N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

• Klíčová slova
• Channelopathy, Endogenous neuroactive steroid, GRIN2 genes, Glutamate receptors, Rescue pharmacology, Surface expression,
• MeSH
• elektrofyziologické jevy MeSH
• lidé MeSH
• neurosteroidy * MeSH
• receptory N-methyl-D-aspartátu * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• N-methyl D-aspartate receptor subtype 2A MeSH Prohlížeč
• neurosteroidy * MeSH
• NR2B NMDA receptor MeSH Prohlížeč
• receptory N-methyl-D-aspartátu * MeSH

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.

• Klíčová slova
• glutamate receptors, neurosteroids, patch clamp, structure,
• MeSH
• alanin genetika   MeSH
• buněčná membrána účinky léků   MeSH
• cholesterol metabolismus   MeSH
• elektrofyziologické jevy MeSH
• HEK293 buňky MeSH
• konformace proteinů MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• pregnenolon farmakologie   MeSH
• receptory N-methyl-D-aspartátu účinky léků   MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• cholesterol MeSH
• NMDA receptor A1 MeSH Prohlížeč
• NR2B NMDA receptor MeSH Prohlížeč
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH

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.

• MeSH
• aminokyselinové motivy MeSH
• lidé MeSH
• mutace * MeSH
• pregnanolon * chemie   metabolismus   MeSH
• receptory N-methyl-D-aspartátu * antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• vestibulární aparát * chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pregnanolon * MeSH
• receptory N-methyl-D-aspartátu * MeSH

NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) action at NMDA receptors. 3alpha5betaS was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3alpha5betaS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3alpha5betaS in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3alpha5betaS at NMDA receptors. In accordance with the model, 3alpha5betaS was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3alpha5betaS is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.

• MeSH
• buněčné linie MeSH
• hipokampus cytologie   fyziologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály účinky léků   fyziologie   MeSH
• metoda terčíkového zámku MeSH
• modely neurologické MeSH
• neokortex fyziologie   MeSH
• novorozená zvířata MeSH
• pregnany farmakologie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   MeSH
• techniky in vitro MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 20-oxo-5beta-pregnan-3alpha-yl sulfate MeSH Prohlížeč
• pregnany MeSH
• receptory N-methyl-D-aspartátu MeSH

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission and synaptic plasticity as well as excitotoxicity. They have several binding sites for allosteric modulators, including neurosteroids, endogenous compounds synthesized by the nervous tissue and expected to act locally. Whole-cell patch-clamp recording from human embryonic kidney 293 cells expressing NR1-1a/NR2B receptors revealed that neurosteroid pregnenolone sulfate (PS) (300 microm), when applied to resting NMDA receptors, potentiates the amplitude of subsequent responses to 1 mm glutamate fivefold and slows their deactivation twofold. The same concentration of PS, when applied during NMDA receptor activation by 1 mm glutamate, has only a small effect. The association and dissociation rate constants of PS binding and unbinding from resting NMDA receptors are estimated to be 3.3 +/- 2.0 mm(-1)sec(-1) and 0.12 +/- 0.02 sec(-1), respectively, corresponding to an apparent affinity K(d) of 37 microm. The results of experiments indicate that the molecular mechanism of PS potentiation of NMDA receptor responses is attributable to an increase in the peak channel open probability (P(o)). Responses to glutamate recorded in the continuous presence of PS exhibit marked time-dependent decline. Our results indicate that the decline is induced by a change of the NMDA receptor affinity for PS after receptor activation. These results suggest that the PS is a modulator of NMDA receptor P(o), the effectiveness of which is lowered by glutamate binding. This modulation may have important consequences for the neuronal excitability.

• MeSH
• biologické modely MeSH
• gating iontového kanálu účinky léků   MeSH
• hipokampus cytologie   MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• kyselina glutamová farmakologie   MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• neurony účinky léků   metabolismus   MeSH
• novorozená zvířata MeSH
• pravděpodobnost MeSH
• pregnenolon farmakologie   MeSH
• receptory N-methyl-D-aspartátu agonisté   účinky léků   metabolismus   MeSH
• rekombinantní proteiny agonisté   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina glutamová MeSH
• NR1 NMDA receptor MeSH Prohlížeč
• NR2B NMDA receptor MeSH Prohlížeč
• pregnenolon MeSH
• pregnenolone sulfate MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH
• rekombinantní proteiny MeSH