1. Chapter 12 The metabotropic glutamate receptors
From Cellular and Molecular Neurophysiology, Fourth Edition.
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2. From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure 12.1 Glutamate activates G-protein-coupled receptors producing the second messenger inositol tri-phosphates (IP3).
(a) Metabotropic glutamate receptors (mGluRs) activate the enzyme PLC by stimulating specific G proteins. This enzyme degrades the membrane phospholipid phosphatidyl-inositol-biphosphate (PIP2) to produce IP3 and di-acylglycerol (DAG). IP3 then acts on intracellular receptor channels and allows the release of Ca2+ from intracellular stores. (b) The first evidence for the existence of mGlu receptors came when glutamate was found to stimulate the production of IP3 in cultured neurons and brain tissues. (c) Further evidence for the existence of PLC-coupled mGluRs was the observation that glutamate activates chloride currents through the release of Ca2+ from intracellular stores in Xenopus oocytes injected with rat brain mRNA (scale bars: vertical: 200 nA; horizontal: 20 s).
Part (b) adapted from Sladeczek F, Pin J-P, Recasens M et al. (1985) Glutamate stimulates inositol phosphate formation in striatal neurons. Nature317, 717–719, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
Copyright © 2015 Elsevier Ltd. All rights reserved.

3. Figure 12.2 Classification, coupling properties and pharmacology of mGluRs.
(a) mGluRs can be divided into three groups based on amino-acid sequence similarity (left), signaling properties (center) and pharmacology (right). Agonists and antagonists selective for each group of mGluRs are indicated. The recently identified negative and positive modulators are indicated. Note these modulators are selective for a single mGluR subtype. (b) Schematic representation of the well characterized mGluR variants resulting from alternative splicing of the pre-mRNA. The 7TM coding region is indicated with the seven red vertical bars. Identical mRNA sequences are linked by dotted lines.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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4. Figure 12.2 Classification, coupling properties and pharmacology of mGluRs.(cont.)
(a) mGluRs can be divided into three groups based on amino-acid sequence similarity (left), signaling properties (center) and pharmacology (right). Agonists and antagonists selective for each group of mGluRs are indicated. The recently identified negative and positive modulators are indicated. Note these modulators are selective for a single mGluR subtype. (b) Schematic representation of the well characterized mGluR variants resulting from alternative splicing of the pre-mRNA. The 7TM coding region is indicated with the seven red vertical bars. Identical mRNA sequences are linked by dotted lines.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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5. Figure 12.3 Determination of the major functional domains of mGluRs using the chimeric approach.
(a) Swapping the two third N-terminal portions of the extracellular domain of the group-I mGluR1 (red) with those of the group-II mGluR2 (green) generated a chimeric receptor that couples to PLC, like the group-I mGluR1, but is activated by the group-II agonist DCG-IV. This indicates that the N-terminal domain of mGluRs is responsible for agonist recognition. (b) Swapping most intracellular parts, except the second intracellular loop, of the adenylyl-cyclase-coupled mGluR3 (green) with those of the PLC-coupled mGluR1 (red) was not sufficient to allow the chimeric receptor to activate PLC and generate intracellular Ca2+ signals (central). However, swapping of the second intracellular loop and other intracellular parts resulted in chimeric receptors that activate PLC-like mGluR1 (right). This indicates that the second intracellular loop plays a critical role in specifying PLC activation by mGluR1, whereas the other intracellular parts are required for an efficient coupling to PLC.
Part (a) adapted from Takahashi K, Tsuchida K, Taneba Y et al. (1993) Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination. J. Biol. Chem. 266, 19341–19345, with permission. Part (b) adapted from Gomeza J, Joly C, Kuhn R et al. (1996) The second intracellular loop of metabotropic glutamate receptor 1 cooperates with other intracellular domain to control coupling to G proteins. J. Biol. Chem.271, 2199–2205, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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6. 6 Figure 12.4 Structure and activation mechanism of mGluRs.
