Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors

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Abstract
NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single‐channel properties examined using two‐electrode voltage‐clamp and patch‐clamp recording techniques. Glutamate dose–response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600‐fold reduction in glutamate potency compared to wild‐type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild‐type or mutant receptors. The mutation did not affect the potency of the co‐agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate‐site agonists such as aspartate or NMDA were 30‐ to 60‐fold compared to wild‐type. Single‐channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild‐type NR2D‐containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D‐containing NMDA receptors. Rapid applications of glutamate on outside‐out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60‐fold faster than wild‐type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.