Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel.

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs, there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here, we have characterized the structure and dynamics of DeCLIC through cryoelectron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium-binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid-body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium-binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering.

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel.

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

The nematode serotonin-gated chloride channel MOD-1: A novel target for anthelmintic therapy.

Anthelmintics are used to treat human and veterinary parasitic diseases and to reduce crop and livestock production loss associated with parasitosis. The free-living nematode Caenorhabditis elegans, a model system for anthelmintic drug discovery, has a serotonin (5-HT)-gated chloride channel, MOD-1, which belongs to the Cys-loop receptor family and modulates locomotory and behavioral functions. Since MOD-1 is unique to nematodes, it is emerging as an attractive anthelmintic drug target, but details of MOD-1 function are unclear. Here, we revealed novel aspects of MOD-1 function from the molecular level to the organism level and identified compounds targeting this receptor, which may provide new directions for anthelmintic drug discovery. We used whole-cell current recordings from heterologously expressed MOD-1 to show that tryptamine (Tryp), a weak partial agonist of vertebrate serotonin type 3 (5-HT3) receptors, efficaciously activates MOD-1. A screen for modulators revealed that GABAergic ligands piperazine (PZE) and muscimol reduce 5-HT-elicited currents, thus identifying novel MOD-1 allosteric inhibitors. Next, we performed locomotor activity assays, and we found 5-HT and Tryp rapidly decrease worm motility, which is reversible only at low 5-HT concentrations. Mutants lacking MOD-1 are partially resistant to both drugs, demonstrating its role in locomotion. Acting as an antagonist of MOD-1, we showed PZE reduces the locomotor effects of exogenous 5-HT. Therefore, Tryp- and PZE-derived compounds, acting at MOD-1 through different molecular mechanisms, emerge as promising anthelmintic agents. This study enhances our knowledge of the function and drug selectivity of Cys-loop receptors and postulates MOD-1 as a potential target for anthelmintic therapy.

Cannabidiol as a modulator of α7 nicotinic receptors.

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health.

Cryo-EM structures of a lipid-sensitive pentameric ligand-gated ion channel embedded in a phosphatidylcholine-only bilayer.

The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC (Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C-at the agonist-binding sites-and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD-TMD coupling in such a way that the "conformational wave" of liganded-receptor gating takes place in the ECD and the interfacial M2-M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand's fluctuations, and thus delays its escape into bulk solvent.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks.

BACKGROUND: Ticks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick's central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied. RESULTS: We carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls). CONCLUSIONS: We established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

Tyrosine phosphorylation differentially fine-tunes ionotropic and metabotropic responses of human α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.

PharmacoGenetic targeting of a C. elegans essential neuron provides an in vivo screening for novel modulators of nematode ion channel function.

Chemical or drug treatments are successfully used to treat parasitic nematode infections that impact human, animal and plant health. Many of these exert their effects through modifying neural function underpinning behaviours essential for parasite viability. Selectivity against the parasite may be achieved through distinct pharmacological properties of the parasite nervous system, as exemplified by the success of the ivermectin which target a glutamate-gated chloride channel found only in invertebrates. Despite the success of the ivermectins, emerging resistance and concerns around eco-toxicity are driving the search for new nematocidal chemicals or drugs. Here, we describe the potential of a 5-HT-gated chloride channel MOD-1, which is involved in vital parasite behaviours with constrained distribution in the invertebrate phyla. This ion channel has potential pharmacophores that could be targeted by new nematocidal chemicals and drugs. We have developed a microtiter based bioassay for MOD-1 pharmacology based on its ectopic expression in the Caenorhabditis elegans essential neuron M4. We have termed this technology 'PhaGeM4' for 'Pharmacogenetic targeting of M4 neuron'. Exposure of transgenic worms harbouring ectopically expressed MOD-1 to 5-HT results in developmental arrest. By additional expression of a fluorescence marker in body wall muscle to monitor growth we demonstrate that this assay is suitable for the identification of receptor agonists and antagonists. Indeed, the developmental progression is a robustly quantifiable bioassay that resolves MOD-1 activation by quipazine, 5-carboxyamidotryptamine and fluoxetine and highlight methiothepin as a potent antagonist. This assay has the intrinsic ability to highlight compounds with optimal bioavailability and furthermore to filter out off-target effects. It can be extended to the investigation of other classes of membrane receptors and modulators of neuronal excitation. This approach based on heterologous modulation of the essential M4 neuron function offers a route to discover new effective and selective anthelmintics potentially less confounded by disruptive environmental impact.

