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 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 Ca 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 of modulation of NMDARs by an endogenous neurosteroid, which has implications for identifying state-dependent molecules.

The NMDA receptor intracellular C-terminal domains reciprocally interact with allosteric modulators

“…A prominent role of the NMDAR CTD in PAM activity, particularly at GluN2A-containing receptors, is consistent with observations that dialysis of cells during whole cell recording or upon forming outside-out patches eliminates the PAM activity of PS and UBP684 [17,18,25] at GluN1/GluN2A receptors. Thus, soluble intracellular ions, phosphorylating/dephosphorylating enzymes, or disrupted protein-protein interactions may account for loss of PAM activity in dialyzed cells.…”

“…For example, a NMDAR PAM, unlike an agonist, could enhance weak NMDAR signals in pathological conditions of NMDAR hypofunction without causing activation of other NMDARs that should stay inactive. However, there are incidental observations that the activity of some allosteric modulators can be lost or enhanced due to a change in the intracellular environment such as when whole cell recordings or outside-out patch recordings are generated [17][18][19]. Given the therapeutic potential of the allosteric modulators, it is important to understand the conditions in which these agents may gain or lose activity so that the appropriate receptor populations can be targeted in disease.…”

Pregnenolone Inhibits Osteoclast Differentiation and Protects Against Lipopolysaccharide-Induced Inflammatory Bone Destruction and Ovariectomy-Induced Bone Loss

“…However, a specific receptor for Preg has yet to be identified. Studies have shown that Preg can acts as an modulator of NMDA receptor and as an agonist of pregnane X receptor (PXR) (Malayev et al, 2002;Shizu et al, 2013;Smith et al, 2014;Chopra et al, 2015). Interestingly, both NMDA receptor and PXR have been shown to play a role in osteoclast formation and function.…”

Mechanism and properties of positive allosteric modulation of N -methyl- d -aspartate receptors by 6-alkyl 2-naphthoic acid derivatives

“…Whole-cell electrophysiology was conducted as previously described (Chopra et al, 2015). Briefly, mice (~30–35 day old) were anesthetized by isoflurane and decapitated in accordance with the approved protocols of Creighton University IACUC.…”

“…The alkyl-naphthoic acid PAMs characterized here add to the pharmacodynamic diversity of the rapidly expanding list of NMDAR PAMs such as PS (Chopra et al, 2015; Horak et al, 2006; Horak et al, 2004; Jang et al, 2004; Kostakis et al, 2011; Wu et al, 1991), UBP512, UBP646 (Costa et al, 2010), UBP714 (Irvine et al, 2012), CIQ (Mullasseril et al, 2010), PYD106 (Khatri et al, 2014), SGE201 (Linsenbardt et al, 2014; Paul et al, 2013), and GNE6901 (Hackos et al, 2016). These agents differ in their subtype-selectivity, N-terminal insert-sensitivity, pH-sensitivity, use/disuse-dependency, and their effects on agonist potency, efficacy and deactivation.…”