Channel Blockers Acting atN-Methyl-d-aspartate Receptors: Differential Effects of Mutations in the Vestibule and Ion Channel Pore

Abstract

A large number of structurally diverse compounds act as openchannel blockers of NMDA receptors. They may share discrete or overlapping binding sites within the channel. In this study, the effects of mutations in and around the membrane-spanning and pore-forming regions of NMDA receptor subunits were studied with three blockers, MK-801, memantine, and TB-3-4, using recombinant NMDA receptors expressed in Xenopus laevis oocytes. Mutations at the critical asparagine residues in the M2 loop of NR1 and NR2B and at a tryptophan residue in M2 of NR2B reduced block by MK-801, memantine, and TB-3-4. Mutations at residues in the pre-M1, M1, M3, post-M3, and post-M4 regions had differential effects on the three blockers. Many mutations in these regions reduced block by MK-801 and TB-3-4 but had no effect on block by memantine. The differential effects on block by memantine and MK-801 are unlikely to be caused by differences in the size of these blockers. Benzyl rings in MK-801 and TB-3-4 may make hydrophobic interactions with aromatic and hydrophobic amino acid residues in the pore. Some mutations in the pre-M1 and M3 regions generated constitutively open channels, characterized by large holding currents. The effects of the various mutants are discussed in the context of models based on the known structure of the pore of the KcsA potassium channel and on previous studies dealing with solvent accessible residues in NMDA receptor subunits as determined by modification after cysteine mutagenesis.

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors

“…1A and B, 13A and B). Consistent with previous reports (Mori et al, 1992;Sakurada et al, 1993;Kawajiri and Dingledine, 1993;Ferrer-Montiel et al, 1995;Kashiwagi et al, 2002), we found that both the N616Q and N616R mutants decreased the affinity for MK-801. In sharp contrast, the N616 mutations did not affect the binding affinity of [ 3 H]DX suggesting that this ligand does not derive binding energy from the interaction with N616.…”

“…Disruptive mutation of N650, a putative residue of the extracellular vestibule, reduced the affinity for both [ 3 H]MK-801 and [ 3 H]DX approximately four-to fivefold. Consistent with our results, a shift of the MK-801 IC 50 value from 2 nM to 13 nM for blockade of glutamate-induced currents has been reported for NR1-1a (N650A)/NR2B receptors (Kashiwagi et al, 2002). Since substitution at N650 does not entirely eliminate the binding of either [ 3 H]MK-801 or [ 3 H]DX, it is reasonable to propose that the residue is either a site of interaction for both ligands or confers topology to the pore/vestibule region that influences the affinity of both ligands.…”

“…The A645S mutation reduced the affinity of [ 3 H]MK-801 by approximately sixfold. This reduction of high-affinity binding of [ 3 H]MK-801 is comparable to the substantial reduction of the MK-801 blockade of inward currents induced by glutamate reported for NR1-1a (A645S)/NR2B receptors (Kashiwagi et al, 2002). Sequence and SCAM analysis has predicted that A645 is part of the α-helical region of M3 with this amino acid associated with the interior of the protein (Sprengel et al, 2001;Beck et al, 1999).…”

“…It has been demonstrated that W611 is essential to high-affinity PCP binding, yet has no corresponding effect on that of MK-801 (Ferrer-Montiel et al, 1995). Kashiwagi et al (2002) demonstrated the insensitivity of MK-801, memantine and Mg 2+ blockade to a disruptive mutation of W611. While W611 is not directly involved in MK-801 binding, it may contribute to the overall structure of the pore via an effect on subunit interaction that is essential for PCP binding (Ferrer-Montiel et al, 1995).…”

“…Kashiwagi et al (2002) has reported differential effects of mutations on the blockade of three open-channel blockers of the NMDAR. Evaluation of residues associated with the M2 region of the NMDAR revealed that mutation of the critical asparagine (N616) reduced the block of memantine, MK-801, N 1 -N 4 -N 8 tribenzylspermidine and Mg 2+ (Kashiwagi et al, 2002). Conversely, many mutations in the pre-M1, M1, M3 and post-M3 regions had little or no effect on the block by memantine, while reducing that of MK-801 and TB-3-4 (Kashiwagi et al, 2002).…”