Α1 Subunits Research Articles

Modulation of relapse-like drinking in male Sprague-Dawley rats by ligands targeting the α5GABAA receptor

Preclinical evidence suggests a key role for GABAA receptors containing the α5 subunit (i.e., α5GABAA receptors) in the abuse-related effects of alcohol, including the reinforcing and discriminative stimulus effects, as well as cue-induced alcohol-seeking behavior. However, the contribution of this GABAA receptor subtype to relapse-like drinking behavior remains unknown. The present study evaluated the capacity of ligands targeting α5GABAA receptors to modulate the alcohol deprivation effect (ADE), a model of relapse-like drinking. Groups of Sprague-Dawley rats underwent repeated cycles of long-term access to alcohol solutions (5%, 10%, 20% v/v) and water in the home cage followed by water only deprivation periods. Upon evidence that the ADE could be reliably expressed across cycles, drug treatment was initiated. One group received the α5GABAA receptor-preferring agonist QH-ii-066 and the other group received the α5GABAA receptor-selective inverse agonist L-655,708. At the end of ADE testing, rats underwent testing in the elevated zero maze under vehicle or L-655,708 treatment for assessment of anxiety-like behavior. The ADE was reliably expressed across repeated cycles of alcohol access/deprivation in a subset of rats. Low doses of QH-ii-066 enhanced expression of the ADE; whereas, L-655,708 dose-dependently inhibited expression of the ADE. L-655,708 did not engender anxiogenic effects in the elevated zero maze under the conditions evaluated. These findings suggest a key role for α5GABAA receptor mechanisms in relapse-like drinking. Moreover, they suggest that α5GABAA receptors may represent a novel pharmacological target for the development of medications to prevent or reduce alcohol relapse.

Unconditioned and learned morphine tolerance influence hippocampal-dependent short-term memory and the subjacent expression of GABA-A receptor alpha subunits.

ɣ-aminobutyric acid (GABA) facilitator valproic acid may be able to curb memory disruption induced by morphine exposure. The effects of the GABA facilitator valproic acid on the behavioral tolerance induced by morphine were investigated. Then hippocampal-dependent tasks named spatial-working and short-term memory procedures using the Y-maze apparatus were examined in morphine tolerant rats. Finally, the changes in the expression of hippocampal GABA-A receptors underlying morphine tolerance were also examined. Rats were treated with daily morphine injections, with or without distinct contextual pairing. To examine the effect of valproic acid on morphine tolerance expression, valproic acid was pretreated an hour before morphine. Spatial-working and short-term memory procedures using the Y-maze apparatus were examined in morphine tolerant rats. Afterwards the changes in the expression of hippocampal GABAα receptors using the quantitative real-time PCR and western blot techniques to detect GABArα subunits mRNAs and protein level were studied. Our results showed that both learned and non-associative morphine tolerance influence short-term memory and the subjacent expression of GABArα mRNAs and protein level. Despite its attenuating effects on the development and expression of both learned and non-associative morphine tolerance, only associative morphine tolerance-induced memory dysfunction was ameliorated by valproic acid pretreatment. We also found that the expression of GABArα1, α2, α5 subunits mRNAs and GABAα protein level were affected heavier in associative morphine tolerant rats. Our data supports the hypothesis that unconditioned and learned morphine tolerance influences short-term memory and the expression of GABArα 1, α2, α5 mRNAs and GABArα protein level differently, and adds to our understanding of the behavioral and molecular aspects of the learned tolerance to morphine effects.

