Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule

Abstract

N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor’s ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system.

Preferential Inhibition of Tonically over Phasically Activated NMDA Receptors by Pregnane Derivatives

“…Due to the nature of the steroid-receptor interaction, the onset of inhibition is slow (hundreds of milliseconds) compared with the relatively fast onset of the NMDAR responses induced by 1 mM glutamate (tens of milliseconds). This difference, together with the fact that PA-S binding to the NMDAR can occur only in the presence of the agonist (i.e., it is use dependent; Petrovic et al, 2005;Kussius et al, 2009;Vyklicky et al, 2015), underlie the lower sensitivity of the peak versus the steady-state NMDAR response to inhibitory steroids.…”

“…Even though both memantine and steroids are use-dependent inhibitors, they differ in the voltage dependency of block and in the trapping versus foot-in-the-door mechanism of action (Blanpied et al, 1997;Petrovic et al, 2005;Vyklicky et al, 2015). Figure 3A shows that both memantine (10 M) and PA-S (300 M) inhibited steady-state GluN1/ GluN2B receptor responses and exhibited pharmacodynamic similarities characterized by a slow offset of inhibition.…”

“…These data indicate that PA-hPim is flexible and tends to adopt a more compact conformation inside the NMDAR through an intramolecular interaction of the aliphatic chain to the steroid skeleton. This intramolecular interaction has an adverse energy effect, but facilitates steroid passage to the gating Thr residues (Beck et al, 1999;Sobolevsky et al, 2002;Vyklicky et al, 2015). In contrast, the conformation of PA-hOxa inside the channel vestibule remains mostly extended (Fig.…”

“…Remarkably, PA-hPim, the synthetic derivative with the longest substituent, exhibited no effect at phasically activated receptors while inhibiting tonically activated receptors. To avoid the effect of slow steroid diffusion to the site of action at the NMDAR Vyklicky et al, 2015), the inhibition of tonically and phasically activated NMDAR was estimated from responses made in the continuous presence of the steroid. Due to the nature of the steroid-receptor interaction, the onset of inhibition is slow (hundreds of milliseconds) compared with the relatively fast onset of the NMDAR responses induced by 1 mM glutamate (tens of milliseconds).…”

“…Theoretical considerations predict that, although NMDAR inhibitors with use-dependent onset and offset of inhibition ("trapping" blockers), exemplified by memantine, would show some preference for tonically versus phasically activated NMDARs, the strongest selectivity would be achieved by use-dependent inhibitors with slow onset and useindependent offset of inhibition ("foot-in-the-door" block; . Because an endogenous neurosteroid, 20-oxo-5␤-pregnan-3␣-yl sulfate (PA-S; 3␣,5␤-pregnanolone sulfate), is an NMDAR inhibitor with a foot-in-the-door mechanism of block (Petrovic et al, 2005;Vyklicky et al, 2015), we have explored the effect of PA-S and novel synthetic PA-S analogs on synaptically activated NMDARs and on receptors activated by sustained glutamate applications.…”