N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

] Institute of Physiology CAS Videnska 1083 142 20 Prague 4 Czech Republic [2] School of Pharmacy and Biomedical Sciences University of Central Lancashire Preston PR1 2HE UK [3] Institute of Medical Physiology School of Medicine University of Belgrade Visegradska 26 2 11000 Beograd Srbija

Charles University Prague Faculty of Science Albertov 6 128 43 Prague 2 Czech Republic

Institute of Chemical Technology Prague Technicka 5 166 28 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry CAS Flemingovo nam 2 166 10 Prague 2 Czech Republic

Institute of Physiology CAS Videnska 1083 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

Lynch M. A. Long-term potentiation and memory. Physiological reviews 84, 87–136 (2004). PubMed

Traynelis S. F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacological reviews 62, 405–496 (2010). PubMed PMC

Mota S. I., Ferreira I. L. & Rego A. C. Dysfunctional synapse in Alzheimer’s disease - A focus on NMDA receptors. Neuropharmacology 76 Pt A, 16–26 (2014). PubMed

Sheng M., Sabatini B. L. & Südhof T. C. Synapses and Alzheimer’s disease. Cold Spring Harb. Perspect. Biol. 4, 1–18 (2012). PubMed PMC

Korinek M. et al. Neurosteroid modulation of N-methyl-D-aspartate receptors: molecular mechanism and behavioral effects. Steroids 76, 1409–1418 (2011). PubMed

Dubrovsky B. O. Steroids, neuroactive steroids and neurosteroids in psychopathology. Progress in neuro-psychopharmacology & biological psychiatry 29, 169–192 (2005). PubMed

Lapchak P. A. The neuroactive steroid 3-alpha-ol-5-beta-pregnan-20-one hemisuccinate, a selective NMDA receptor antagonist improves behavioral performance following spinal cord ischemia. Brain research 997, 152–158 (2004). PubMed

Weaver C. E. et al. Geometry and charge determine pharmacological effects of steroids on N- methyl-D-aspartate receptor-induced Ca(2+) accumulation and cell death. The Journal of pharmacology and experimental therapeutics 293, 747–754 (2000). PubMed

Rambousek L. et al. Cellular and behavioural effects of a new steroidal inhibitor of the N-methyl-d-aspartate receptor 3alpha5beta-pregnanolone glutamate. Neuropharmacology 61, 61–68 (2011). PubMed

Park-Chung M., Wu F. S. & Farb D. H. 3 alpha-Hydroxy-5 beta-pregnan-20-one sulfate: a negative modulator of the NMDA-induced current in cultured neurons. Molecular pharmacology 46, 146–150 (1994). PubMed

Vales K. et al. 3alpha5beta-Pregnanolone glutamate, a use-dependent NMDA antagonist, reversed spatial learning deficit in an animal model of schizophrenia. Behavioural brain research 235, 82–88 (2012). PubMed

Borovska J. et al. Access of inhibitory neurosteroids to the NMDA receptor. British journal of pharmacology 166, 1069–1083 (2012). PubMed PMC

Petrovic M., Sedlacek M., Horak M., Chodounska H. & Vyklicky L. Jr. 20-oxo-5beta-pregnan-3alpha-yl sulfate is a use-dependent NMDA receptor inhibitor. The Journal of neuroscience 25, 8439–8450 (2005). PubMed PMC

Malayev A., Gibbs T. T. & Farb D. H. Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids. British journal of pharmacology 135, 901–909 (2002). PubMed PMC

Park-Chung M. et al. Distinct sites for inverse modulation of N-methyl-D-aspartate receptors by sulfated steroids. Molecular pharmacology 52, 1113–1123 (1997). PubMed

Sobolevskii A. I. & Khodorov B. I. Blocker studies of the functional architecture of the NMDA receptor channel. Neuroscience and behavioral physiology 32, 157–171 (2002). PubMed

Kussius C. L., Kaur N. & Popescu G. K. Pregnanolone sulfate promotes desensitization of activated NMDA receptors. The Journal of neuroscience 29, 6819–6827 (2009). PubMed PMC

Chen G. Q., Cui C., Mayer M. L. & Gouaux E. Functional characterization of a potassium-selective prokaryotic glutamate receptor. Nature 402, 817–821 (1999). PubMed

Smothers C. T. & Woodward J. J. Expression of glycine-activated diheteromeric NR1/NR3 receptors in human embryonic kidney 293 cells Is NR1 splice variant-dependent. The Journal of pharmacology and experimental therapeutics 331, 975–984 (2009). PubMed PMC

