMPARs. E.g. Stargazin Principles of Neural Science 5th edition © The McGraw-Hill Companies © Arruda Carvalho UTSC 30  2024-10-09 AMPAR Trafficking is Complex and Tightly Controlled Jacobi and von Engelhardt, Curr Op Neurobiol, 2017 © Arruda Carvalho UTSC TARP Composition Differs Across the Brain Jacobi and von Engelhardt, Curr Op Neurobiol, 2017 © Arruda Carvalho UTSC 31  2024-10-09 TARPs: Stargazin stg mice lack Stargazin © Arruda Carvalho UTSC TARPs: Stargazin stg WT GluA1 GluA2 GluA GluA stg mice show lack of functional AMPARs in cerebellum despite normal expression of GluA © Arruda Carvalho UTSC 32  2024-10-09 TARPs: Stargazin Increasing expression of Stargazin increases the presence of AMPAR in the membrane Exogenous glutamate (whole neuron) Bath application Evoked response Neurotransmitter release in the synapse Control = normal levels of stargazin Stg = overexpression of stargazin Schnell et al., PNAS, 2002 © Arruda Carvalho UTSC TARPs: Stargazin Increasing expression of Stargazin increases the presence of AMPAR in the membrane Bath application Evoked response Increased presence of AMPARs in the membrane leads to an increase in response to bath application of glutamate (H), but no change in the evoked response (E), as these receptors are not in the synapse Schnell et al., PNAS, 2002 © Arruda Carvalho UTSC 33  2024-10-09 TARPs: Stargazin Increasing expression of PSD-95 increases the presence of AMPAR in the synapse Bath application Evoked response Schnell et al., PNAS, 2002 © Arruda Carvalho UTSC (1) (2) Stargazin guides AMPARs to membrane (1) and anchors them to the synapse via its PDZ domain-mediated interaction with PSD95 (2) Tomita et al., J. Cell Biol 2001 © Arruda Carvalho UTSC 34  2024-10-09 synaptic Bath application (exogenous glutamate) Spontaneous Transmission stg/stg = stargazin knockout Stargazin C = Removal of C terminal PDZ binding domain (disrupts binding to PSD-95) © Arruda Carvalho UTSC Tomita et al., J. Cell Biol 2001 “The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins is required for targeting the AMPA receptor to synapses” © Arruda Carvalho UTSC 35  2024-10-09 AMPAR Auxiliary Subunits: Stargazin Stargazin Kato et al., TINS, 2010 Stargazin increases GluA surface expression, glutamate affinity, and attenuates the rate and extent of desensitization The stargazin-mediated enhancement of glutamate evoked currents reflects both an increase in GluA surface expression and an augmentation of ion flow © Arruda Carvalho UTSC Lecture Outline Overview Ionotropic Receptor Families nACh receptor 5-HT3 Receptor GABAA and Glycine Receptors Purinergic Receptors Glutamatergic Ionotropic Receptors AMPA/Kainate NMDA Receptor Localization in the PSD © Arruda © 2018 Arruda Carvalho Carvalho UTSC winterUTSC term 36