Speculation on How RIC-3 and Other Chaperones Facilitate α7 Nicotinic Receptor Folding and Assembly.

The process of how multimeric transmembrane proteins fold and assemble in the endoplasmic reticulum is not well understood. The alpha7 nicotinic receptor (α7 nAChR) is a good model for multimeric protein assembly since it has at least two independent and specialized chaperones: Resistance to Inhibitors of Cholinesterase 3 (RIC-3) and Nicotinic Acetylcholine Receptor Regulator (NACHO). Recent cryo-EM and NMR data revealed structural features of α7 nAChRs. A ser-ala-pro (SAP) motif precedes a structurally important but unique "latch" helix in α7 nAChRs. A sampling of α7 sequences suggests the SAP motif is conserved from C. elegans to humans, but the latch sequence is only conserved in vertebrates. How RIC-3 and NACHO facilitate receptor subunits folding into their final pentameric configuration is not known. The artificial intelligence program AlphaFold2 recently predicted structures for NACHO and RIC-3. NACHO is highly conserved in sequence and structure across species, but RIC-3 is not. This review ponders how different intrinsically disordered RIC-3 isoforms from C. elegans to humans interact with α7 nAChR subunits despite having little sequence homology across RIC-3 species. Two models from the literature about how RIC-3 assists α7 nAChR assembly are evaluated considering recent structural information about the receptor and its chaperones.

Cholinergic transmission in C. elegans: Functions, diversity, and maturation of ACh-activated ion channels.

Acetylcholine is an abundant neurotransmitter in all animals. Effects of acetylcholine are excitatory, inhibitory, or modulatory depending on the receptor and cell type. Research using the nematode C. elegans has made ground-breaking contributions to the mechanistic understanding of cholinergic transmission. Powerful genetic screens for behavioral mutants or for responses to pharmacological reagents identified the core cellular machinery for synaptic transmission. Pharmacological reagents that perturb acetylcholine-mediated processes led to the discovery and also uncovered the composition and regulators of acetylcholine-activated channels and receptors. From a combination of electrophysiological and molecular cellular studies, we have gained a profound understanding of cholinergic signaling at the levels of synapses, neural circuits, and animal behaviors. This review will begin with a historical overview, then cover in-depth current knowledge on acetylcholine-activated ionotropic receptors, mechanisms regulating their functional expression and their functions in regulating locomotion.

Extrasynaptic δ-subunit containing GABAA receptors.

γ-Aminobutyric acid type A receptors (GABAARs) are GABA gated heteropentameric chloride channels responsible for the adult brain's primary inhibition. In specific brain cells, such as in the hippocampus, one of the subtypes of GABAARs, the δ subunit containing GABAARs (δ-GABAARs), is predominantly expressed and located in extrasynaptic or perisynaptic positions. δ-GABAARs mediate a slow constant inhibitory current called tonic inhibition. While δ-GABAARs and tonic inhibition is critical for the excitability of single neurons, accumulating data suggest that the function of δ-GABAARs are broader and includes an integrative role in the network oscillations. While these open new horizons on the neurobiology of δ-GABAARs, the complexity continues to challenge the analysis of GABAARs and their subtypes. This review will summarize the current knowledge of molecular, cellular and physiological characteristics of δ-GABAARs during health and disease.

A Missense Mutation A384P Associated with Human Hyperekplexia Reveals a Desensitization Site of Glycine Receptors.

