Abstract
Glutamate receptors play a major role in neural cell plasticity, growth, and maturation. The degree to which ionotropic glutamate receptors (iGluR) conduct current is dependent on binding of extracellular ligands, of which glutamate is the native agonist. Although the glutamate binding site of the GluR2 class of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) iGluR has been structurally characterized, the allosteric sites attributed to neurosteroid binding have yet to be localized. Here, using intrinsic tryptophan fluorescence spectroscopy, we show that the extracellular glutamate binding core of the GluR2 class of AMPA receptors also binds to two neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate, both of which negatively modulate its activity. Interest in these sulfated neurosteroids stems from their differential modulation of other members of the iGluR family and their potential use as endogeneous agents for stroke therapy. In particular, whereas PS inhibits AMPA and other non-N-methyl-d-aspartate (NMDA) family members, it activates the NMDA receptor. In addition to providing evidence for binding of these neurosteroids to the glutamate binding core of the GluR2 class of AMPA receptors, our data suggests that both neurosteroids bind in a similar manner, consistent with their modulation of activity of this class of iGluR. Interestingly, the conformational change induced upon binding of these neurosteroids is distinct from that induced upon glutamate binding.
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Abbreviations
- AMPA:
-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
- CD:
-
circular dichroism
- HPLC-SEC:
-
HPLC size-exclusion chromatography
- iGluR:
-
ionotropic glutamate receptors
- LB:
-
Luria broth
- NMDA:
-
N-methyl-d-aspartate
- PregaS:
-
3α-hydroxy-5β-pregnan-20-one sulfate
- PS:
-
pregnenolone sulfate
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Spivak, V., Lin, A., Beebe, P. et al. Identification of a neurosteroid binding site contained within the GluR2-S1S2 domain. Lipids 39, 811–819 (2004). https://doi.org/10.1007/s11745-004-1301-8
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DOI: https://doi.org/10.1007/s11745-004-1301-8