Sprengel R. et al. Glutamate receptor channel signatures. Trends Pharmacol Sci 22, 7–10 (2001). PubMed

Sobolevsky A. I., Rosconi M. P. & Gouaux E. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462, 745–756 (2009). PubMed PMC

Sobolevsky A. I., Beck C. & Wollmuth L. P. Molecular rearrangements of the extracellular vestibule in NMDAR channels during gating. Neuron 33, 75–85 (2002). PubMed

Beck C., Wollmuth L. P., Seeburg P. H., Sakmann B. & Kuner T. NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines. Neuron 22, 559–570 (1999). PubMed

Zuo J. et al. Neurodegeneration in Lurcher mice caused by mutation in delta2 glutamate receptor gene. Nature 388, 769–773 (1997). PubMed

Kashiwagi K. et al. Channel blockers acting at N-methyl-D-aspartate receptors: differential effects of mutations in the vestibule and ion channel pore. Molecular pharmacology 61, 533–545 (2002). PubMed

Sobolevsky A. I., Prodromou M. L., Yelshansky M. V. & Wollmuth L. P. Subunit-specific contribution of pore-forming domains to NMDA receptor channel structure and gating. The Journal of general physiology 129, 509–525 (2007). PubMed PMC

Chang H. R. & Kuo C. C. The activation gate and gating mechanism of the NMDA receptor. The Journal of neuroscience 28, 1546–1556 (2008). PubMed PMC

Murthy S. E., Shogan T., Page J. C., Kasperek E. M. & Popescu G. K. Probing the activation sequence of NMDA receptors with lurcher mutations. The Journal of general physiology 140, 267–277 (2012). PubMed PMC

Kemp J. A. et al. 7-Chlorokynurenic acid is a selective antagonist at the glycine modulatory site of the N-methyl-D-aspartate receptor complex. Proc Natl Acad Sci U S A 85, 6547–6550 (1988). PubMed PMC

Evans R. H., Francis A. A., Jones A. W., Smith D. A. & Watkins J. C. The effects of a series of omega-phosphonic alpha-carboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparations. British journal of pharmacology 75, 65–75 (1982). PubMed PMC

Xu M., Smothers C. T., Trudell J. & Woodward J. J. Ethanol inhibition of constitutively open N-methyl-D-aspartate receptors. The Journal of pharmacology and experimental therapeutics 340, 218–226 (2012). PubMed PMC

McNaught A. D. & Wilkinson A. in IUPAC, Compendium of Chemical Terminology, 2nd edn (Blackwell Science, 1997).

Schreier S., Malheiros S. V. & de Paula E. Surface active drugs: self-association and interaction with membranes and surfactants. Physicochemical and biological aspects. Biochim Biophys Acta 1508, 210–234 (2000). PubMed

Vyklicky L. Jr., Krusek J. & Edwards C. Differences in the pore sizes of the N-methyl-D-aspartate and kainate cation channels. Neurosci Lett 89, 313–318 (1988). PubMed

Villarroel A., Burnashev N. & Sakmann B. Dimensions of the narrow portion of a recombinant NMDA receptor channel. Biophysical journal 68, 866–875 (1995). PubMed PMC

Zarei M. M. & Dani J. A. Structural basis for explaining open-channel blockade of the NMDA receptor. The Journal of neuroscience 15, 1446–1454 (1995). PubMed PMC

Dai J. & Zhou H. X. An NMDA receptor gating mechanism developed from MD simulations reveals molecular details underlying subunit-specific contributions. Biophysical journal 104, 2170–2181 (2013). PubMed PMC

Kazi R. et al. Asynchronous movements prior to pore opening in NMDA receptors. The Journal of neuroscience 33, 12052–12066 (2013). PubMed PMC

Havlikova H. et al. Serum profiles of free and conjugated neuroactive pregnanolone isomers in nonpregnant women of fertile age. The Journal of clinical endocrinology and metabolism 91, 3092–3099 (2006). PubMed

Kancheva R. et al. Relationships of circulating pregnanolone isomers and their polar conjugates to the status of sex, menstrual cycle, and pregnancy. The Journal of endocrinology 195, 67–78 (2007). PubMed

Baulieu E. E. Neurosteroids: of the nervous system, by the nervous system, for the nervous system. Recent Prog Horm Res 52, 1–32 (1997). PubMed

Mellon S. H. & Griffin L. D. Synthesis, regulation, and function of neurosteroids. Endocr Res 28, 463 (2002). PubMed

