The role of histidines in neurosteroid binding of NMDA NR2B and D subunits

Implicit in cellular mechanisms essential for learning and memory, NMDA receptors are members of the family of ionotropic receptors. Activation of these receptors through the binding of glutamate results in the opening of ion channels, triggering an excitatory pathway which can be moderated by certain endogenous and exogenous compounds. Previous research with the endogenous neurosteroids pregnenolone sulfate (PS) and 3‐α‐hydroxy‐5‐β‐20‐ one sulfate (PregS) have indicated that the binding of these neurosteroids to NMDA receptors containing the NR2C and NR2D isoforms of the NR2 subunit decreases current flow through NMDA receptors. Binding of PS to NMDA receptors containing NR2A or NR2B subunits causes an increased amount of current flow through the channel, whereas binding of PregS to these subunits decreases the current flow through these channel. Binding studies from our lab have determined that the neurosteroids PS and PregS bind to the amino terminal domain (ATD) of both NR2B and NR2D subunits. Binding of both neurosteroids was monitored between pH 6.0 and 8.0 in 0.5 pH increments. Results demonstrated that the binding of PS to NR2B and D ATDS occurred only between pH 6 and 6.5. PregS showed a similar pH dependence for the NR2B ATD, although binding was restored at pH 8. However, binding of PregS to the NR2D ATD occurred over a wider range, from pH 6–8 with the exception of pH 7.5. The pH dependence of neurosteroid binding suggests the importance of a protonated histidine residue in each binding interaction. The NR2B ATD domain (residues 121–292) contains one histidine (residue 127), whereas the similar NR2D ATD domain contains 3 histidines (residues 143, 182, 202). Results from His to Ala and Gln mutations and chemical modification studies will be presented.