The interplay between α7 nicotinic acetylcholine receptors, pannexin-1 channels and P2X7 receptors elicit exocytosis in chromaffin cells.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

Interaction of the α7-nicotinic subunit with its human-specific duplicated dupα7 isoform in mammalian cells: Relevance in human inflammatory responses.

The α7 nicotinic receptor subunit and its partially duplicated human-specific dupα7 isoform are coexpressed in neuronal and non-neuronal cells. In these cells, α7 subunits form homopentameric α7 nicotinic acetylcholine receptors (α7-nAChRs) implicated in numerous pathologies. In immune cells, α7-nAChRs are essential for vagal control of inflammatory response in sepsis. Recent studies show that the dupα7 subunit is a dominant-negative regulator of α7-nAChR activity in Xenopus oocytes. However, its biological significance in mammalian cells, particularly immune cells, remains unexplored, as the duplicated form is indistinguishable from the original subunit in standard tests. Here, using immunocytochemistry, confocal microscopy, coimmunoprecipitation, FRET, flow cytometry, and ELISA, we addressed this challenge in GH4C1 rat pituitary cells and RAW264.7 murine macrophages transfected with epitope- and fluorescent protein-tagged α7 or dupα7. We used quantitative RT-PCR of dupα7 gene expression levels in peripheral blood mononuclear cells (PBMCs) from patients with sepsis to analyze its relationship with PBMC α7 mRNA levels and with serum concentrations of inflammatory markers. We found that a physical interaction between dupα7 and α7 subunits in both cell lines generates heteromeric nAChRs that remain mainly trapped in the endoplasmic reticulum. The dupα7 sequestration of α7 subunits reduced membrane expression of functional α7-nAChRs, attenuating their anti-inflammatory capacity in lipopolysaccharide-stimulated macrophages. Moreover, the PBMC's dupα7 levels correlated inversely with their α7 levels and directly with the magnitude of the patients' inflammatory state. These results indicate that dupα7 probably reduces human vagal anti-inflammatory responses and suggest its involvement in other α7-nAChR-mediated pathophysiological processes.

Molecular mode of action of CGS 9895 at α1 ß2 γ2 GABAA receptors.

γ-aminobutyric type A (GABAA ) receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics, and anesthetics. Previously, pyrazoloquinoline 2-p-methoxyphenylpyrazolo [4,3-c] quinolin-3(5H)-one (CGS 9895) was described as a positive allosteric modulator acting through the α+/ß- interface in the extracellular domain of GABAA receptors. The localization of the binding site was based on a steric hindrance approach, rather than on direct effects of point mutations. In this study we further characterized modulation by this compound which seems to have multiple sites of action. We investigated GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. We have identified the α1 Y209 residue at the α+/ß- interface as a key residue for CGS 9895 modulation. In addition, the interaction between this residue and various structural analogs was characterized, allowing tentative positioning of CGS 9895 versus α1 Y209 (rat sequence). Not all compounds were found to be sensitive to mutations at the α1 Y209 residue. In addition, the interaction of CGS 9895 with flurazepam was characterized. Flurazepam is hypothesized to act at the same subunit interface in the extracellular domain. We also provide evidence that the GABAA receptor harbors additional modulatory sites for CGS 9895 at each of the subunit interfaces in the transmembrane domain. GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We have identified the α1 Y209 residue present at the extracellular α+/ß- subunit interface as a key residue for the positive allosteric modulation of the GABAA receptor by CGS 9895. This receptor harbors additional modulatory sites for this compound at subunit interfaces in the transmembrane domain.

Usefulness of α7 nicotinic receptor messenger RNA levels in peripheral blood mononuclear cells as a marker for cholinergic antiinflammatory pathway activity in septic patients: results of a pilot study.

BACKGROUND: Stimulation of the vagus nerve in the so-called cholinergic antiinflammatory pathway (CAP) attenuates systemic inflammation, improving survival in animal sepsis models via α7 nicotinic acetylcholine receptors on immunocompetent cells. Because the relevance of this regulatory pathway is unknown in human sepsis, this pilot study assessed whether the α7 gene expression level in septic patients' peripheral blood mononuclear cells (PBMC) might be used to assess CAP activity and clinical outcome. METHODS: The PBMCs α7 messenger RNA levels were determined by real-time quantitative reverse-transcription polymerase chain reaction in 33 controls and 33 patients at enrollment and after their hospital discharge. Data were analyzed to find significant associations between α7 level, vagally mediated heart rate variability as an indirect reflection of CAP activity, serum concentrations of different inflammation markers, and clinical course. RESULTS: Septic patients' α7 levels were significantly increased and returned to control values after recovery. These α7 levels correlated directly with the vagal heart input and inversely with the magnitude of the patient's inflammatory state, disease severity, and clinical outcome. CONCLUSIONS: This study reveals that the PBMC α7 gene expression level is a clinically relevant marker for CAP activity in sepsis: the higher the α7 expression, the better the inflammation control and the prognosis.

α subunits in GABAA receptors are dispensable for GABA and diazepam action.

The major isoform of the GABAA receptor is α1ß2γ2. The binding sites for the agonist GABA are located at the ß2+/α1- subunit interfaces and the modulatory site for benzodiazepines at α1+/γ2-. In the absence of α1 subunits, a receptor was formed that was gated by GABA and modulated by diazepam similarly. This indicates that alternative subunits can take over the role of the α1 subunits. Point mutations were introduced in ß2 or γ2 subunits at positions homologous to α1- benzodiazepine binding and GABA binding positions, respectively. From this mutation work we conclude that the site for GABA is located at a ß2+/ß2- subunit interface and that the diazepam site is located at the ß2+/γ2- subunit interface. Computational docking leads to a structural hypothesis attributing this non-canonical interaction to a binding mode nearly identical with the one at the α1+/γ2- interface. Thus, the ß2 subunit can take over the role of the α1 subunit for the formation of both sites, its minus side for the GABA binding site and its plus side for the diazepam binding site.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments.

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16. The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Functional sites involved in modulation of the GABAA receptor channel by the intravenous anesthetics propofol, etomidate and pentobarbital.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs. Among the many modulatory compounds are also the intravenous anesthetics propofol and etomidate, and barbiturates. The mechanism of receptor modulation by these compounds is of mayor relevance. The site of action of these compounds has been located to subunit interfaces in the intra-membrane region of the receptor. In α1ß2γ2 GABAA receptors there are five such interfaces, two ß+/α- and one each of α+/ß-, α+/γ- and γ+/ß- subunit interfaces. We have used reporter mutations located in the second trans-membrane region in different subunits to probe the effects of changes at these subunit interfaces on modulation by propofol, etomidate and pentobarbital. We provide evidence for the fact that each of these compounds either modulates through a different set of subunit interfaces or through the same set of subunit interfaces to a different degree. As a GABAA receptor pentamer harbors two ß+/α- subunit interfaces, we used concatenated receptors to dissect the contribution of individual interfaces and show that only one of these interfaces is important for receptor modulation by etomidate.

Novel positive allosteric modulators of GABAA receptors with anesthetic activity.

GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1ß2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1ß2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two ß+/α- subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual ß+/α- interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the ß subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

A new IRAK-M-mediated mechanism implicated in the anti-inflammatory effect of nicotine via α7 nicotinic receptors in human macrophages.

Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the 'endotoxin tolerant' phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.