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

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented. SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.