Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine

Background and purpose: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. Experimental approach: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. Key results: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N + 1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N + 1) sites but not at the NR1(N0) site significantly reduced Mg2+ block. Conclusion and implications: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.

Selective loss of NMDA receptor NR1 subunit isoforms in Alzheimer's disease

Previous work had shown that the ratio of NMDA receptor NR1 subunit mRNA transcripts containing an N-terminal splice cassette to those that do not is markedly lower in regions of the Alzheimer's disease (AD) brain that are susceptible to pathological damage, compared with spared regions in the same cases or homotropic regions in controls. To elucidate the origins of this difference in proportionate expression, we measured the absolute levels of each of the eight NR1 transcripts by quantitative internally standardized RT-PCR assay. Expression of transcripts with the cassette was strongly attenuated in susceptible regions of Alzheimer's brain, whereas expression of non-cassette transcripts differed little from that in controls. The expression of other NR1 splice variants was not associated with pathology relevant to disease status, although some combinations of splice cassettes were well maintained in AD cases. The population profile of NR1 transcripts in occipital cortex differed from the profiles in other brain regions studied. Western analysis confirmed that the expression of protein isoforms containing the N-terminal peptide was very low in susceptible areas of the Alzheimer's brain. Cells that express NR1 subunits with the N-terminal cassette may be selectively vulnerable to toxicity in AD.

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer ( FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine

1 The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. 2 In the absence of external Mg2+ ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination. 3 The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per similar to20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. 4 The inhibition of the open NMDA receptor by external Mg2+ and 5-HT was not additive, suggesting competition between Mg2+ and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg2+. 5 The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT = 5-methyltryptamine > tryptamine > 7-methyltryptamine > 5-HTmuch greater than tryptophan melatonin. 6 Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration.

Ethanol prevents NMDA receptor reduction by maternal separation in neonatal rat hippocampus

We measured the effects of ethanol on glutamate receptor levels in the hippocampus of neonatal Wistar rats using a vapor chamber model. Two control groups were used; a normal suckle group and a maternal separation group. Levels of NMDA receptors were not significantly altered in ethanol-treated animals compared to the normal suckle control group, as shown by [H-3]MK-801 binding and Western blot analysis. However, MK-801 binding and NR1 subunit immunoreactivity were greatly reduced in the hippocampus of separation control animals. Neither ethanol treatment nor maternal separation altered levels of GluR1 or GluR2(4). These results have serious implications for the importance of maternal contact for normal brain development.

NMDA receptor dysfunction in Alzheimer's disease

The inherent neurotoxic potential ofthe endogenous excitatory amino acid glutamate, may be causally related to the pathogenesis ofAD neurodegeneration disorders. Neuronal excitotoxicity is conceivably mediated by the N-methyl-D-aspartate-(NMDA)-Ca2+- ionotropic receptor. NMDA receptors exist as multimeric complexes comprising proteins from two families – NR1 and NR2(A-D). The polyamines, spermine and spermidine bind to, and modulate NMDA receptor efficacy via interaction with exon 5, an alternatively-spliced, 21 amino acid, N-terminal cassette. AD associated cognitive impairment may therefore occur via subunitspecific NMDA receptor dysfunction effecting regional selectivity of neuronal degradation.

NMDA receptor variant responses to conantokins

Excitotoxicity may have role in neuronal death in many disorders including Alzheimer disease. Sensitivity of a cell to excitotoxicity may depend on its subtype of NMDA receptors. A drug that selectively reduced such overstimulation could limit susceptibility to damage. We examined the pharmacology of NMDA receptor subtypes in response to the agonists glutamate and glycine, the modulator spermine, and the antagonists conantokin-G and its Ala(7) analogue in Xenopus oo¨ cytes. Cells were injected with capped RNA coding for NMDA NR1 and NR2 subunits. Membrane currents induced by rapid application of agonists were recorded under two-electrode voltageclamp. Conantokins were bath-applied to give cumulative concentration responses. Spermine gave slightly different shifts in glutamate affinity when different NR1 splice variants were combined with NR2A subunits. In the presence of spermine, both an increase and a decrease in affinity for glutamate were seen with differing subunit combinations that could not be explained by the absence or presence of the N-terminal 23-amino-acid insert.

NMDA receptor dysfunction in Alzheimer's disease

The inherent neurotoxic potential ofthe endogenous excitatory amino acid glutamate, may be causally related to the pathogenesis ofAD neurodegeneration disorders. Neuronal excitotoxicity is conceivably mediated by the N-methyl-D-aspartate-(NMDA)-Ca2+- ionotropic receptor. NMDA receptors exist as multimeric complexes comprising proteins from two families – NR1 and NR2(A-D). The polyamines, spermine and spermidine bind to, and modulate NMDA receptor efficacy via interaction with exon 5, an alternatively-spliced, 21 amino acid, N-terminal cassette. ADassociated cognitive impairment may therefore occur via subunitspecific NMDA receptor dysfunction effecting regional selectivity ofneuronal degradation. Total RNA was prepared from pathologically spared and susceptible regions from AD cases and matched controls. Quantitation was performed using standard curve methodology in which a known amount ofa synthetic ribonucleic acid competitor deletion construct was co-amplified against total RNA. Expression profile analysis oftwo NR1 mRNA subsets has revealed significant differences in NR11XX mRNA levels in cingulate gyrus, P.