Hana Chodounska

Subtype-dependence of N-methyl-D-aspartate receptor modulation by pregnenolone sulfate.

N-methyl-D-aspartate receptors play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits. The subunit composition determines the biophysical and pharmacological properties of the N-methyl-D-aspartate receptor channel complex. In this study, we report that responses mediated by recombinant rat N-methyl-D-aspartate receptors expressed in human embryonic kidney HEK293 cells are differentially affected by naturally occurring neurosteroid pregnenolone sulfate. We show that responses induced by 1mM glutamate in NR1-1a/NR2A and NR1-1a/NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR1-1a/NR2C and NR1-1a/NR2D receptors with no differences in the concentration of pregnenolone sulfate that produced 50% potentiation. In addition to potentiation, pregnenolone sulfate also has an inhibitory effect at recombinant N-methyl-D-aspartate receptors, with values of the concentration of pregnenolone sulfate that produces 50% inhibition of NR1/NR2D=NR1/NR2C<NR1/NR2B<NR1/NR2A. In addition, we show that the structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit is critical for both the potentiating and inhibitory effects of pregnenolone sulfate. The modulatory effects of pregnenolone sulfate are consistent with a model in which this neurosteroid acts at two distinct binding sites on the N-methyl-D-aspartate receptor. These data provide insight into the mechanisms by which pregnenolone sulfate and related sulfated neurosteroids modulate activity of N-methyl-D-aspartate receptor channels.

Molecular Mechanism of Pregnenolone Sulfate Action at NR1/NR2B Receptors

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 μm), 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-1sec-1 and 0.12 ± 0.02 sec-1, respectively, corresponding to an apparent affinity Kd of 37 μm. 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 (Po). 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 Po, the effectiveness of which is lowered by glutamate binding. This modulation may have important consequences for the neuronal excitability.

20-Oxo-5β-Pregnan-3α-yl Sulfate Is a Use-Dependent NMDA Receptor Inhibitor

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-5β-pregnan-3α-yl sulfate (3α5βS) action at NMDA receptors. 3α5βS 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 3α5βS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3α5βS 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 3α5βS at NMDA receptors. In accordance with the model, 3α5βS 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 3α5βS 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.

Neurosteroid modulation of N-methyl-D-aspartate receptors: Molecular mechanism and behavioral effects

Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-D-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases.

Access of inhibitory neurosteroids to the NMDA receptor

NMDA receptors are glutamatergic ionotropic receptors involved in excitatory neurotransmission, synaptic plasticity and excitotoxic cell death. Many allosteric modulators can influence the activity of these receptors positively or negatively, with behavioural consequences. 20‐Oxo‐5β‐pregnan‐3α‐yl sulphate (pregnanolone sulphate; PA‐6) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective. We tested the hypothesis that the interaction of PA‐6 with the plasma membrane is critical for its inhibitory effect at NMDA receptors.

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

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.

Cellular and behavioural effects of a new steroidal inhibitor of the N-methyl-D-aspartate receptor 3α5β-pregnanolone glutamate

Preclinical studies have demonstrated a considerable role for N-methyl-d-aspartate (NMDA) receptors in excitotoxicity and the concurrent neuroprotective effect of NMDA receptor antagonists. Because NMDA receptors are one of the most widespread receptors in the central nervous system, application of their antagonist often leads to serious side effects ranging from motor impairment to induction of schizophrenic-like psychosis. Therefore, we have initiated development and testing of a novel synthetic NMDA receptor antagonist derived from naturally occurring neurosteroids. 20-oxo-5β-pregnan-3α-yl-l-glutamyl-1-ester (3α5βP-Glu) is a novel synthetic steroidal inhibitor of the NMDA receptor. Our results show that 3α5βP-Glu preferentially inhibits tonically activated NMDA receptors, is able to cross the blood brain barrier, does not induce psychotomimetic symptoms (such as hyperlocomotion and sensorimotor gating deficit) and reduced an excitotoxic damage of brain tissue and subsequent behavioural impairment in rats. In particular, 3α5βP-Glu significantly ameliorated neuronal damage in the dentate gyrus and subiculum, and improved behavioural performance in active allothetic place avoidance tasks (AAPA, also known as the carousel maze) after bilateral NMDA-induced lesions to the hippocampi. These findings provide a possible new therapeutic approach for the treatment of diseases induced by NMDA receptor overactivation.

Stereoselectivity of sodium borohydride reduction of saturated steroidal ketones utilizing conditions of Luche reduction.

A series of keto steroids were reduced with sodium borohydride in the presence of cerium(III) chloride or samarium(III) iodide (Luche reduction). The ratios of axial and equatorial alcohols were determined by HPLC and the results were compared with those obtained by a standard sodium borohydride reduction. The best results were obtained with the 2-keto derivative 1, 7-keto derivatives 5 and 6, and 12-keto derivative 8 where the cerium(III) ion addition resulted in the inversion of the axial/equatorial ratios. The Luche reduction of the 20-keto derivative 11 improved the proportion of the (20S)-alcohol in a mixture of (20S)/(20R) alcohols up to 35% from 5% in a standard sodium borohydride reduction.

Synthesis of C3, C5, and C7 pregnane derivatives and their effect on NMDA receptor responses in cultured rat hippocampal neurons

The synthesis of several novel 5alpha- and 5beta-20-oxo-pregnane derivatives substituted in the position 3 and 7 of the steroid skeleton is described. Activity of synthesized compounds was studied in voltage-clamped cultured rat hippocampal neurons. Substituted derivatives inhibited NMDA-elicited neuronal activity. The relationship between biological activity and structure is discussed.

Effects of steroids on NMDA receptors and excitatory synaptic transmission in neonatal motoneurons in rat spinal cord slices.

The effect of steroids on NMDA receptors and excitatory postsynaptic transmission was studied in fluorescence‐labelled motoneurons in thin spinal cord slices. In outside‐out patches, NMDA‐induced responses were potentiated by 79% in the presence of 20‐oxopregn‐5‐en‐3β‐yl sulfate (PS), while in the presence of 20‐oxo‐5α‐pregnan‐3α‐yl sulfate (3α5αS) and 20‐oxo‐5β‐pregnan‐3α‐yl sulfate (3α5βS) they were diminished by 57% and 66%, respectively. PS and 3α5βS had no effect on the amplitude of single NMDA receptor channel openings, however, both compounds altered relative distribution of the openings to individual conductance levels. In control cases, the most frequent openings of the NMDA receptor channels were at the 70 pS conductance level, while in the presence of PS or 3α5βS, the most frequent openings were at the 55 pS conductance level. Analysis of the mean current transferred by NMDA receptor channel openings at individual conductance levels indicated that in the presence of PS, the mean current induced by 55 pS conductance openings was significantly increased. In the presence of 3α5βS, the mean currents induced by 55 pS and 70 pS conductance openings were significantly decreased. The amplitude of NMDA receptor‐mediated EPSCs was potentiated by 54% in the presence of PS and the deactivation kinetics slowed. Neither the amplitude nor the kinetics of NMDA receptor‐mediated EPSCs was significantly changed in the presence of 3α5βS. The results of our experiments indicate that neurosteroids affect NMDA receptors in motoneurons. The effect appears to be influenced by the receptor subunit composition.