Hyperekplexia, an inherited neuronal disorder characterized by exaggerated startle responses with unexpected sensory stimuli, is caused by dysfunction of glycinergic inhibitory transmission. From analysis of newly identified human hyperekplexia mutations in the glycine receptor (GlyR) α1 subunit, we found that an alanine-to-proline missense mutation (A384P) resulted in substantially higher desensitization level and lower agonist sensitivity of homomeric α1 GlyRs when expressed in HEK cells. The incorporation of the ß subunit fully reversed the reduction in agonist sensitivity and partially reversed the desensitization of α1A384P The heteromeric α1A384Pß GlyRs showed enhanced desensitization but unchanged agonist-induced maximum responses, surface expression, main channel conductance, and voltage dependence compared with that of the wild-type α1ß (α1WTß) GlyRs. Coexpression of the R392H and A384P mutant α1 subunits, which mimic the expression of the compound heterozygous mutation in a hyperekplexia patient, resulted in channel properties similar to those with α1A384P subunit expression alone. In comparison, another human hyperekplexia mutation α1P250T, which was previously reported to enhance desensitization, caused a strong reduction in maximum currents in addition to the altered desensitization. These results were further confirmed by overexpression of α1P250T or α1A384P subunits in cultured neurons isolated from SD rats of either sex. Moreover, the IPSC-like responses of cells expressing α1A384Pß induced by repeated glycine pulses showed a stronger frequency-dependent reduction than those expressing α1WTß. Together, our findings demonstrate that A384 is associated with the desensitization site of the α1 subunit and its proline mutation produced enhanced desensitization of GlyRs, which contributes to the pathogenesis of human hyperekplexia.SIGNIFICANCE STATEMENT Human startle disease is caused by impaired synaptic inhibition in the brainstem and spinal cord, which is due to either direct loss of GlyR channel function or reduced number of synaptic GlyRs. Considering that fast decay kinetics of GlyR-mediated inhibitory synaptic responses, the question was raised whether altered desensitization of GlyRs will cause dysfunction of glycine transmission and disease phenotypes. Here, we found that the α1 subunit mutation A384P, identified from startle disease patients, results in enhanced desensitization and leads to rapidly decreasing responses in the mutant GlyRs when they are activated repeatedly by the synaptic-like simulation. These observations suggest that the enhanced desensitization of postsynaptic GlyRs could be the primary pathogenic mechanism of human startle disease.

LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors.

The endoplasmic reticulum-Golgi intermediate compartment protein-53 (ERGIC-53, aka LMAN1), which cycles between the endoplasmic reticulum (ER) and Golgi, is a known cargo receptor for a number of soluble proteins. However, whether LMAN1 plays a role as a trafficking factor in the central nervous system is largely unknown. Here, we determined the role of LMAN1 on endogenous protein levels of the Cys-loop superfamily of neuroreceptors, including gamma-aminobutyric acid type A receptors (GABAARs), 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and nicotinic acetylcholine receptors (nAChRs). Knockdown of LMAN1 reduces the surface trafficking of endogenous ß3 subunits of GABAARs in mouse hypothalamic GT1-7 neurons. Furthermore, Western blot analysis of brain homogenates from LMAN1 knockout mice demonstrated that loss of LMAN1 decreases the total protein levels of 5HT3A receptors and γ2 subunits of GABAARs. LMAN1 knockout regulates the ER proteostasis network by upregulating ERP44 without changing calnexin levels. Interestingly, despite the critical role of the glycan-binding function of LMAN1 in its other known cargo clients, LMAN1 interacts with GABAARs in a glycan-independent manner. In summary, LMAN1 is a trafficking factor for certain neuroreceptors in the central nervous system. This is the first report of LMAN1 function in membrane protein trafficking.

Molecular function of the novel α7ß2 nicotinic receptor.

The α7 nicotinic receptor is a promising drug target for neurological and inflammatory disorders. Although it is the homomeric member of the family, a novel α7ß2 heteromeric receptor has been discovered. To decipher the functional contribution of the ß2 subunit, we generated heteromeric receptors with fixed stoichiometry by two different approaches comprising concatenated and unlinked subunits. Receptors containing up to three ß2 subunits are functional. As the number of ß2 subunits increases in the pentameric arrangement, the durations of channel openings and activation episodes increase progressively probably due to decreased desensitization. The prolonged activation episodes conform the kinetic signature of α7ß2 and may have an impact on neuronal excitability. For activation of α7ß2 receptors, an α7/α7 binding-site interface is required, thus indicating that the three ß2 subunits are located consecutively in the pentameric arrangement. α7-positive allosteric modulators (PAMs) are emerging as novel therapeutic drugs. The presence of ß2 in the pentamer affects neither type II PAM potentiation nor activation by an allosteric agonist whereas it impairs type I PAM potentiation. This first single-channel study provides fundamental basis required to decipher the role and function of the novel α7ß2 receptor and opens doors to develop selective therapeutic drugs.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel.

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and ß subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at ß/α intersubunit interfaces, where the GluClß and GluClα subunits, respectively, contribute the "principal" and "complementary" components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits' cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two ß subunits arranged in a counterclockwise ß-α-ß-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer.