(a) Schematic view of the structural domains of mGluRs as determined by sequence homology searches: from the N-terminal end (left) to the C-terminus (right): signal peptide (in black), the Venus flytrap domain (gray), the cysteine-rich domain (hatched), the seven transmembrane domain (with seven vertical bars) and the intracellular C-terminal segment (white). (b) Structural view of an mGluR based on the solved structure of the Venus flytrap domain of mGluR1, on the 3D model of the cysteine-rich region, and the solved structure of the prototypical G-protein-coupled receptor rhodopsin. Note the receptor is a constitutive dimer: the subunit in the front is colored according to its secondary structure (helices in red, strands in yellow and loops in gray), while the other subunit in the back is in blue. The dimer of the Venus flytrap domain is that observed in the absence of bound agonist (inactive state), while the dimer of 7TMs is based on the proposed dimerization mode of rhodopsin in its inactive state. (c) The three identified conformations of the dimer of Venus flytrap of mGluR1: top, the inactive unliganded form; upon binding of at least one molecule of glutamate, closure of the yellow subunit results in a major change in the relative orientation of the domains, leading to a partial activation of PLC; bottom, upon binding of glutamate in the blue subunit and cation (such as Gd3+) binding at the interface between the two subunits, a third state is obtained in which both domains are closed, and this corresponds to the fully active state of the receptor.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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7. Figure 12.5 Modulation of mGluR function by positive and negative modulators.
On the left, a scheme of the general structure of an mGluR subunit shows that whereas agonists and antagonists bind to the Venus flytrap domain, allosteric modulators (both positive and negative) bind to the 7TM domain. On the right, the effects of the positive and negative modulators on the dose effect of an agonist are illustrated. The positive modulators increase both the potency (the dose–response curve is shifted to the left) and efficacy (increase in the maximal effect of agonist) of agonists. The negative modulators act as non-competitive antagonists, decreasing the maximal effect of agonists.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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8. Figure 12.6 Schematic representation of a glutamatergic synapse with the modulatory roles of mGluRs.
Note group-I mGluRs (1 and 5) are mostly located in the postsynaptic spines, on the side of the postsynaptic density, from where they regulate postsynaptic ionotropic glutamate receptors, as well as Ca2+ and K+ channels. Group-II (mGluR2) and group-III (mGluR4, 7 and 8) are mostly presynaptic, regulating neurotransmitter release by, at least, inhibiting Ca2+ channels.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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9. 9 From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure 12.7 mGluR-mediated inhibition and activation of voltage-sensitive Ca2+ currents in neurons.
(a) The upper panel shows a recording of macroscopic Ca2+ currents from a rat CA3 pyramidal cell outside-out patch. The membrane was held at −80 mV and stepped to +10 mV, resulting in activation of voltage-sensitive Ca2+ channels. Application of the group-I mGluR agonist, t-ACPD, to the surface of the patch induced in a reversible reduction of the Ca2+ current. (b) However, when macroscopic Ca2+ currents were recorded in the cell-attached mode and the agonist was applied outside the patch, t-ACPD had no effect. These findings suggest that group-I mGluRs acted through a membrane delimited mechanism rather than readily diffusible second messenger. (c) In cerebellar granule cells, mGluR1 triggers a tight coupling between ryanodine-sensitive receptors (RyR) and membrane L-type Ca2+ channels (LCC), in a G-protein-dependent manner. The release of Ca2+ from intracellular ryanodine-sensitive stores activates a Ca2+-dependent K+ conductance (CSKC) located in close proximity to LCC and RYRs. (d) The opening probability of L-type Ca2+ channels (NPo) was monitored in a cell-attached patch and after excision of the patch into the inside-out configuration. The agonist, t-ACPD (100 μM), induced opening of LCC recorded in the cell-attached configuration. LCC remained active in the excised patch and the activity was blocked by ryanodine, but not the IP3 receptor antagonist heparin, when applied to the intracellular side of the recorded patch. This experiment confirmed the model described in (c).
Part (b) adapted from Swartz KJ, Bean BP (1992) Inhibition of Ca2+ channels in rat CA3 pyramidal neurons by a metabotropic glutamate receptor. J. Neurosci.12, 4358–4371, with permission. Part (d) modified from Fagni L, Chavis P, Ango F (2000) Complex interactions mGluRs, intracellular Ca2+ stores and ion channels in neurons. Trends Neurosci.23, 80–88, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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10. Figure 12.8 Current models for post- and presynaptic multiprotein mGluR complexes.
