Site-Specific Fluorescence Reveals Distinct Structural Changes Induced in the Human ρ1 GABA Receptor by Inhibitory NeurosteroidsS⃞

The ρ1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236–247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the ρ1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (pregnanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, allopregnanolone sulfate, and β-estradiol) and probed how these steroids, as well as the nonsteroidal inhibitor picrotoxinin, modify GABA-elicited fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The fluorophore responds with changes in quantum yield to changes in the environment, allowing it to probe for structural changes taking place during channel activation or modulation. The results indicate that the modulators have specific effects on fluorescence changes suggesting that distinct conformational changes accompany inhibition. The findings are consistent with the steroids acting as allosteric inhibitors of the ρ1 GABA receptor and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the ρ1 GABA receptor

Structure–Activity Relationships and Pharmacophore Model of a Noncompetitive Pyrazoline Containing Class of GluN2C/GluN2D Selective Antagonists

Here we describe the synthesis and structure–activity relationship for a class of pyrazoline-containing dihydroquinolone negative allosteric modulators of the NMDA receptor that show strong subunit selectivity for GluN2C- and GluN2D-containing receptors over GluN2A- and GluN2B-containing receptors. Several members of this class inhibit NMDA receptor responses in the nanomolar range and are more than 50-fold selective over GluN1/GluN2A and GluN1/GluN2B NMDA receptors, as well as AMPA, kainate, GABA, glycine, nicotinic, serotonin, and purinergic receptors. Analysis of the purified enantiomers of one of the more potent and selective compounds shows that the <i>S</i>-enantiomer is both more potent and more selective than the <i>R-</i>enantiomer. The <i>S</i>-enantiomer had an IC₅₀ of 0.17–0.22 μM at GluN2D- and GluN2C-containing receptors, respectively, and showed over 70-fold selectivity over other NMDA receptor subunits. The subunit selectivity of this class of compounds should be useful in defining the role of GluN2C- and GluN2D-containing receptors in specific brain circuits in both physiological and pathophysiological conditions

GluN2D-Containing N-methyl-D-Aspartate Receptors Mediate Synaptic Transmission in Hippocampal Interneurons and Regulate Interneuron Activity

International audienceN-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamatergic receptors that have been implicated in learning, development, and neuropathological conditions. They are typically composed of GluN1 and GluN2A-D subunits. Whereas a great deal is known about the role of GluN2A- and GluN2B-containing NMDARs, much less is known about GluN2D-containing NMDARs. Here we explore the subunit composition of synaptic NMDARs on hippocampal interneurons. GluN2D mRNA was detected by single-cell PCR and in situ hybridization in diverse interneuron subtypes in the CA1 region of the hippocampus. The GluN2D subunit was detectable by immunoblotting and immunohistochemistry in all subfields of the hippocampus in young and adult mice. In whole-cell patch-clamp recordings from acute hippocampal slices, (+)-CIQ, the active enantiomer of the positive allosteric modulator CIQ, significantly enhanced the amplitude of the NMDAR component of miniature excitatory postsynaptic currents (mEPSCs) in CA1 interneurons but not in pyramidal cells. (+)-CIQ had no effect in slices from Grin2d-/- mice, suggesting that GluN2D-containing NMDARs participate in excitatory synaptic transmission onto hippocampal interneurons. The time course of the NMDAR component of the mEPSC was unaffected by (1)-CIQ potentiation and was not accelerated in slices from Grin2d-/- mice compared with wild-type, suggesting that GluN2D does not detectably slow the NMDAR EPSC time course at this age. (+)-CIQ increased the activity of CA1 interneurons as detected by the rate and net charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal cells. These data provide evidence that interneurons contain synaptic NMDARs possessing a GluN2D subunit, which can influence interneuron function and signal processing

Design, Synthesis, and Structure–Activity Relationship of a Novel Series of GluN2C-Selective Potentiators

NMDA receptors are tetrameric complexes composed of GluN1 and GluN2A–D subunits that mediate a slow Ca²⁺<b>-</b>permeable component of excitatory synaptic transmission. NMDA receptors have been implicated in a wide range of neurological diseases and thus represent an important therapeutic target. We herein describe a novel series of pyrrolidinones that selectively potentiate only NMDA receptors that contain the GluN2C subunit. The most active analogues tested were over 100-fold selective for recombinant GluN2C-containing receptors over GluN2A/B/D-containing NMDA receptors as well as AMPA and kainate receptors. This series represents the first class of allosteric potentiators that are selective for diheteromeric GluN2C-containing NMDA receptors

Structural Rearrangements in Loop F of the GABA Receptor Signal Ligand Binding, Not Channel Activation

Structure-function studies of the Cys loop family of ionotropic neurotransmitter receptors (GABA, nACh, 5-HT3, and glycine receptors) have resulted in a six-loop (A–F) model of the agonist-binding site. Key amino acids have been identified in these loops that associate with, and stabilize, bound ligand. The next step is to identify the structural rearrangements that couple agonist binding to channel opening. Loop F has been proposed to move upon receptor activation, although it is not known whether this movement is along the conformational pathway for channel opening. We test this hypothesis in the GABA receptor using simultaneous electrophysiology and site-directed fluorescence spectroscopy. The latter method reveals structural rearrangements by reporting changes in hydrophobicity around an environmentally sensitive fluorophore attached to defined positions of loop F. Using a series of ligands that span the range from full activation to full antagonism, we show there is no correlation between the rearrangements in loop F and channel opening. Based on these data and agonist docking simulations into a structural model of the GABA binding site, we propose that loop F is not along the pathway for channel opening, but rather is a component of the structural machinery that locks ligand into the agonist-binding site