Alcohol abuse and alcoholism are major health problems and one of the leading preventable causes of death. Before achieving better treatments for alcoholism, it is necessary to understand the critical actions of alcohol on membrane proteins that regulate fundamental functions in the central nervous system. After generating a genetically modified knock-in (KI) mouse having a glycine receptor (GlyR) with phenotypical silent mutations at KK385/386AA, we studied its cellular and in vivo ethanol sensitivity. Analyses with western blotting and immunocytochemistry indicated that the expression of a1 GlyRs in nervous tissues and spinal cord neurons (SCNs) were similar between WT and KI mice. The analysis of synaptic currents recorded from KI mice showed that the glycinergic synaptic transmission had normal properties, but the sensitivity to ethanol was significantly reduced. Furthermore, the glycine-evoked current in SCNs from KI was resistant to ethanol and G-protein activation by GTP-g-S. In behavioral studies, KI mice did not display the foot-clasping behavior upon lifting by the tail and lacked an enhanced startle reflex response that are characteristic of other glycine KI mouse lines with markedly impaired glycine receptor function. The most notable characteristic of the KI mice was their significant lower sensitivity to ethanol (B40%), expressed by shorter times in loss of righting reflex (LORR) in response to a sedative dose of ethanol (3.5 g/Kg). These data provide the first evidence to link a molecular site in the GlyR with the sedative effects produced by intoxicating doses of ethanol.

In recent years there has been an increase in the understanding of ethanol actions on the type A γ-aminobutyric acid chloride channel (GABAAR), a member of the pentameric ligand gated ion channels (pLGICs). However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR), another member of the pLGIC family, as a suitable target for the development of new pharmacological tools.

Failure of neural tube closure leads to neural tube defects (NTDs), which can have serious neurological consequences or be lethal. Use of antiepileptic drugs (AEDs) during pregnancy increases the incidence of NTDs in offspring by unknown mechanisms. Here we show that during neural tube formation, neural plate cells exhibit spontaneous calcium dynamics that are partially mediated by glutamate signaling. We demonstrate that NMDA receptors are important for the formation of the neural tube and that the loss of their function induces an increase in neural plate cell proliferation and impairs neural cell migration, which result in NTDs. We present evidence that the AED valproic acid perturbs glutamate signaling, leading to NTDs that are rescued with varied efficacy by preventing DNA synthesis, activating NMDA receptors, or recruiting the NMDA receptor target ERK1/2. These findings may prompt mechanistic identification of AEDs that do not interfere with neural tube formation. Neural tube defects are one of the most common birth defects. Clinical investigations have determined that the use of antiepileptic drugs during pregnancy increases the incidence of these defects in the offspring by unknown mechanisms. This study discovers that glutamate signaling regulates neural plate cell proliferation and oriented migration and is necessary for neural tube formation. We demonstrate that the widely used antiepileptic drug valproic acid interferes with glutamate signaling and consequently induces neural tube defects, challenging the current hypotheses arguing that they are side effects of this antiepileptic drug that cause the increased incidence of these defects. Understanding the mechanisms of neurotransmitter signaling during neural tube formation may contribute to the identification and development of antiepileptic drugs that are safer during pregnancy.

The a1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular Gbg actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the a1-GlyR, which includes the large intracellular domain and provides new information on structural requirements for allosteric modulation by ethanol and Gbg. The data strongly suggest the existence of an a-helical conformation in the regions near transmembrane (TM)-3 and TM4 of the large intracellular domain. The secondary structure in the N-terminal region of the large intracellular domain near TM3 appeared critical for ethanol action, and this was tested using the homologous domain of the g2-subunit of the GABA A receptor predicted to have little helical conformation. This region of g2 was able to bind Gbg and form a functional channel when combined with a1-GlyR, but it was not sensitive to ethanol. Mutations in the N-and C-terminal regions introduced to replace corresponding amino acids of the a1-GlyR sequence restored the ability to be modulated by ethanol and Gbg. Recovery of the sensitivity to ethanol was associated with the existence of a helical conformation similar to a1-GlyR, thus being an essential secondary structural requirement for GlyR modulation by ethanol and G protein.

Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y 2 receptor (P2Y 2 R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y 2 Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y 2 R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y 2 R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10–100 μM, but inhibiting at 300 μM ATP. On the other hand, 1–300 μM UTP, a P2Y 2 R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y 2 R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y 2 R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

The purinergic P2X2 receptor (P2X2R) is an ATP-gated ion channel widely expressed in the nervous system. Here, we identified a putative Cdk5 phosphorylation site in the full-size variant P2X2aR (372TPKH375), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine-phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5-activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells, and P2X2/3R mediated increases of intracellular Ca2+ in trigeminal neurons were Cdk5-dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

Glycine receptors (GlyRs) are members of the pentameric ligandgated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood.To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α 1 GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α 1 GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKAdependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKAtargeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLic and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs. Glycine receptors (GlyRs) belong to the pentameric ligand-gated ion channel (pLGIC) family. GlyRs are anion-permeable channels, allowing the fast influx of chloride and the control of neuronal excitability. An individual GlyR subunit is composed by an extracellular domain (ECD), four transmembrane domains (TM1-4) and an intracellular domain between the TM3 and TM4 domains (ICD) 1-4. To date, a single β subunit and four α subunits (α1-4) has been described. The α subunits share a high degree of sequence identity (≈75%). Nevertheless, they exhibit important differences in their biophysical and pharmacological properties as well as in their distribution along the CNS 1,3,4. In the mammalian CNS, GlyR activity critically controls neurophysiological functions such as motor coordination, respiratory control, muscle tone, as well as pain processing 2,3,5-13. The importance of glycinergic inhibition was first recognized in studies using the GlyR antagonist strychnine 14,15. Later, genetic studies found that mutations in the GlyR α1 and β genes are linked to hyperekplexia in humans 16. More recent evidence has shown that specific GlyR subunits may play key roles in several diseases. For example, while the α1 subunit has been linked to tumorigenesis and alcohol intoxication 17,18 , mutations in the α2 subunit have been linked to autism 19. Alterations in the RNA processing of α3 subunits generates hyperactive receptors, which have been rela...