title: Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.
abstract: Pregnenolone sulfate (PS), one of the most commonly occurring neurosteroids in the central nervous system, influences the function of several receptors. PS modulates N-methyl-D-aspartate receptors (NMDARs) and has been shown to have both positive and negative modulatory effects on NMDAR currents generally in a subtype-selective manner. We assessed the gating mechanism of PS modulation of GluN1/GluN2A receptors transiently expressed in human embryonic kidney 293 cells using whole-cell and single-channel electrophysiology. Only a modest effect on the whole-cell responses was observed by PS in dialyzed (nonperforated) whole-cell recordings. Interestingly, in perforated conditions, PS was found to increase the whole-cell currents in the absence of nominal extracellular Ca(2+), whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca(2+). The Ca(2+)-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca(2+)-dependent bidirectional effect of PS. Single-channel cell-attached analysis demonstrated that PS primarily affected the mean open time to produce its effects: positive modulation mediated by an increase in duration of open time constants, and negative modulation mediated by a reduction in the time spent in a long-lived open state of the receptor. Further kinetic modeling of the single-channel data suggested that the positive and negative modulatory effects are mediated by different gating steps which may represent GluN2 and GluN1 subunit-selective conformational changes, respectively. Our studies provide a unique mechanism of modulation of NMDARs by an endogenous neurosteroid, which has implications for identifying state-dependent molecules.
authors.affiliation: Department of Pharmacology, Creighton University, Omaha, Nebraska (D.A.C., S.M.D.); and Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska (D.T.M.).
authors.forename: Divyan A
authors.initials: DA
authors.lastname: Chopra
authors.affiliation: Department of Pharmacology, Creighton University, Omaha, Nebraska (D.A.C., S.M.D.); and Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska (D.T.M.).
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Monaghan
authors.affiliation: Department of Pharmacology, Creighton University, Omaha, Nebraska (D.A.C., S.M.D.); and Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska (D.T.M.) shashankdravid@creighton.edu.
authors.forename: Shashank M
authors.initials: SM
authors.lastname: Dravid
mesh_terms: D002118:Calcium / Q000378:metabolism*; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism* / Q000494:pharmacology; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002118:Calcium; D009419:Nerve Tissue Proteins; D011284:Pregnenolone; D016194:Receptors, N-Methyl-D-Aspartate; C485668:GRIN1 protein, human; C018370:pregnenolone sulfate