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

Abstract

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.

Analysis of M4 transmembrane domains in NMDA receptor function: a negative allosteric modulation site at the GluN1 M4 is determining the efficiency of neurosteroid modulation

“…Consequently, we attribute the complete lack of a positive-modulatory effect of PS in GluN1/GluN3A receptors to the unique design of the M4 interface of the GluN3 subunit. Our results are consistent with other studies of conventional GluN1/GluN2 NMDA receptors, in which it has been shown that the M4 and its linker region of GluN2 subunits, in particular, control the subunit-specific PS action by determining the positive-modulatory effect of PS (Jang et al, 2004;Krausova et al, 2020). However, the exact mechanism that couples the modulatory properties of the M4 domain to channel activation and the importance of these TM interfaces in subunit-specific PS regulation are still unknown.…”

“…In contrast, studies on conventional GluN1/GluN2 NMDA receptors led to the view that the M4 domain is more required for the formation of GluN1/GluN2 heterodimers and, in the case of the GluN2B subunit, also for masking ER retention signals in GluN1 (Cao et al, 2011;Horak et al, 2008). We can show that no M4 deletion affects the assembly and surface expression of the conventional GluN1/GluN2A NMDA receptor, supporting the view that the M4 of glutamate-gated NMDARs are primarily structural determinants that are more involved in the allosteric regulation of ion channel opening by modulatory compounds (Krausova et al, 2020). The absence of M4 in prokaryotic GluR0 also supports the idea that this transmembrane region may not be essential for iGluR assembly and that its presence in eukaryotic iGluRs is predominantly required for modulating or fine-tuning the kinetic properties of the channel.…”

“…Recent findings through molecular dynamics simulations and alanine screening indicate that the binding site responsible for positive modulation of GluN1/GluN2A/B indeed appears to be localized at the GluN2 M1/M4 interface. These simulations also raise the prospect of another binding site at the appropriate positions for the GluN1 M1/M4 interface that does not carry the PAM effect (Krausova et al, 2020).…”

“…To investigate the importance of our identified interactions of the M4 domains with the M1 and M3 domains of the neighboring subunit for the functional modulation of GluN1/GluN2A receptors, we performed Imax measurements in the absence and presence of the neurosteroid pregnenolone sulfate (20oxo-5-pregnen-3β-yl sulfate, abbreviated PS; (Horak et al, 2006;Jang et al, 2004;Krausova et al, 2020;Wilding et al, 2016)) after expression of the wt-GluN1/GluN2A receptor and the GluN1 ΔM4 +M4 N1 /GluN2A and GluN1/GluN2A ΔM4 +M4 N2A constructs at saturating agonist concentrations. Remarkably, when Imax values were compared in the absence and presence of PS for the different constructs, a difference in the modulation of whole-cell currents was immediately apparent.…”

Comparison of pregnenolone sulfate, pregnanolone and estradiol levels between patients with menstrually-related migraine and controls: an exploratory study

“…Therefore, it would also be important to study neuroactive steroids with excitatory functions such as pregnenolone sulfate (pregn-5-en-3β-ol-20-one 3β-sulfate). Actually, this neurosteroid acts as a positive allosteric N-methyl-D-aspartate (NMDA) receptor modulator [ 6 , 7 ] and is a potent stimulator of transient receptor potential melastatin-3 (TRPM3) channels, that function as nociceptors in the somatosensory system [ 8 ]. Moreover, at the molecular level, stimulation of TRPM3 channels has been shown to be involved in calcitonin gene-related peptide (CGRP) exocytosis [ 9 , 10 ].…”