Abstract

NMDA receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies reveal reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell surface NMDARs in cortical neurons increases 60 -100% within 10 min of exposure to PregS as shown by surface biotinylation and affinity purification. Delayed onset potentiation is reversible and selective for expressed receptors containing the NR2A or NR2B, but not NR2C or NR2D, subunits. Moreover, substitution of NR2B J-K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed onset potentiation by PregS occurs via a non- canonical, pertussis toxin sensitive G-protein coupled and Ca²⁺ dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.