Dataset Information

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 whole-cell 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(2+), whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca(2+). The Ca(2+)-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca(2+)-dependent bidirectional effect of PS. Single-channel cell-attached analysis demonstrated that PS primarily affected the mean open time to produce its effects: positive modulation mediated by an increase in duration of open time constants, and negative modulation mediated by a reduction in the time spent in a long-lived open state of the receptor. Further kinetic modeling of the single-channel 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.

SUBMITTER: Chopra DA

PROVIDER: S-EPMC4576688 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.

Chopra Divyan A DA Monaghan Daniel T DT Dravid Shashank M SM

Molecular pharmacology 20150710 4

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 whole-cell and single- ...[more]

PMID: 26162866

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Project description:NMDA receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic currents. These receptors are involved in several important brain functions, including learning and memory, and have also been implicated in neuropathological conditions and acute central nervous system injury, which has driven therapeutic interest in their modulation. The EU1794 series of positive and negative allosteric modulators of NMDA receptors has structural determinants of action near the preM1 helix that is involved in channel gating. Here, we describe the effects of the negative allosteric modulator EU1794-4 on GluN1/GluN2A channels studied in excised outside-out patches. Coapplication of EU1794-4 with a maximally effective concentration of glutamate and glycine increases the fraction of time the channel is open by nearly 1.5-fold, yet reduces single-channel conductance by increasing access of the channel to several subconductance levels, which has the net overall effect of reducing the macroscopic current. The lack of voltage-dependence of negative modulation suggests this is unrelated to a channel block mechanism. As seen with other NMDA receptor modulators that reduce channel conductance, EU1794-4 also reduces the Ca2+ permeability relative to monovalent cations of GluN1/GluN2A receptors. We conclude that EU1794-4 is a prototype for a new class of NMDA receptor negative allosteric modulators that reduce both the overall current that flows after receptor activation and the flux of Ca2+ ion relative to monovalent cations. SIGNIFICANCE STATEMENT: NMDA receptors are implicated in many neurological conditions but are challenging to target given their ubiquitous expression. Several newly identified properties of the negative allosteric modulator EU1794-4, including reducing Ca2+ flux through NMDA receptors and attenuating channel conductance, explain why this modulator reduces but does not eliminate NMDA receptor function. A modulator with these properties could have therapeutic advantages for indications in which attenuation of NMDA receptor function is beneficial, such as neurodegenerative disease and acute injury.

Project description:BACKGROUND:Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, gamma-aminobutyric acid type A (GABA(A)) receptors, and sigma(1) receptors, and voltage-gated Ca(2+) and K(+) channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K(+) (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K(+) homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders. METHODOLOGY/PRINCIPAL FINDINGS:The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels. CONCLUSIONS/SIGNIFICANCE:The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.

Dataset Information

Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.

Publications

Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.

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