A Novel Alcohol-Sensitive Site in the M3 Domain of the NMDA Receptor GluN2A Subunit

Accumulating studies have demonstrated that the N-methyl-D-aspartate receptor is one of the most important targets of ethanol in the central nervous system. Previous studies from this laboratory have found that one position in the third (F637) and two positions in the fourth (M823 and A825) membrane-associated (M) domains of the N-methyl-D-aspartate receptor GluN2A subunit modulate alcohol action and ion channel gating. Using site-directed mutagenesis and whole-cell patch-clamp recording, we have found an additional position in M3 of the GluN2A subunit, F636, which significantly influences ethanol sensitivity and functionally interacts with F637. Tryptophan substitution at F636 significantly decreased the ethanol IC50, decreased both peak and steady-state glutamate EC50, and altered agonist deactivation and apparent desensitization. There was a significant correlation between steadystate: peak current ratio, a measure of desensitization, and ethanol IC50 values for a series of mutants at this site, raising the possibility that changes in ethanol sensitivity may be secondary to changes in desensitization. Mutant cycle analysis revealed a significant interaction between F636 and F637 in regulating ethanol sensitivity. Our results suggest that F636 in the M3 domain of the GluN2A subunit not only influences channel gating and agonist potency, but also plays an important role in mediating the action of ethanol. These studies were supported by grants R01 AA015203-01A1 and AA015203-06A1 from the NIAAA to R.W.P

A Site of Alcohol Action at the NMDA Receptor M3-M4 Domain Interface

The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The structural model of the NMDA receptor, predicted from the structure of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We investigated possible interactions between the M3 and M4 domain positions of the two subunit types affecting the ethanol sensitivity of the receptor by using dual substitution mutants. In an initial screen of single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(G638) and GluN2A(F636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition were observed at four pairs of positions in GluN1/GluN2A: G638/M823, F639/L824, M818/F636, and L819/F637. Two of these interactions involve a position in M4 of both subunits, GluN1(M818) and GluN2A(L824), that does not by itself alter ethanol sensitivity, and one of the previously identified positions affecting ethanol sensitivity, GluN2A(A825), did not appear to interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, and support the existence of four sites of alcohol action on the NMDA receptor at the M3-M4 domain intersubunit interfaces. These studies were supported by grants R01 AA015203-01A1 and AA015203-06A1 from the NIAAA to R.W.P

Interactions Among Positions in the Third and Fourth Membrane-Associated Domains at the Intersubunit Interface of the N-Methyl-D-Aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-d-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces

β-N-methylamino-L-alanine Enhances Neurotoxicity Through Multiple Mechanisms

The idea that the environmental toxin β-N-methylamino-l-alanine (BMAA) is involved in neurodegenerative diseases on Guam has risen and fallen over the years. The theory has gained greater interest with recent reports that BMAA is biomagnified, is widely distributed around the planet, and is present in the brains of Alzheimer\u27s patients in Canada. We provide two important new findings. First, we show that BMAA at concentrations as low as 10 μM can potentiate neuronal injury induced by other insults. This is the first evidence that BMAA at concentrations below the mM range can enhance death of cortical neurons and illustrates potential synergistic effects of environmental toxins with underlying neurological conditions. Second, we show that the mechanism of BMAA toxicity is threefold: it is an agonist for NMDA and mGluR5 receptors, and induces oxidative stress. The results provide further support for the hypothesis that BMAA plays a role in neurodegenerative diseases

Role of Membrane GM1 on Early Neuronal Membrane Actions of Aβ During Onset of Alzheimer\u27s Disease

The ability of beta-amyloid peptide (Aβ) to disrupt the plasma membrane through formation of pores and membrane breakage has been previously described. However, the molecular determinants for these effects are largely unknown. In this study, we examined if the association and subsequent membrane perforation induced by Aβ was dependent on GM1levels. Pretreatment of hippocampal neurons with D-PDMP decreased GM1 and Aβ clustering at the membrane (Aβ fluorescent-punctas/20 μm, control = 16.2 ± 1.1 vs. D-PDMP = 6.4 ± 0.4, p \u3c 0.001). Interestingly, membrane perforation with Aβ occurred with a slower time course when the GM1 content was diminished (time to establish perforated configuration (TEPC) (min): control = 7.8 ± 2 vs. low GM1 = 12.1 ± 0.5, p \u3c 0.01), suggesting that the presence of GM1 in the membrane can modulate the distribution and the membrane perforation by Aβ. On the other hand, increasing GM1 facilitated the membrane perforation (TEPC: control = 7.8 ± 2 vs. GM1 = 6.2 ± 1 min, p \u3c 0.05). Additionally, using Cholera Toxin Subunit-B (CTB) to block the interaction of Aβ with GM1 attenuated membrane perforation significantly. Furthermore, pretreatment with CTB decreased the membrane association of Aβ (fluorescent-punctas/20 μm, Aβ: control = 14.8 ± 2.5 vs. CTB = 8 ± 1.4, p \u3c 0.05), suggesting that GM1 also plays a role in both association of Aβ with the membrane and in perforation. In addition, blockade of the Aβ association with CTB inhibited synaptotoxicity. Taken together, our results strongly suggest that membrane lipid composition can affect the ability of Aβ to associate and subsequently perforate the plasma membrane thereby modulating its neurotoxicity in hippocampal neurons

Two Adjacent Phenylalanines In the NMDA Receptor GluN2A Subunit M3 Domain Interactively Regulate Alcohol Sensitivity and Ion Channel Gating

