title: Access of inhibitory neurosteroids to the NMDA receptor.
abstract: OBJECTIVE
  NMDA receptors are glutamatergic ionotropic receptors involved in excitatory neurotransmission, synaptic plasticity and excitotoxic cell death. Many allosteric modulators can influence the activity of these receptors positively or negatively, with behavioural consequences. 20-Oxo-5β-pregnan-3α-yl sulphate (pregnanolone sulphate; PA-6) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective. We tested the hypothesis that the interaction of PA-6 with the plasma membrane is critical for its inhibitory effect at NMDA receptors.
  
  
  METHODS
  Electrophysiological recordings and live microscopy were performed on heterologous HEK293 cells expressing GluN1/GluN2B receptors and cultured rat hippocampal neurons.
  
  
  RESULTS
  Our experiments showed that the kinetics of the steroid inhibition were slow and not typical of drug-receptor interaction in an aqueous solution. In addition, the recovery from steroid inhibition was accelerated by β- and γ-cyclodextrin. Values of IC(50)  assessed for novel synthetic C3 analogues of PA-6 differed by more than 30-fold and were positively correlated with the lipophilicity of the PA-6 analogues. Finally, the onset of inhibition induced by C3 analogues of PA-6 ranged from use-dependent to use-independent. The onset and offset of cell staining by fluorescent analogues of PA-6 were slower than those of steroid-induced inhibition of current responses mediated by NMDA receptors.
  
  
  CONCLUSIONS
  We conclude that steroid accumulation in the plasma membrane is the route by which it accesses a binding site on the NMDA receptor. Thus, our results provide a possible structural framework for pharmacologically targeting the transmembrane domains of the receptor.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic.
authors.forename: Jirina
authors.initials: J
authors.lastname: Borovska
authors.forename: Vojtech
authors.initials: V
authors.lastname: Vyklicky
authors.forename: Eva
authors.initials: E
authors.lastname: Stastna
authors.forename: Vojtech
authors.initials: V
authors.lastname: Kapras
authors.forename: Barbora
authors.initials: B
authors.lastname: Slavikova
authors.forename: Martin
authors.initials: M
authors.lastname: Horak
authors.forename: Hana
authors.initials: H
authors.lastname: Chodounska
authors.forename: Ladislav
authors.initials: L
authors.lastname: Vyklicky
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D015394:Molecular Structure; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000494:pharmacology*; D011278:Pregnanes / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: C508346:20-oxo-5beta-pregnan-3alpha-yl sulfate; D018691:Excitatory Amino Acid Antagonists; D018696:Neuroprotective Agents; D018377:Neurotransmitter Agents; D011278:Pregnanes; D016194:Receptors, N-Methyl-D-Aspartate