Voltage-gated calcium channels (Ca<sub>V</sub>) in GtoPdb v.2021.3

Ca2+ channels are voltage-gated ion channels present in the membrane of most excitable cells. The nomenclature for Ca2+channels was proposed by [127] and approved by the NC-IUPHAR Subcommittee on Ca2+ channels [70]. Most Ca2+ channels form hetero-oligomeric complexes. The α1 subunit is pore-forming and provides the binding site(s) for practically all agonists and antagonists. The 10 cloned α1-subunits can be grouped into three families: (1) the high-voltage activated dihydropyridine-sensitive (L-type, CaV1.x) channels; (2) the high- to moderate-voltage activated dihydropyridine-insensitive (CaV2.x) channels and (3) the low-voltage-activated (T-type, CaV3.x) channels. Each α1 subunit has four homologous repeats (I-IV), each repeat having six transmembrane domains (S1-S6) and a pore-forming region between S5 and S6. Voltage-dependent gating is driven by the membrane spanning S4 segment, which contains highly conserved positive charges that respond to changes in membrane potential. All of the α1-subunit genes give rise to alternatively spliced products. At least for high-voltage activated channels, it is likely that native channels comprise co-assemblies of α1, β and α2-δ subunits. The γ subunits have not been proven to associate with channels other than the α1s skeletal muscle Cav1.1 channel. The α2-δ1 and α2-δ2 subunits bind gabapentin and pregabalin.

Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2021.3

Nicotinic acetylcholine (ACh) receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [215, 3, 159, 225, 259]. All nicotinic receptors are pentamers in which each of the five subunits contains 4 TM domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [120]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [159]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). Nicotinic ACh receptors contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the 4 TM domains [264, 87]; see also [106]). The high resolution crystal structure of the molluscan ACh binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[35]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94Â resolution [55], has revealed the orthosteric binding site in detail (reviewed in [215, 120, 39, 198]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and β(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [96, 93, 159]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [159]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [159] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [263]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [159]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [158, 9, 118]).The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally- and/or heterologously-expressed nACh receptor subtypes [143]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses- see also [46]).

Glycine receptors in GtoPdb v.2021.3

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors and Zn2+- activated channels. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [33, 7], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [83, 93, 21]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the adult form of the receptor contains α1 (or α3) and β subunits whereas the immature form is mostly composed of only α2 subunits. The &a;pha;4 subunit is a pseudogene in humans. High resolution molecular structures are available for the α1 and α3 homomeric receptors [50, 20]. As in other Cys-loop receptors, the orthosteric binding site for agonists and the competitive antagonist strychnine is formed at the interfaces between the subunits’ extracellular domains. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. This a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [56, 54, 88]. G protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [124, 123]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [96]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [27]. Extracellular Zn2+ potentiates GlyR function at nanomolar concentrations [86]. and causes inhibition at higher micromolar concentrations (17).

GABAA receptors in GtoPdb v.2021.3.

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABAA, slow' [45]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [278, 235, 236, 283]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, β2-, β3- and γ2), along with RNA editing of the α3 subunit [71]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [359, 50]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [359], but they are classified as GABA A receptors by NC-IUPHAR on the basis of structural and functional criteria [16, 235, 236]. Many GABAA receptor subtypes contain α-, β- and γ-subunits with the likely stoichiometry 2α.2β.1γ [168, 235]. It is thought that the majority of GABAA receptors harbour a single type of α- and β - subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4- α5-or α 6-subunit, or the β1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [209, 272, 83, 19, 288]. GABA binding occurs at the β+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/β- interface ([254]; reviewed by [282]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by 'classical' benzodiazepines, such as flunitrazepam (but see [356]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [52, 140, 188, 316] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the δ subunit appear to be exclusively extrasynaptic. NC-IUPHAR [16, 235, 3, 2] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [235, 236]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [16, 95, 168, 173, 143, 278, 216, 235, 236] and [9, 10]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [359, 50, 145, 223]. Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [198].

Homomeric and Heteromeric α7 Nicotinic Acetylcholine Receptors in Health and Some Central Nervous System Diseases.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels involved in the modulation of essential brain functions such as memory, learning, and attention. Homomeric α7 nAChR, formed exclusively by five identical α7 subunits, is involved in rapid synaptic transmission, whereas the heteromeric oligomers composed of α7 in combination with β subunits display metabotropic properties and operate in slower time frames. At the cellular level, the activation of nAChRs allows the entry of Na+ and Ca2+; the two cations depolarize the membrane and trigger diverse cellular signals, depending on the type of nAChR pentamer and neurons involved, the location of the intervening cells, and the networks of which these neuronal cells form part. These features make the α7 nAChR a central player in neurotransmission, metabolically associated Ca2+-mediated signaling, and modulation of diverse fundamental processes operated by other neurotransmitters in the brain. Due to its ubiquitous distribution and the multiple functions it displays in the brain, the α7 nAChR is associated with a variety of neurological and neuropsychiatric disorders whose exact etiopathogenic mechanisms are still elusive.