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity

Differential Actions of Ethanol and Trichloroethanol at Sites in the M3 and M4 Domains of the NMDA Receptor GluN2A (NR2A) Subunit

Background and purpose:  Alcohol produces its behavioural effects in part due to inhibition of N-methyl-d-aspartate (NMDA) receptors in the CNS. Previous studies have identified amino acid residues in membrane-associated domains 3 (M3) and 4 (M4) of the NMDA receptor that influence ethanol sensitivity. In addition, in other alcohol-sensitive ion channels, sedative-hypnotic agents have in some cases been shown to act at sites distinct from the sites of ethanol action. In this study, we compared the influence of mutations at these sites on sensitivity to ethanol and trichloroethanol, a sedative-hypnotic agent that is a structural analogue of ethanol. Experimental approach:  We constructed panels of mutants at ethanol-sensitive positions in the GluN2A (NR2A) NMDA receptor subunit and transiently expressed these mutants in human embryonic kidney 293 cells. We used whole-cell patch-clamp recording to assess the actions of ethanol and trichloroethanol in these mutant NMDA receptors. Key results:  Ethanol sensitivity of mutants at GluN2A(Ala825) was not correlated with any physicochemical measures tested. Trichloroethanol sensitivity was altered in two of three ethanol-insensitive mutant GluN2A subunits: GluN2A(Phe637Trp) in M3 and GluN2A(Ala825Trp) in M4, but not GluN2A(Met823Trp). Trichloroethanol sensitivity decreased with increasing molecular volume at Phe637 or increasing hydrophobicity at Ala825 and was correlated with ethanol sensitivity at both sites. Conclusions and implications:  Evidence obtained to date is consistent with a role of GluN2A(Ala825) as a modulatory site for ethanol and trichloroethanol sensitivity, but not as a binding site. Trichloroethanol appears to inhibit the NMDA receptor in a manner similar, but not identical to, that of ethanol

The Level of NMDA Receptor in the Membrane Modulates Amyloid-β Association and Perforation

Alzheimer’s disease is a neurodegenerative disorder that affects mostly the elderly. The main histopathological markers are the senile plaques formed by amyloid-β peptide (Aβ) aggregates that can perforate the plasma membrane of cells, increasing the intracellular calcium levels and releasing synaptic vesicles that finally lead to a delayed synaptic failure. Several membrane proteins and lipids interact with Aβ affecting its toxicity in neurons. Here, we focus on NMDA receptors (NMDARs) as proteins that could be modulating the association and neurotoxic perforation induced by Aβ on the plasma membrane. In fact, our results showed that decreasing NMDARs, using enzymatic or siRNA approaches, increased the association of Aβ to the neurons. Furthermore, overexpression of NMDARs also resulted in an enhanced association between NMDA and Aβ. Functionally, the reduction in membrane NMDARs augmented the process of membrane perforation. On the other hand, overexpressing NMDARs had a protective effect because Aβ was now unable to cause membrane perforation, suggesting a complex relationship between Aβ and NMDARs. Because previous studies have recognized that Aβ oligomers are able to increase membrane permeability and produce amyloid pores, the present study supports the conclusion that NMDARs play a critical protective role on Aβ actions in hippocampal neurons. These results could explain the lack of correlation between brain Aβ burden and clinically observed dementia

Positions in the N‐methyl‐D‐aspartate Receptor GluN2C Subunit M3 and M4 Domains Regulate Alcohol Sensitivity and Receptor Kinetics

Background Alcohol alters synaptic transmission in the brain. The N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR), a subtype of glutamate‐gated ion channel, is an important synaptic target of alcohol in the brain. We and others have previously identified 4 alcohol‐sensitive positions in the third and fourth membrane‐associated (M) domains, designated M31‐2and M41‐2, of the GluN1, GluN2A, and GluN2B NMDAR subunits. In the present study, we tested whether the corresponding positions in the GluN2C subunit also regulate alcohol sensitivity and ion channel gating. Methods We performed alanine‐ and tryptophan‐scanning mutagenesis in the GluN2C subunit followed by expression in HEK 293 cells and electrophysiological patch‐clamp recording. Results Alanine substitution at the M31 (F634) and M41‐2 (M821 and M823) positions did not alter ethanol (EtOH) sensitivity, whereas substitution of alanine at the M32 position (F635) yielded nonfunctional receptors. Tryptophan substitution at the M31‐2 positions did not change EtOH sensitivity, whereas tryptophan substitution at the M41 position increased, and at the M42 position decreased, EtOH sensitivity. The increased EtOH sensitivity of the tryptophan mutant at M41 is in marked contrast to previous results observed in the GluN2A and GluN2B subunits. In addition, this mutant exhibited increased desensitization, but to a much lesser extent compared to the corresponding mutations in GluN2A and GluN2B. A series of mutations at M41 altered EtOH sensitivity, glutamate potency, and desensitization. Seven amino acid substitutions (of 15 tested) at this position yielded nonfunctional receptors. Among the remaining mutants at M41, EtOH sensitivity was not significantly correlated with hydrophobicity, molecular volume, or polarity of the substituent, or with glutamate EC50 values, but was correlated with maximal steady‐state‐to‐peak current ratio, a measure of desensitization. Conclusions The identity and characteristics of alcohol‐sensitive positions in the GluN2C subunit differ from those previously reported for GluN2A and GluN2B subunits, despite the high homology among these subunits