Park-Chung M., Malayev A., Purdy R. H., Gibbs T. T. & Farb D. H. Sulfated and unsulfated steroids modulate gamma-aminobutyric acidA receptor function through distinct sites. Brain research 830, 72–87 (1999). PubMed

Weaver C. E. Jr., Marek P., Park-Chung M., Tam S. W. & Farb D. H. Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci U S A 94, 10450–10454 (1997). PubMed PMC

Stastna E. et al. Synthesis of C3, C5, and C7 pregnane derivatives and their effect on NMDA receptor responses in cultured rat hippocampal neurons. Steroids 74, 256–263 (2009). PubMed

Cerny I. et al. Neuroactive steroids with perfluorobenzoyl group. Steroids 77, 1233–1241 (2012). PubMed

Wollmuth L. P. & Sobolevsky A. I. Structure and gating of the glutamate receptor ion channel. Trends in neurosciences 27, 321–328 (2004). PubMed

Kashiwabuchi N. et al. Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR delta 2 mutant mice. Cell 81, 245–252 (1995). PubMed

Nowak L., Bregestovski P., Ascher P., Herbet A. & Prochiantz A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307, 462–465 (1984). PubMed

Mayer M. L., Westbrook G. L. & Guthrie P. B. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309, 261–263 (1984). PubMed

Benveniste M. & Mayer M. L. Trapping of glutamate and glycine during open channel block of rat hippocampal neuron NMDA receptors by 9-aminoacridine. The Journal of physiology 483 (Pt 2), 367–384 (1995). PubMed PMC

Hollmann M. et al. Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron 10, 943–954 (1993). PubMed

Smothers C. T. & Woodward J. J. Expression of glycine-activated diheteromeric NR1/NR3 receptors in human embryonic kidney 293 cells Is NR1 splice variant-dependent. Journal of Pharmacology and Experimental Therapeutics 331, 975–984 (2009). PubMed PMC

Sucher N. J. et al. Developmental and regional expression pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain. The Journal of neuroscience 15, 6509–6520 (1995). PubMed PMC

Horak M., Chang K. & Wenthold R. J. Masking of the endoplasmic reticulum retention signals during assembly of the NMDA receptor. The Journal of neuroscience 28, 3500–3509 (2008). PubMed PMC

Abdrachmanova G., Teisinger J. & Vyklicky L. Jr. Axotomy-induced changes in the properties of NMDA receptor channels in rat spinal cord motoneurons. The Journal of physiology 538, 53–63 (2002). PubMed PMC

Arnostova L. M., Pouzar V. & Drasar P. Synthesis of the sulfates derived from 5 alpha-cholestane-3 beta,6 alpha-diol. Steroids 57, 233–235 (1992). PubMed

Karakas E. & Furukawa H. Crystal structure of a heterotetrameric NMDA receptor ion channel. Science 344, 992–997 (2014). PubMed PMC

Lee C. H. et al. NMDA receptor structures reveal subunit arrangement and pore architecture. Nature 511, 191–197 (2014). PubMed PMC

Sali A. & Blundell T. L. Comparative protein modelling by satisfaction of spatial restraints. Journal of molecular biology 234, 779–815 (1993). PubMed

Larkin M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). PubMed

Sanner M. F. Python: a programming language for software integration and development. Journal of molecular graphics & modelling 17, 57–61 (1999). PubMed

Lomize M. A., Lomize A. L., Pogozheva I. D. & Mosberg H. I. OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006). PubMed

Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology

Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids

Novel neurosteroid pregnanolone pyroglutamate suppresses neurotoxicity syndrome induced by tetramethylenedisulfotetramine but is ineffective in a rodent model of infantile spasms

Altered Steroidome in Women with Gestational Diabetes Mellitus: Focus on Neuroactive and Immunomodulatory Steroids from the 24th Week of Pregnancy to Labor

Pitfalls of NMDA Receptor Modulation by Neuroactive Steroids. The Effect of Positive and Negative Modulation of NMDA Receptors in an Animal Model of Schizophrenia

Palmitoylation Controls NMDA Receptor Function and Steroid Sensitivity

Site of Action of Brain Neurosteroid Pregnenolone Sulfate at the N-Methyl-D-Aspartate Receptor

NMDA Receptor Opening and Closing-Transitions of a Molecular Machine Revealed by Molecular Dynamics

Strong Inhibitory Effect, Low Cytotoxicity and High Plasma Stability of Steroidal Inhibitors of N-Methyl-D-Aspartate Receptors With C-3 Amide Structural Motif

The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate

Surface Expression, Function, and Pharmacology of Disease-Associated Mutations in the Membrane Domain of the Human GluN2B Subunit

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives