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's 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 three fold: 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.

The mechanisms that induce Alzheimer's disease (AD) are largely unknown thereby deterring the development of disease-modifying therapies. One working hypothesis of AD is that Aβ excess disrupts membranes causing pore formation leading to alterations in ionic homeostasis. However, it is largely unknown if this also occurs in native brain neuronal membranes. Here we show that similar to other pore forming toxins, Aβ induces perforation of neuronal membranes causing an increase in membrane conductance, intracellular calcium and ethidium bromide influx. These data reveal that the target of Aβ is not another membrane protein, but that Aβ itself is the cellular target thereby explaining the failure of current therapies to interfere with the course of AD. We propose that this novel effect of Aβ could be useful for the discovery of anti AD drugs capable of blocking these “Aβ perforates”. In addition, we demonstrate that peptides that block Aβ neurotoxicity also slow or prevent the membrane-perforating action of Aβ.

The current understanding about ethanol effects on the ligand-gated ion channel (LGIC) superfamily has been restricted to identify potential binding sites within transmembrane (TM) domains in the Cys-loop family. Here, we demonstrate a key role of the TM3-4 intracellular loop and G␤␥ signaling for potentiation of glycine receptors (GlyRs) by ethanol. We discovered 2 motifs within the large intracellular loop of the GlyR ␣1 subunit that are critical for the actions of pharmacological concentrations of ethanol. Significantly, the sites were ethanol-specific because they did not alter the sensitivity to general anesthetics, neurosteroids, or longer n-alcohols. Furthermore, G␤␥ scavengers selectively attenuated the ethanol effects on recombinant and native neuronal GlyRs. These results show a selective mechanism for low-ethanol concentration effects on the GlyR and provide a mechanism on ethanol pharmacology, which may be applicable to other LGIC members. Moreover, these data provide an opportunity to develop new genetically modified animal models and novel drugs to treat alcohol-related medical concerns.pharmacology ͉ signal transduction ͉ glycine receptor ͉ alcoholism ͉ G proteins

Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Although ethanol is known to inhibit NMDA receptors by influencing ion-channel gating, its molecular site of action and the mechanism underlying this effect have not been established. We have previously identified a conserved methionine residue in the fourth membrane-associated domain of the NMDA receptor NR2A subunit (Met 823 ) that influences desensitization and gating of the ion channel. Here we report that this residue plays an important role in mediating the effect of ethanol on the NMDA receptor. Ethanol IC 50 values among functional substitution mutants at this position varied over the range ϳ130 -225 mM. There was a weak correlation between ethanol IC 50 and mean open time of NR2A(Met 823 ) mutants that was dependent on inclusion of the value for the tryptophan mutant. In the absence of this value, there was no trend toward a correlation among the remaining mutants. Desensitization appeared to influence the action of ethanol, because ethanol IC 50 of the mutants was correlated with the steadystate to peak current ratio. With the exception of tryptophan, ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent. The relation between ethanol sensitivity and the molecular volume and hydrophobicity at this position suggests that this residue interacts with or forms part of a site of ethanol action and that the presence of a tryptophan residue in this site disrupts its ability to interact with ethanol.Ethyl alcohol or ethanol, one of the oldest and most widely abused drugs, produces a well known spectrum of behavioral effects primarily through actions on ion channels in the nervous system. The N-methyl-D-aspartate (NMDA) 1 receptor, a subtype of receptor for the major excitatory neurotransmitter glutamate, is thought to be of particular importance in mediating the effects of alcohols in the mammalian brain. Ethanol inhibits NMDA-activated current in central neurons (1, 2) as well as NMDA-evoked Ca 2ϩ flux, cyclic GMP production, and neurotransmitter release (3-6). Studies using in vivo electrophysiological techniques have also reported ethanol inhibition of NMDA receptors at relevant concentrations (7). Ethanol appears to interact with a novel allosteric site, which is independent of the recognition site for the agonist glutamate or coagonist glycine (6, 8 -12), and reduces the mean open time and opening frequency of the channel (13,14).Presumed alcohol binding sites have been identified on a small number of receptors and ion channels, but the location and nature of these sites vary considerably. Sites of alcohol interaction have been reported to be located in the ion-channel lumen of nicotinic acetylcholine receptors from muscle (15) in a cytoplasmic loop of a Drosophila potassium channel (16), in the cytoplasmic C terminus of G-protein-coupled inwardly rectifying potassium channels (17), and in ...

It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult ␣ 1 and embryonic ␣ 2 subunits, can be modified through selective mutations that rescued or impaired G␤␥ modulation. Even though both isoforms were able to physically interact with G␤␥, only the ␣ 1 GlyR was functionally modulated by G␤␥ and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in ␣ 2 GlyRs was enough to generate GlyRs modulated by G␤␥ and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a preopen-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective G␤␥ modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.Glycine receptors (GlyRs) 4 are members of the ligand-gated ion receptor (LGIC) superfamily, which includes the Cys-loop family composed of the inhibitory ␥-aminobutyric acid receptors and GlyRs and the excitatory nicotinic acetylcholine (nAChR) and 5-hydroxytryptamine receptors. These ionotropic receptors mediate fast synaptic transmission in the central nervous system (1, 2). Specifically, inhibitory GlyRs are critical for the control of excitability in the mammalian spinal cord and brain stem, regulating important physiological functions such as pain transmission, respiratory rhythms, motor coordination, and neuronal development (3-7).Like all Cys-loop receptors, GlyRs are heteropentameric complexes composed of ␣ and ␤ subunits, which can assemble to form homomeric (5␣) or heteromeric (2␣3␤) channels. To date, molecular cloning studies have demonstrated four isoforms of the ␣ GlyRs (␣ 1-4 ) and one ␤ isoform. Homomeric and heteromeric receptors share most of the GlyR general features, including a high percentage of identity between ␣ GlyRs (Ϸ75%). Nevertheless, biochemical, immunocytochemical, and in situ hybridization studies have shown that the expression of the subunits are developmentally and regionally regulated (3,4,8). For example, the ␣ 1 subunit expression increases after birth, whereas expression of the ␣ 2 subunit appears mainly restricted to early...

