Gonzalo E. Yevenes

GABA-A Receptors as Molecular Sites of Ethanol Action. Direct or Indirect Actions?

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 ethanols 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 studying the interaction between the receptor and ethanol is through the combined use of recombinant receptors and overexpression in neurons.

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein βγ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR α1 subunit that are critical for binding and functional modulation by Gβγ. Mutations within these sequences demonstrated that all of the residues detected are important for Gβγ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are α-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein βγ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling.

A Single Phenylalanine Residue in the Main Intracellular Loop of α1 γ-Aminobutyric Acid Type A and Glycine Receptors Influences Their Sensitivity to Propofol

Background: The intravenous anesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and &ggr;-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, the influence of the large intracellular loop in propofol sensitivity of both receptors was explored. Methods: The large intracellular loop of &agr;1 GlyRs and &agr;1&bgr;2 GABAARs was screened using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with wild type or mutant receptors. Results: Alanine mutation of a conserved phenylalanine residue within the &agr;1 large intracellular loop significantly reduced propofol enhancement in both GlyRs (360 ± 30 vs. 75 ± 10%, mean ± SEM) and GABAARs (361 ± 49% vs. 80 ± 23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane. At the single-channel level, the ability of propofol to increase open probability was significantly reduced in both &agr;1 GlyR (189 ± 36 vs. 22 ± 13%) and &agr;1&bgr;2 GABAAR (279 ± 29 vs. 29 ± 11%) mutant receptors. Conclusion: In this study, it is demonstrated that the large intracellular loop of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Results suggest a new role of the large intracellular loop in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol.

Blockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.

The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with Gβγ. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gβγ to 21 ± 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gβγ binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gβγ dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of Gβγ to GlyRs to 57 ± 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gβγ-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 ± 14% of control) and attenuated the GlyR potentiation by ethanol (51 ± 10% versus 10 ± 3%).

Activated G Protein αs Subunits Increase the Ethanol Sensitivity of Human Glycine Receptors

It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through Gβγ subunits. However, more specific roles of G protein α subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human α1 GlyR is selectively enhanced by the expression of Gαs Q-L. For example, constitutively active Gαs, but not Gαq or Gαi, was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 ± 4 versus 48 ± 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the Gαs Q-L effect was not dependent on cAMP/PKA-dependent signaling. On the other hand, the effect of Gαs Q-L was blocked by a Gβγ scavenger (9 ± 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with Gβγ were not affected by Gαs Q-L, suggesting that Gβγ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated Gαs can facilitate the Gβγ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of Gαs is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration.

The Basic Property of Lys385 Is Important for Potentiation of the Human α1 Glycine Receptor by Ethanol

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the α1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gβγ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 ± 1 versus 41 ± 0.5 μM) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human α1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gβγ, is critical for ethanol potentiation of the receptor.

Prevention of Synaptic Alterations and Neurotoxic Effects of PAMAM Dendrimers by Surface Functionalization

One of the most studied nanocarriers for drug delivery are polyamidoamine (PAMAM) dendrimers. However, the alterations produced by PAMAM dendrimers in neuronal function have not been thoroughly investigated, and important aspects such as effects on synaptic transmission remain unexplored. We focused on the neuronal activity disruption induced by dendrimers and the possibility to prevent these effects by surface chemical modifications. Therefore, we studied the effects of fourth generation PAMAM with unmodified positively charged surface (G4) in hippocampal neurons, and compared the results with dendrimers functionalized in 25% of their surface groups with folate (PFO25) and polyethylene glycol (PPEG25). G4 dendrimers significantly reduced cell viability at 1 µM, which was attenuated by both chemical modifications, PPEG25 being the less cytotoxic. Patch clamp recordings demonstrated that G4 induced a 7.5-fold increment in capacitive currents as a measure of membrane permeability. Moreover, treatment with this dendrimer increased intracellular Ca2+ by 8-fold with a complete disruption of transients pattern, having as consequence that G4 treatment increased the synaptic vesicle release and frequency of synaptic events by 2.4- and 3-fold, respectively. PFO25 and PPEG25 treatments did not alter membrane permeability, total Ca2+ intake, synaptic vesicle release or synaptic activity frequency. These results demonstrate that cationic G4 dendrimers have neurotoxic effects and induce alterations in normal synaptic activity, which are generated by the augmentation of membrane permeability and a subsequent intracellular Ca2+ increase. Interestingly, these toxic effects and synaptic alterations are prevented by the modification of 25% of PAMAM surface with either folate or polyethylene glycol.