Unbalanced Regulation of α7 nAChRs by Ly6h and NACHO Contributes to Neurotoxicity in Alzheimer's Disease.

α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain where they promote fast cholinergic synaptic transmission and serve important neuromodulatory functions. However, their high permeability to Ca2+ also predisposes them to contribute to disease states. Here, using transfected HEK-tsa cells and primary cultured hippocampal neurons from male and female rats, we demonstrate that two proteins called Ly6h and NACHO compete for access to α7 subunits, operating together but in opposition to maintain α7 assembly and activity within a narrow range that is optimal for neuronal function and viability. Using mixed gender human temporal cortex and cultured hippocampal neurons from rats we further show that this balance is perturbed during Alzheimer's disease (AD) because of amyloid β (Aβ)-driven reduction in Ly6h, with severe reduction leading to increased phosphorylated tau and α7-mediated neurotoxicity. Ly6h release into human CSF is also correlated with AD severity. Thus, Ly6h links cholinergic signaling, Aβ and phosphorylated tau and may serve as a novel marker for AD progression.SIGNIFICANCE STATEMENT One of the earliest and most persistent hypotheses regarding Alzheimer's disease (AD) attributes cognitive impairment to loss of cholinergic signaling. More recently, interest has focused on crucial roles for amyloid β (Aβ) and phosphorylated tau in Alzheimer's pathogenesis. Here, we demonstrate that these elements are linked by Ly6h and its counterpart, NACHO, functioning in opposition to maintain assembly of nicotinic acetylcholine receptors (nAChRs) within the physiological range. Our data suggests that Aβ shifts the balance away from Ly6h and toward NACHO, resulting in increased assembly of Ca2+-permeable nAChRs and thus a conversion of basal cholinergic to neurotoxic signaling.

ПРОГНОСТИЧЕСКИЕ МАРКЕРЫ У ПАЦИЕНТОВ С ТИМУСНЕЗАВИСИМОЙ И ТИМУСЗАВИСИМОЙ МИАСТЕНИЕЙ

Objective. To assess the presence of specific markers in patients with thymus-independent and thymus-dependent myasthenia gravis for choosing treatment tactics. Methods. The presence of specific markers was assessed in 138 patients with thymus-independent (M - myasthenia gravis without thymus changes) and thymus-dependent (MH - myasthenia gravis with thymus hyperplasia, MT - myasthenia gravis with thymoma). The method ELISA (the content of antibodies to subunits 1 and 7 nAChR in blood serum, to 7 nAChR subunit in thymocyte mitochondria, a detectablelevel of antinuclear antibody(ANA), immunofluorescence (ANA glow) and flow cytometry (expression of CD14+CD11c+and CD14 + HLA-DR +) has been used. Results. The relationship between the clinical phenotypes of myasthenia gravis and the variants of HLA diplotypes was revealed: in young patients with thymus-independent myasthenia gravis (M), a high heterogeneity of the genotypic markers HLA-DR (DR1, DR2, DR3, DR5, DR7) was detected. Patients with thymus-dependent myasthenia (MT) had only the HLA DR2 and HLA DR7 diplo- and haplotypes. The presence of HLA DR2 and HLA DR7 haplotypes in some young patients with progressive thymus-independent myasthenia gravis (M) led to the development of myasthenia gravis with thymoma (MT) in the elderly people. The pathogenic role also belongs to infection (СMV, EBV, HBV, HCV, HSV-1, HSV-2, HHV-6, mycoplasma) and food intolerance (IgE and IgG4) in the development and progression of myasthenia gravis. A four-fold prevalence of α7 subunit nicotinic acetylcholine receptors on the thymocyte mitochondria as an additional targets of autoimmune aggression in myasthenia gravis was determined. Specific antinuclear antibodies to centromere chromosome proteins were visualized in the elderly people with thymoma. Conclusion. The prognosis of the myasthenia gravis progression and the development of remission can be made using genomic (the presence of certain HLA-DR haplotypes) and molecular (ANA antibodies to centromere chromosome proteins, expression of CD20+, CD14+CD11c+, CD14+HLA-DR+) biomarkers, that can be used for the choice of treatment tactics. What this paper adds The change of complex biomarkers has been firstly studied for prognosis and choice of complex treatment tactics for young patients with progressive thymus-independent myasthenia gravis and for the elderly patients with thymus-dependent myasthenia gravis. In the presence of certain HLA-DR haplotypes, antinuclear antibodies to centromere chromosome proteins, an increasing expression of CD20+, CD14+CD11c+ and CD14+HLADR+ in some young patients with thymus-independent myasthenia gravis, analogically to biomarkers in thymus-dependent myasthenia gravis with thymoma, to perform a thymectomy is of high dangerous. potentialrisksof the procedure.