As the number of carbon atoms in an aliphatic n-alcohol is increased from one to five, intoxicating potency, lipid solubility, and membrane lipid disordering potency all increase in a similar exponential manner. However, the potency of aliphatic n-alcohols for producing intoxication reaches a maximum at six to eight carbon atoms and then decreases. The molecular basis of this "cutoff" effect is not understood, as it is not correlated with either the lipid solubility or the membrane disordering potency of the alcohols, which continue to increase exponentially. Since it has been suggested that inhibition of N-methyl-D-aspartate (NMDA) receptors by alcohols may play a role in alcohol intoxication, we investigated whether a series of aliphatic n-alcohols would exhibit a cutoff in potency for inhibition of NMDA receptors. We found that although potency for inhibition of NMDA receptors increased exponentially for alcohols with one to five carbon atoms, potency for inhibition of NMDA receptors reached a maximum at six to eight carbon atoms and then abruptly disappeared. This cutoff for alcohol inhibition of NMDA receptors is consistent with an interaction of the alcohols with a hydrophobic pocket on the receptor protein. In addition, the similarity of the cutoffs for alcohol inhibition of NMDA receptors and alcohol intoxication suggests that the cutoff for NMDA receptor inhibition may contribute to the cutoff for alcohol intoxication, which is consistent with an important role of NMDA receptors in alcohol intoxication.The molecular basis of alcohol's behavioral effects is poorly understood. Since the turn of the century, it has been widely accepted that alcohols produce their central nervous system effects by acting on the lipids of neuronal membranes (1-8). According to this "lipid theory," the effects of alcohols on the function of membrane proteins, such as receptors and ion channels, are secondary to their perturbation of membrane lipids. Consistent with this hypothesis, the intoxicating potency of aliphatic n-alcohols with up to five carbon atoms is correlated with both their lipid solubility (membrane/buffer partition coefficient) and their membrane lipid disordering potency (9, 10). However, as the number of carbon atoms is increased from six to eight, the intoxicating potency of aliphatic nalcohols reaches a plateau and then decreases (9, 10). This cutoff effect for intoxication is difficult to reconcile with the lipid theory of alcohol action, because it is not correlated with either membrane/buffer partition coefficient or potency for disordering membrane lipids, which both continue to increase exponentially with increasing carbon chain length (9-12).Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. N-Methyl-D-aspartate (NMDA) receptors are a subtype of glutamate receptors that appear to be involved in a variety of neural phenomena including nervous system excitability, cognitive function, and motor coordination (13-15). Since NMDA receptor function is inhibit...

Despite the fact that ethanol is one of the most widely used psychoactive agents, the mechanisms and sites of action by which it modifies brain functions are only now being elucidated. Studies over the last decade have shown that ethanol can specifically alter the function of several ligand-activated ion channels including N-methyl-D-aspartate (NMDA), serotonin (5-HT(3)), glycine and GABA(A) receptors. After several years of extensive research in this field, the resolution of what, where and how ethanol modifies GABA(A) receptors continues to be controversial. For example, after demonstrating that ethanol was able to alter Cl(-) flux in synaptoneurosomes and cultured neurons, several electrophysiological studies were unable to show enhancement of the GABA(A) receptor current in single neurons. The lack of positive results with low ethanol concentrations was interpreted as being due to receptor heterogeneity and differences in intracellular modulation by protein kinases and calcium. The existence of high receptor heterogeneity with respect to ethanol sensitivity has been supported by studies done in a variety of cell types which showed that ethanol potentiated some, but not other neurons. Adding to this complexity, it was shown that while some hippocampal GABA(A) receptors can be affected by ethanol concentrations between 1 and 100 mM, others are only sensitive to concentrations above 200 mM. The curve of the relationship between low ethanol concentrations and current enhancement suggests a high degree of complexity in the molecular interaction because of its steepness and "inverted" U shape. Similarly, the effects of ethanol on GABA(A) receptors seems much more complex than those of benzodiazepines, barbiturates and neurosteroids. The major problem encountered in advancing understanding of the mechanism of ethanol action in native neuronal receptors has been the large variability detected in ethanol sensitivity. For example, several studies have shown that only some groups of neurons are sensitive to pharmacologically relevant concentrations of ethanol (1-100 mM). This receptor sensitivity variability has not been resolved using recombinant expression systems. For example, studies performed in recombinant receptors, although important for elucidating molecular requirements, have shown that they are less sensitive to ethanol suggesting that neuronal substrates are important for ethanol actions. In this review, we discuss the possibility that ethanol's action on the GABA(A) receptor may not be due solely to a direct interaction with the receptor protein, but that its effects could also be modulated by intracellular regulation, and that this latter effect is the more physiologically relevant one. Data in cortical and hippocampal neurons suggest that ethanol action on the receptor is labile, and that it also depends on repetitive stimulation and neuron integrity. In addition, the action of ethanol can be modified by activation of protein kinases and neuronal development. Finally, we discuss that the best approach for...