In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2ß2 nAChR and validated the functional importance of specific residues in α2 and ß2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.

Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the δ subunit.

The γ-Aminobutyric acid type A receptors (GABAARs) are heteropentameric chloride channels responsible for primary inhibition in the mammalian brain. Studies have shown the expression of recombinant GABAAR subunits tagged with the green fluorescent protein (GFP), a 26.9 kDa protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. This allows the formation of recombinant proteins essential for the development of relevant in-vitro and in-vivo methodologies. Among the GABAAR subunits, the δ subunit was never tagged in its cytoplasmic domain, an evolutionary conserved domain found in between the third and the fourth transmembrane domains. In this study, first, we have cloned the mouse cDNAs encoding for the δ, α1, ß2 subunits of GABAARs, and then developed two fusion proteins of δ subunit each tagged with the GFP variant, EGFP (enhanced GFP) at unique sites in the cytoplasmic domain. The recombinant proteins were expressed alone or in combination with α1 and/or ß2 subunits in neuroblastoma 2a cells. Live cell confocal microscopy indicated that the cytoplasmically tagged δ subunits were targeted to the cell membrane when expressed in the presence of α1 and ß2 subunits in neuroblastoma 2a cells. However, this was not observed when they were expressed alone or only with α1 or ß2 subunits in the same cell line. These results confirm the general oligomerization and targeting pattern of GABAAR subtypes described in the other in-vitro studies in the literature. Thus, our results suggest that the EGFP tagging in the ctoplasmic domain did not interfere with the oligomerization and cell surface expression of recombinant δ subunits. To our knowledge, this is the first study showing the generation, expression and preliminary analysis of the δ-GABAARs tagged in the cytoplasmic domain of the δ subunit which can be further elaborated to probe intracellular protein interactions of GABAARs via the δ subunit.

Molecular function of α7 nicotinic receptors as drug targets.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in many physiological and pathological processes. In vertebrates, there are seventeen different nAChR subunits that combine to yield a variety of receptors with different pharmacology, function, and localization. The homomeric α7 receptor is one of the most abundant nAChRs in the nervous system and it is also present in non-neuronal cells. It plays important roles in cognition, memory, pain, neuroprotection, and inflammation. Its diverse physiological actions and associated disorders have made of α7 an attractive novel target for drug modulation. Potentiation of the α7 receptor has emerged as a novel therapeutic strategy for several neurological diseases, such as Alzheimer's and Parkinson's diseases, and inflammatory disorders. In contrast, increased α7 activity has been associated with cancer cell proliferation. The presence of different drug target sites offers a great potential for α7 modulation in different pathological contexts. In particular, compounds that target allosteric sites offer significant advantages over orthosteric agonists due to higher selectivity and a broader spectrum of degrees and mechanisms of modulation. Heterologous expression of α7, together with chaperone proteins, combined with patch clamp recordings have provided important advances in our knowledge of the molecular basis of α7 responses and their potential modulation for pathological processes. This review gives a synthetic view of α7 and its molecular function, focusing on how its unique activation and desensitization features can be modified by pharmacological agents. This fundamental information offers insights into therapeutic strategies.

The dual-gate model for pentameric ligand-gated ion channels activation and desensitization.

Pentameric ligand-gated ion channels (pLGICs) mediate fast neurotransmission in the nervous system. Their dysfunction is associated with psychiatric, neurological and neurodegenerative disorders such as schizophrenia, epilepsy and Alzheimer's disease. Understanding their biophysical and pharmacological properties, at both the functional and the structural level, thus holds many therapeutic promises. In addition to their agonist-elicited activation, most pLGICs display another key allosteric property, namely desensitization, in which they enter a shut state refractory to activation upon sustained agonist binding. While the activation mechanisms of several pLGICs have been revealed at near-atomic resolution, the structural foundation of desensitization has long remained elusive. Recent structural and functional data now suggest that the activation and desensitization gates are distinct, and are located at both sides of the ion channel. Such a 'dual gate mechanism' accounts for the marked allosteric effects of channel blockers, a feature illustrated herein by theoretical kinetics simulations. Comparison with other classes of ligand- and voltage-gated ion channels shows that this dual gate mechanism emerges as a common theme for the desensitization and inactivation properties of structurally unrelated ion channels.