Effect of handling on ATP utilization of cerebral Na,K-ATPase in rats with trimethyltin-induced neurodegeneration.

Previously it was shown that for reduction of anxiety and stress of experimental animals, preventive handling seems to be one of the most effective methods. The present study was oriented on Na,K-ATPase, a key enzyme for maintaining proper concentrations of intracellular sodium and potassium ions. Malfunction of this enzyme has an essential role in the development of neurodegenerative diseases. It is known that this enzyme requires approximately 50% of the energy available to the brain. Therefore in the present study utilization of the energy source ATP by Na,K-ATPase in the frontal cerebral cortex, using the method of enzyme kinetics was investigated. As a model of neurodegeneration treatment with trimethyltin (TMT) was applied. Daily handling (10min/day) of healthy rats and rats suffering neurodegeneration induced by administration of TMT in a dose of (7.5mg/kg), at postnatal days 60-102 altered the expression of catalytic subunits of Na,K-ATPase as well as kinetic properties of this enzyme in the frontal cerebral cortex of adult male Wistar rats. In addition to the previously published beneficial effect on spatial memory, daily treatment of rats was accompanied by improved maintenance of sodium homeostasis in the frontal cortex. The key system responsible for this process, Na,K-ATPase, was able to utilize better the energy substrate ATP. In rats, manipulation of TMT-induced neurodegeneration promoted the expression of the α2 isoform of the enzyme, which is typical for glial cells. In healthy rats, manipulation was followed by increased expression of the α3 subunit, which is typical of neurons.

Evaluation of commercially available antibodies and fluorescent conotoxins for the detection of surface ganglionic acetylcholine receptor on the neuroblastoma cell line, IMR-32 by flow cytometry

Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use (Keefe et al., 2009; Leite et al., 2008; Lozier et al., 2015). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes (Garg and Loring, 2017).We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) (Urriola et al., 2021) that utilises the monoclonal antibody mab35(Urriola et al., 2021) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits (Vernino et al., 1998). Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use.We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.

Is neuronal inhibition or excitability controlled by Na+K+2CL– transporters?

Is neuronal inhibition or excitability controlled by Na⁺K⁺2CL^– transporters?

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

AbstractThe αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Five New Cases of Megacystis-Microcolon-Intestinal Hypoperistalsis Syndrome (MMIHS), with One Case Showing a Novel Mutation

Background Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a lethal congenital disorder characterized by a large, non-obstructed bladder, microcolon, and lack of proper peristalsis. Materials and methods Five cases of MMIHS were identified, confirmed histologically and were predominantly female (F:M, 4:1). DNA sequencing was also performed. Results Four cases showed mutations in the α3 and β4 nicotinic acetylcholine receptor (ηAChR) subunits (CHRNA3 and CHRNB4, respectively) on chromosome 15q24. The 5th case had a delayed clinical presentation of intussusception at 11 months and showed a novel missense mutation in ATP2B4 on Chromosome 1q32. Conclusion The first four patients showed a previously identified mutation. The 5th patient shows a novel mutation in ATP2B4. This novel gene was associated with a less severe presentation and increases success of multiorgan transplant than the other four patients. This highlights how identifying various mutations may impact prognosis and clinical treatment plans for MMIHS patients.

Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

SummaryAmyloid β-protein (Aβ) may contribute to worsening of Alzheimer’s disease (AD) through vascular dysfunction, but the molecular mechanism involved is unknown. Using ex vivo blood vessels and primary endothelial cells from human brain microvessels, we show that patient-derived Aβ assemblies, termed amylospheroids (ASPD), exist on the microvascular surface in patients’ brains and inhibit vasorelaxation through binding to the α3 subunit of sodium, potassium-ATPase (NAKα3) in caveolae on endothelial cells. Interestingly, NAKα3 is also the toxic target of ASPD in neurons. ASPD-NAKα3 interaction elicits neurodegeneration through calcium overload in neurons, while the same interaction suppresses vasorelaxation by increasing the inactive form of endothelial nitric oxide synthase (eNOS) in endothelial cells via mitochondrial ROS and protein kinase C, independently of the physiological relaxation system. Thus, ASPD may contribute to both neuronal and vascular pathologies through binding to NAKα3. Therefore, blocking the ASPD-NAKα3 interaction may be a useful target for AD therapy.

Synaptic Components, Function and Modulation Characterized by GCaMP6f Ca2+ Imaging in Mouse Cholinergic Myenteric Ganglion Neurons.

The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca2+ indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca2+ fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f+/ChAT+) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and β4 subunits, and most express ionotropic serotonin receptors (5-HT3Rs). Cholinergic MG neurons also display small, spontaneous Ca2+ transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na+ channel dependent impulses, presynaptic Ca2+ channels and postsynaptic receptor function reveal that the Ca2+ transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT3Rs. Electrical stimulation of axonal connectives to MG evoked Ca2+ responses in the neurons that similarly depended on nAChRs or/and 5-HT3Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca2+ transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca2+ transients and stimulus evoked Ca2+ responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT3Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca2+ imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.

Gabra6100Q allele Sprague-Dawley rats have a higher sensitivity to hypnosis induced by isoflurane and ethanol than the wild type rats

BackgroundThe neurobiological mechanisms underlying how general anesthetics render a patient’s unconsciousness (hypnosis) remains elusive. The role of the cerebellum in hypnosis induced by general anesthetics is unknown. Gabra6100Q allele Sprague-Dawley (SD) rats have a naturally occurring single nucleotide polymorphism in the GABAA receptor α6 subunit gene that is expressed exclusively in cerebellum granule cells. MethodsWe examined the loss of righting reflex (LORR) induced by isoflurane, and ethanol in Gabra6100Q rats compared with those in wild type (WT) SD rats. We also examined the change of c-Fos expression induced by isoflurane exposure in cerebellum granule cells of both mutant and WT rats. ResultsGabra6100Q rats are more sensitive than WT rats to the LORR induced by isoflurane and ethanol. Moreover, isoflurane exposure induced a greater reduction in c-Fos expression in cerebellum granule cells of Gabra6100Q rats than WT rats. ConclusionsBased on these data, we speculate that cerebellum may be involved in the hypnosis induced by some general anesthetics and thus may represent a novel target of general anesthetics.

Neuropathic Injury-Induced Plasticity of GABAergic System in Peripheral Sensory Ganglia.

GABA is a major inhibitory neurotransmitter in the mammalian central nervous system (CNS). Inhibitory GABAA channel circuits in the dorsal spinal cord are the gatekeepers of the nociceptive input from the periphery to the CNS. Weakening of these spinal inhibitory mechanisms is a hallmark of chronic pain. Yet, recent studies have suggested the existence of an earlier GABAergic “gate” within the peripheral sensory ganglia. In this study, we performed systematic investigation of plastic changes of the GABA-related proteins in the dorsal root ganglion (DRG) in the process of neuropathic pain development. We found that chronic constriction injury (CCI) induced general downregulation of most GABAA channel subunits and the GABA-producing enzyme, glutamate decarboxylase, consistent with the weakening of the GABAergic inhibition at the periphery. Strikingly, the α5 GABAA subunit was consistently upregulated. Knock-down of the α5 subunit in vivo moderately alleviated neuropathic hyperalgesia. Our findings suggest that while the development of neuropathic pain is generally accompanied by weakening of the peripheral GABAergic system, the α5 GABAA subunit may have a unique pro-algesic role and, hence, might represent a new therapeutic target.

