Synthetic approaches for noncentrosymmetric molybdates.

The use of second-order Jahn-Teller active Mo (VI) centers and chiral organic amines is discussed as an approach to crystallographic noncentrosymmetry. Several series of reactions, conducted under mild hydrothermal conditions, were designed to probe important reaction variables. Correlations between reagent and solvent concentrations and the molybdate structure were investigated using composition space analysis, which allows for the isolation of specific reaction variables. The effects of amine structure variation were probed using multiple series of related amines, which consisted of either linear diamines or ethylenediamine derivatives. The addition of fluoride results in the loss of amine-based structural variations. Chiral organic amines were used to demonstrate the viability of using such components to control the three-dimensional symmetry in new materials. The synthesis, structure, and characterization of eight new organically templated polyoxomolybdates and polyoxofluoromolybdates are reported.

Single-channel analysis of a point mutation of a conserved serine residue in the S2 ligand-binding domain of the NR2A NMDA receptor subunit.

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 +/- 0.5 and 29.6 +/- 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 +/- 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling.

We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N-methyl-D-aspartate (NMDA) receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, (R,S)-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by methanethiosulfonate ethylammonium potentiation of agonist-evoked currents. In addition, none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. The modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors, as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why "charge conserving" mutations of two residues in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA is not an agonist at AMPA receptors.

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.