(a) Homer proteins bind to Ca2+-permeable store-operated TRPC1 channel, postsynaptic mGluR1a and mGluR5, as well as two sites of IP3 and ryanodine receptors (IP3R/RyR). Homer can also interact with the shank protein, which associates with GKAP-PSD95-NMDA-receptor complex through a PDZ interaction. The confinement of these receptors and channels by homer-based protein–protein interactions increases the functionality of the system and optimizes mGluR1a mGluR5 intracellular Ca2+ signaling. (b) The adaptor protein PICK1 interacts with the C-terminus of mGluR7 and PKCa via its PDZ domain and dimerizes through its coiled-coil domain. Thus, it physically links mGluR7 to PKCa. Stimulation of mGluR7 releases G-protein βγ-subunits, which results in phospholipase C (PLC) activation in neurons. The PLC pathway triggers PKC activity, which then directly or indirectly inhibits P/Q-type Ca2+ channels. Proper functioning of this cascade requires the integrity of the mGluR7–PICK1–PKCa complex, presumably because of the necessity for mGluR7 to be in close proximity to its effector, PKCa (see Perrog et al. (2002) EMBO J.21, 2990–2999). (c) Ca2+–calmodulin complex and G-protein βγ-subunits undergo competitive binding on the C-terminus of presynaptic mGluR7. The Ca2+ influx from voltage-gated channels activates calmodulin, which then binds to mGluR7 and releases pre-bound G-protein βγ-subunits from the mGluR7 C-terminus. Free G-protein βγ-subunits are thus available for direct inhibition of P/Q-type Ca2+ channels (see Bertaso et al. (2006) J. Neurochem.99, 288–298). These examples illustrate the importance of mGluR multiprotein complexes in the proper function of these receptors.
From Cellular and Molecular Neurophysiology, Fourth Edition.
Copyright © 2015 Elsevier Ltd. All rights reserved. 10

11. Figure 12.9 Modulation of action potential accommodation by inhibition of a Ca2+-dependent K+ current, rather than Ca2+ influx.
The top row shows the action potential firing pattern elicited by prolonged depolarizing current injection in hippocampal neurons. Only one to two action potentials normally fire even in the presence of a suprathreshold stimulus. Such accommodation is due to depolarization-induced Ca2+ influx and activation of IK,AHP. The middle row shows the late outward K+ current IK,AHP under voltage clamp conditions, while the bottom row shows the Ca2+ influx as measured by fluorescence-based techniques. The spike accommodation is reversibly blocked by the group-I mGluR agonist quisqualate, as is the IK,AHP current. However, the magnitude of the intracellular Ca2+ response is not altered by quisqualate, indicating that group-l mGluRs directly modulate the IK,AHP channel, rather than the Ca2+ influx that activates the channel.
Adapted from Charpak S, Gahwiler BH, Do KQ, Knopfel T (1990) Potassium conductances in hippocampal neurons blocked by excitatory amino acid transmitters. Nature347, 765–767, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
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12. 12 From Cellular and Molecular Neurophysiology, Fourth Edition.
Figure mGluR1 mediates slow EPSCs in hippocampal CA3 pyramidal cells and cerebellar Purkinje cells.
(a) Activity-dependent induction of the slow EPSC in hippocampal CA3 pyramidal cells is induced by repetitive stimulation of afferent mossy fibers. (b) The evoked EPSC amplitude is reduced by mGluR1 antagonists S-MCPG (500 μM), S-4CPG (100 μM) and AIDA (200 μM). (c) (Left) A similar slow EPSC can be elicited in cerebellar Purkinje cells by repetitive (100 Hz) parallel fiber stimulation, in the presence of the AMPA- and GABA-receptor antagonists, CNQX and gabazine, respectively. (Center and right) The slow EPSC evoked by 5 successive pulses was blocked by the mGluR1 antagonist CPCCOEt (100 μM, center) and TRPC1-channel antagonist SKF96365 (30 μM; right). Note that at least three successive stimulation pulses (100 Hz) are required to induce the mGluR1-dependent slow EPSC, in both hippocampal neuron and cerebellar Purkinje cell. The amplitude of the EPSC then increased with the number of pulses.
Part (b) adapted from Heuss C, Scanziani M, Gahwiller BH, Gerber U (1999) G-protein-independent signalling mediated by metabotropic glutamate receptors. Nat. Neurosci.2, 1070–1077, with permission. Part (c) adapted from Kim SJ, Kim YS, Yuan JP, Petralla RS, Worley PF, Linden DJ (2003) Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature426, 285–291, with permission.
From Cellular and Molecular Neurophysiology, Fourth Edition.
Copyright © 2015 Elsevier Ltd. All rights reserved. 12