Characterization of an α 4/7-Conotoxin LvIF from Conus lividus That Selectively Blocks α3β2 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptor (nAChR), a member of pentameric ligand-gated ion channel transmembrane protein composed of five subunits, is widely distributed in the central and peripheral nervous system. The nAChRs are associated with various neurological diseases, including schizophrenia, Alzheimer’s disease, Parkinson’s disease, epilepsy and neuralgia. Receptors containing the α3 subunit are associated with analgesia, generating our interest in their role in pharmacological studies. In this study, α-conotoxin (α-CTx) LvIF was identified as a 16 amino acid peptide using a genomic DNA clone of Conus lividus (C. lividus). The mature LvIF with natural structure was synthesized by a two-step oxidation method. The blocking potency of α-CTx lvIF on nAChR was detected by a two-electrode voltage clamp. Our results showed that α-CTx LvIF was highly potent against rα3β2 and rα6/α3β2β3 nAChR subtypes, The half-maximal inhibitory concentration (IC50) values of α-CTx LvIF against rα3β2 and rα6/α3β2β3 nAChRs expressed in Xenopus oocytes were 8.9 nM and 14.4 nM, respectively. Furthermore, α-CTx LvIF exhibited no obvious inhibition on other nAChR subtypes. Meanwhile, we also conducted a competitive binding experiment between α-CTxs MII and LvIF, which showed that α-CTxs LvIF and MII bind with rα3β2 nAChR at the partial overlapping domain. These results indicate that the α-CTx LvIF has high potential as a new candidate tool for the studying of rα3β2 nAChR related neurophysiology and pharmacology.

Sustained treatment with an α5 GABA A receptor negative allosteric modulator delays excitatory circuit development while maintaining GABAergic neurotransmission

α5 subunit GABA type A receptor (GABAAR) preferring negative allosteric modulators (NAMs) are cognitive enhancers with antidepressant-like effects. α5-NAM success in treating mouse models of neurodevelopmental disorders with excessive inhibition have led to Phase 2 clinical trials for Down syndrome. Despite in vivo efficacy, no study has examined the effects of continued α5-NAM treatment on inhibitory and excitatory synapse plasticity to identify mechanisms of action. Here we used L-655,708, an imidazobenzodiazepine that acts as a highly selective but weak α5-NAM, to investigate the impact of sustained treatment on hippocampal neuron synapse and dendrite development. We show that 2-day pharmacological reduction of α5-GABAAR signaling from DIV12-14, when GABAARs contribute to depolarization, delays dendritic spine maturation and the NMDA receptor (NMDAR) GluN2B/GluN2A developmental shift. In contrast, α5-NAM treatment from DIV19-21, when hyperpolarizing GABAAR signaling predominates, enhances surface synaptic GluN2A while decreasing GluN2B. Despite changes in NMDAR subtype surface levels and localization, total levels of key excitatory synapse proteins were largely unchanged, and mEPSCs were unaltered. Importantly, 2-day α5-NAM treatment does not alter the total surface levels or distribution of α5-GABAARs, reduce the gephyrin inhibitory synaptic scaffold, or impair phasic or tonic inhibition. Furthermore, α5-NAM inhibition of the GABAAR tonic current in mature neurons is maintained after 2-day α5-NAM treatment, suggesting reduced tolerance liability, in contrast to other clinically relevant GABAAR-targeting drugs such as benzodiazepines. Together, these results show that α5-GABAARs contribute to dendritic spine maturation and excitatory synapse development via a NMDAR dependent mechanism without perturbing overall neuronal excitability.