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1 The neurosteroid pregnenolone sulphate (PS) potentiates N-methyl-D-aspartate (NMDA) receptor mediated responses in various neuronal preparations. The NR1 subunit can combine with NR2A, NR2B, NR2C, or NR2D subunits to form functional receptors. Dierential NR2 subunit expression in brain and during development raises the question of how the NR2 subunit in¯uences NMDA receptor modulation by neuroactive steroids. 2 We examined the eects of PS on the four diheteromeric NMDA receptor subtypes generated by co-expressing the NR1 100 subunit with each of the four NR2 subunits in Xenopus oocytes. Whereas PS potentiated NMDA-, glutamate-, and glycine-induced currents of NR1/NR2A and NR1/NR2B receptors, it was inhibitory at NR1/NR2C and NR1/NR2D receptors. 3 In contrast, pregnanolone sulphate (3a5bS), a negative modulator of the NMDA receptor that acts at a distinct site from PS, inhibited all four subtypes, but was approximately 4 fold more potent at NR1/NR2C and NR1/NR2D than at NR1/NR2A and NR1/NR2B receptors. 4 These ®ndings demonstrate that residues on the NR2 subunit are key determinants of modulation by PS and 3a5bS. The modulatory eects of PS, but not 3a5bS, on dose-response curves for NMDA, glutamate, and glycine are consistent with a two-state model in which PS either stabilizes or destabilizes the active state of the receptor, depending upon which NR2 subunit is present. 5 The selectivity of sulphated steroid modulators for NMDA receptors of speci®c subunit composition is consistent with a neuromodulatory role for endogenous sulphated steroids. The results indicate that it may be possible to develop therapeutic agents that target steroid modulatory sites of speci®c NMDA receptor subtypes. British Journal of Pharmacology (2002) 135, 901 ± 909

Steroid sulfation occurs in nervous tissue and endogenous sulfated steroids can act as positive or negative modulators of N-methyl-D-aspartate (NMDA) receptor function. In the current study, structure-activity relationships for sulfated steroids were examined in voltage-clamped chick spinal cord and rat hippocampal neurons in culture and in Xenopus laevis oocytes expressing NR1(100) and NR2A subunits. The ability of pregnenolone sulfate (a positive modulator) and epipregnanolone sulfate (a negative modulator) to compete with each another, as well as with other known classes of NMDA receptor modulators, was examined. The results show that steroid positive and negative modulators act at specific, extracellularly directed sites that are distinct from one another and from the spermine, redox, glycine, Mg2+, MK-801, and arachidonic acid sites. Sulfated steroids are effective as modulators of ongoing glutamate-mediated synaptic transmission, which is consistent with their possible role as endogenous neuromodulators in the CNS.

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Intracellular Ca2+ (Ca(i)) signaling following the binding of surface receptors activates a Ca2+ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pHo) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca(2+)-stores including: (i) dialysis of the cell with 100 microM inositol 1,4,5-triphosphate (IP3), (ii) intracellular dialysis with high concentrations of the Ca2+ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca(2+)-ATPase inhibitor thapsigargin (1 microM). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca2+ selective with a selectivity sequence of Ca2+ > Sr2+ >> Mg2+ = Mn2+ = Ni2+. The Ca2+ influx current was modulated by changes in pHo; modulation was not produced as a consequence of changes in internal pH (pHi). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pHo alone failed to induce current activation. These observations are consistent with a scheme by which changes in pHo, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Cai transient associated with receptor activation by regulating the influx of Ca2+ ions.