Bidirectional effect of pregnenolone sulfate on GluN1/GluN2A N-Methyl-D-aspartate receptor gating depending on extracellular calcium and intracellular milieu

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 wholecell 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 Ca²⁺, whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca²⁺ The Ca²⁺-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca²⁺-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 singlechannel 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 ofmodulation ofNMDARs by an endogenous neurosteroid, which has implications for identifying state-dependent molecules.