A regulatory domain (R1-R2) in the amino terminus of the N-methyl-D-aspartate receptor: effects of spermine, protons, and ifenprodil, and structural similarity to bacterial leucine/isoleucine/valine binding protein.

There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N) reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of N-methyl-D-aspartate channels such as Mg2+ block and Ba2+ permeability. Residues in this region presumably form part of the ifenprodil-binding site. To model this region of NR1 we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1(22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket.

The selectivity filter of the N-methyl-D-aspartate receptor: a tryptophan residue controls block and permeation of Mg2+.

A hallmark feature of N-methyl-D-aspartate (NMDA) receptors is their voltage-dependent block by extracellular Mg2+. The structural basis for Mg2+ block is not fully understood. Although asparagine residues in the pore-forming M2 regions of NR1 and NR2 subunits influence Mg2+ block, it has been speculated that additional residues are likely to be involved. Here, we report the unexpected finding that a tryptophan residue in the M2 region of NR2 subunits controls Mg2+ block. An NR2B(W607L) mutation abolished block and greatly increased permeation of extracellular Mg2+. A similar effect was seen with a mutation at the equivalent residue in NR2A but not with mutations at the equivalent residue or adjacent residues in NR1. In NR2B, mutations that changed NR2B(W607) to asparagine (W607N) or alanine (W607A) also greatly reduced Mg2+ block, whereas mutations that changed W607 to the aromatic residues tyrosine (W607Y) or phenylalanine (W607F) had little or no effect on Mg2+ block. Furthermore, the W607L, W607N, and W607A mutants, but not the W607Y and W607F mutants, decreased Ba2+ permeability of NMDA channels. Thus, residue NR2B(W607) may be involved in binding of divalent cations, in particular Mg2+, through a cation-pi interaction with the electron-rich aromatic ring of the tryptophan. We previously suggested that NR2B(W607) may contribute to the narrow constriction of the NMDA channel. A model is now proposed in which the M2 loop of NR2B is folded in such a way that NR2B(W607) is positioned at the narrow constriction, at a level similar to NR2B(N616) and NR1(N616), with these three residues forming a binding site for Mg2+.

Benzyl-polyamines: novel, potent N-methyl-D-aspartate receptor antagonists.

The effects of benzyl-polyamines were studied at recombinant N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus laevis oocytes. A number of mono-, di- and tri-benzyl polyamines, having benzyl substitutions on the terminal or central amino groups, inhibited responses of NR1/NR2 receptors in oocytes voltage-clamped at -70 mV. Among the most potent compounds was N1,N4, N8-tri-benzyl-spermidine (TB-3-4), which had an IC50 value of 0.2 microM. TB-3-4 was approximately 40-fold more potent at NR1/NR2A and NR1/NR2B receptors than at NR1/NR2C or NR1/NR2D receptors. Block by TB-3-4 was strongly voltage dependent. Using voltage ramps analyzed by the Woodhull model of voltage-dependent channel block, TB-3-4 was found to have a Kd(0) value of 5 microM and a zdelta value of 1.41 at NR1/NR2B channels, whereas the affinity of binding [Kd(0) = 250 microM] but not the degree of voltage-dependence (zdelta = 1.43) was much lower at NR1/NR2D channels. At a concentration of 10 microM, TB-3-4 had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors expressed from the GluR1 subunit, indicating that TB-3-4 is a selective NMDA antagonist. TB-3-4 did not permeate wild-type NMDA channels but could easily permeate channels containing an N616G mutation in the NR1 subunit. This mutation is presumed to increase the size of the narrowest constriction of the NMDA channel, thus allowing passage of TB-3-4. Benzyl-polyamines such as TB-3-4 represent a structurally novel class of NMDA receptor channel blockers.

Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons: role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 subunits.

N-Methyl-D-aspartate (NMDA) receptors are modulated by extracellular spermine and protons and are blocked in a voltage-dependent manner by spermine and polyamine derivatives such as N1-dansyl-spermine (N1-DnsSpm). The effects of mutations in the first and third transmembrane domains (M1 and M3) and the pore-forming loop (M2) of NMDA receptor subunits were studied. Surprisingly, some mutations in M2 and M3 of the NR1 subunit, including mutations at W608 and N616 in M2, reduced spermine stimulation and proton inhibition. These mutations may have long-range allosteric effects or may change spermine- and pH-dependent gating processes rather than directly affecting the binding sites for these modulators because spermine stimulation and proton inhibition are not voltage dependent and are thought to involve binding sites outside the pore-forming regions of the receptor. A number of mutations in M1-M3, including mutations at tryptophan and tyrosine residues near the extracellular sides of M1 and M3, reduced block by spermine and N1-DnsSpm. The effects of these mutants on channel block were characterized in detail by using N1-DnsSpm, which produces block but not stimulation of NMDA receptors. Block by N1-DnsSpm was studied by using voltage ramps analyzed with the Woodhull model of channel block. Mutations at W563 (in M1) and E621 (immediately after M2) in the NR1A subunit and at Y646 (in M3) and N616 (in the M2 loop) in the NR2B subunit reduced the affinity for N1-DnsSpm without affecting the voltage dependence of block. These residues may form part of a binding site for N1-DnsSpm. Mutation of a tryptophan residue at position W607 in the M2 region of NR2B greatly reduced block by N1-DnsSpm, and N1-DnsSpm could easily permeate channels containing this mutation. The results suggest that at least parts of the M1 and M3 segments contribute to the pore or vestibule of the NMDA channel and that a tryptophan in M2 (W607 in NR2B) may contribute to the narrow constriction of the pore.

Influence of extracellular pH on inhibition by ifenprodil at N-methyl-D-aspartate receptors in Xenopus oocytes.

Ifenprodil is an atypical N-methyl-D-aspartate (NMDA) receptor antagonist that selectively blocks receptors containing the NR2B subunit. It has been proposed that ifenprodil may act at a stimulatory polyamine site on NMDA receptors, although interactions between ifenprodil and polyamines are non-competitive. NMDA receptors are also inhibited by extracellular protons, and an interaction between protons and polyamine stimulation has been described. Using voltage-clamp recording of recombinant NR1/NR2B receptors expressed in oocytes, ifenprodil inhibition was found to be pH sensitive with a smaller inhibition at alkaline pH. Similar effects of pH were seen on inhibition by nylidrin, eliprodil, and haloperidol, which are thought to act at the ifenprodil binding site. The pH sensitivity of ifenprodil block occurs at NR1B/NR2B as well as NR1A/NR2B receptors, suggesting that it is not influenced by the exon-5 insert that is present in NR1B but absent in NR1A. Protons may directly affect the ifenprodil binding site or may alter the coupling of ifenprodil binding to inhibition of channel gating.

A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.