Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors.
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.
Chen
Philip E
PE
Division of Neuroscience, University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK.
Johnston
Alexander R
AR
Mok
M H Selina
MH
Schoepfer
Ralf
R
Wyllie
David J A
DJ
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D018698:Glutamic Acid / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000235:genetics* / Q000378:metabolism*; D013912:Threonine / Q000235:genetics; D014982:Xenopus laevis
D008024:Ligands; C473649:NR1A NMDA receptor, rat; C464436:NR2D NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D013912:Threonine; D018698:Glutamic Acid