Identification of novel gating mechanisms underlying pharmacological potentiation of NMDA receptors
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Authors
Chopra, Divyan A.
Issue Date
2015-07-31
Volume
Issue
Type
Thesis
Language
en_US
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Abstract
Excitatory neurotransmission mediated by N-methyl-D-aspartate receptors (NMDARs) is known to play an important role in synaptic plasticity, learning and memory. Moreover, NMDAR dysfunction may contribute to a variety of neuropsychiatric and neurological disorders including schizophrenia, epilepsy, stroke and trauma. Pregnenolone sulfate (PS) is one of the most commonly occurring neurosteroids in the central nervous system and influences function of several receptors. PS modulates 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 HEK 293 cells using whole-cell and single-channel electrophysiology. Only a modest effect on the whole-cell responses was observed by PS in dialyzed (non-perforated) whole-cell recordings. Interestingly, in perforated conditions, PS was found to increase the whole-cell currents in the absence of nominal extracellular Ca2+ whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca2+. The Ca2+-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca2+-dependent bidirectional effect of PS. Single-channel cell-attached analysis demonstrated that PS primarily affected the mean open time to produce its effects, with positive modulation mediated by an increase in duration of open time constants while 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. UBP (University of Bristol Pharmaceuticals) series compounds act at allosteric binding sites on the NMDARs and have been able to achieve subtype-selectivity. We found that UBP684 significantly increased the whole-cell current and mean open time and reduced the mean shut time for GluN1/GluN2A receptors. Mutagenesis studies suggest that UBP684 can stabilize the closed cleft of the LBD and drive the receptor into the more stable open states.
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Publisher
Creighton University
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Copyright is retained by the Author.
A non-exclusive distribution right is granted to Creighton University with the understanding that access to this work is restricted to Creighton University faculty, staff and students. Furthermore, it will not be submitted to ProQuest.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.