The NR1 M3 domain mediates allosteric coupling in the N-methyl-D-aspartate receptor.

N-Methyl-D-aspartate (NMDA) receptors play a critical role in both development of the central nervous system and adult neuroplasticity. However, although the NMDA receptor presents a valuable therapeutic target, the relationship between its structure and functional properties has yet to be fully elucidated. To further explore the mechanism of receptor activation, we characterized two gain-of-function mutations within the NR1 M3 segment, a transmembrane domain proposed to couple ligand binding and channel opening. Both mutants (A7Q and A7Y) displayed significant glycine-independent currents, indicating that their M3 domains may preferentially adopt a more activated conformation. Substituted cysteine modification experiments revealed that the glycine binding clefts of both A7Q and A7Y are inaccessible to modifying reagents and resistant to competitive antagonism. These data suggest that perturbation of M3 can stabilize the ligand binding domain in a closed cleft conformation, even in the absence of agonist. Both mutants also displayed significant glutamate-independent current and insensitivity to glutamate-site antagonism, indicating partial activation by either glycine or glutamate alone. Furthermore, A7Q and A7Y increased accessibility of the NR2 M3 domain, providing evidence for intersubunit coupling at the transmembrane level and suggesting that these NR1 mutations dominate the properties of the intact heteromeric receptor. The equivalent mutations in NR2 did not exhibit comparable phenotypes, indicating that the NR1 and NR2 M3 domains may play different functional roles. In summary, our data demonstrate that the NR1 M3 segment is functionally coupled to key structural domains in both the NR1 and NR2 subunits.

Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds.

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.

Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel.

K+ channels achieve exquisite ion selectivity without jeopardizing efficient permeation by employing multiple, interacting K+-binding sites. Introduction ofa cadmium (Cd2+)-binding site in the external vestibule of Kv2.1 (drk1), allowed us to functionally characterize a binding site for external monovalent cations. Permeant ions displayed higher affinity for this site than non-permeant monovalent cations, although the selectivity profile was different from that of the channel. Point mutations identified the highly conserved aspartate residue immediately following the selectivity filter as a critical determinant of the antagonism between external K+ and Cd2+ ions. A conservative mutation at this position (D378E) significantly affected the open-state stability. Moreover, the mean open time was found to be modulated by external K+ concentration, suggesting a coupling between channel closing and the permeation process. Reducing the Rb+ conductance by mutating the selectivity filter to the sequence found inKv4.1, also significantly reduced the effectiveness ofRb+ ions to antagonize Cd2+ inhibition, thereby implicating the selectivity filter as the site at which K+ions exert their antagonistic effect on Cd2+ block. The equivalent of D378 in KcsA, D80, takes part in an inter-subunit hydrogen-bond network that allows D80to functionally interact with the selectivity filter. The results suggest that external K+ ions antagonize Cd2+inhibition (in I379C) and modulate the mean open time(in the wild-type Kv2.1) by altering the occupancy profile of the K+-binding sites in the selectivity filter.