The beta2 subunit inhibits stimulation of the alpha1/beta1 form of soluble guanylyl cyclase by nitric oxide. Potential relevance to regulation of blood pressure.

Cytosolic guanylyl cyclases (GTP pyrophosphate-lyase [cyclizing; EC 4.6.1.2]), primary receptors for nitric oxide (NO) generated by NO synthases, are obligate heterodimers consisting of an alpha and a beta subunit. The alpha1/beta1 form of guanylyl cyclase has the greatest activity and is considered the universal form. An isomer of the beta1 subunit, i.e., beta2, has been detected in the liver and kidney, however, its role is not known. In this study, we investigated the function of beta2. Immunoprecipitation experiments showed that the beta2 subunit forms a heterodimer with the alpha1 subunit. NO-stimulated cGMP formation in COS 7 cells cotransfected with the alpha1 and beta2 subunits was approximately 1/3 of that when alpha1 and beta1 subunits were cotransfected. The beta2 subunit inhibited NO-stimulated activity of the alpha1/beta1 form of guanylyl cyclase and NO-stimulated cGMP formation in cultured smooth muscle cells. Our results provide the first evidence that the beta2 subunit can regulate NO sensitivity of the alpha1/beta1 form of guanylyl cyclase. Northern analysis for guanylyl cyclase subunits was performed on RNA from kidneys of Dahl salt-sensitive rats, which have been shown to have decreased renal sensitivity to NO. Compared to the Dahl salt-resistant rat, message for beta2 was increased, beta1 was decreased, and alpha1 was unchanged. These results suggest a molecular basis for decreased renal guanylyl cyclase activity, i.e. , an increase in the alpha1/beta2 heterodimer, and decrease in the alpha1/beta1 heterodimer.     

Roles for nicotinic acetylcholine receptor subunit large cytoplasmic loop sequences in receptor expression and function

To evaluate possible physiological roles of the large cytoplasmic loops (C2) and neighboring transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits, we generated novel fusion constructs in which human nAChR alpha4, beta2, or beta4 subunit C2 or C2 and neighboring sequences were replaced by corresponding sequences from the mouse serotonin type 3A (5-HT3A) receptor subunit. Following stable expression in human SH-EP1 cells, we found that extensive sequence substitutions involving third and fourth transmembrane domains and neighboring "proximal" C2 sequences (e.g., beta2 H322-V335 and V449-R460) did not allow functional expression of nAChR containing chimeric subunits. However, expression of functional nAChR was achieved containing wild-type alpha4 subunits and chimeric beta2 (beta2chi) subunits whose "nested" C2 domain sequences K336-S448 were replaced with the corresponding 5-HT3A subunit sequences. Whereas these findings suggested indispensable roles for M3/M4 transmembrane and/or proximal C2 sequences in alpha4beta2-nAChR function, nested C2 sequences in the beta2 subunit are not essential for functional receptor expression. Ligand-binding analyses also revealed only subtle differences in pharmacological profiles of alpha4beta2-nAChR compared with alpha4beta2chi-nAChR. Nevertheless, there was heightened emergence of agonist-mediated self-inhibition of alpha4beta2chi function, greater sensitivity to functional blockade by a number of antagonists, and faster and more complete acute desensitization of alpha4beta2chi-nAChR than for alpha4beta2-nAChR. These studies are consistent with unexpected roles of nested C2 sequences in nAChR function.     

Structural determinants of the pharmacological properties of the GABAA receptor alpha6 subunit

GABA(A) receptors are responsible for fast inhibitory neurotransmission in the mammalian brain and are the targets for many clinical drugs that act as antiepileptics, anxiolytics, and sedatives. The pharmacological characteristics of the receptor are largely determined by its subunit composition. Compared with all other alpha subtypes, the alpha6 subtype confers unique pharmacological properties. In particular, alpha6-containing receptors are more sensitive to both the agonist GABA and the antagonist amiloride. Results from chimeric constructs of the alpha1 and alpha6 subunits suggested that structural differences within the extracellular N-terminal domain were responsible for both these characteristics. Within this domain, we examined 15 amino acid residues unique to the alpha6 subtype. Each of these sites was individually mutated in the alpha6 subunit to the corresponding residue of the alpha1 subunit. The mutated subunits were expressed in human embryonic kidney-293T cells along with wild-type beta3 and gamma2L subunits and sensitivity to GABA and amiloride determined with whole-cell electrophysiological recordings. Serine83 in the alpha6 subunit influenced sensitivity to both GABA and amiloride. Leucine174 and tyrosine175 were also found to contribute to inhibition by amiloride but did not affect GABA sensitivity. These structural differences are at least partly responsible for the unique pharmacological properties associated with the alpha6 subunit.     

Analyses of Na, K-ATPase subunit genes]

Na, K-ATPase is the enzyme responsible for the maintenance of the electrochemical gradients of Na+ and K+ across the plasma membrane. The enzyme consists of a heterodimer of an alpha and a beta subunits. Na, K-ATPase belongs to a multigene family. Three alpha subunit (alpha 1 alpha 2 alpha 3) and three beta subunit (beta 1 beta 2 beta 3) isoforms have been described so far. The gene expression of each subunit is differentially regulated in various tissues and developmental stages. To clarify the molecular mechanisms of the regulation of Na, K-ATPase gene expression and of the coordinated regulation between the alpha and beta subunit genes, extensive analyses of the genes encoding alpha and beta subunit isoforms have been performed. The rapid progress of the study in this field is reviewed.     

LFA-1 immunodeficiency disease. Definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte function-associated antigen 1 (LFA-1) by complementation in hybrid cells

Lymphocyte function associated antigen 1 (LFA-1) is a leukocyte cell adhesion protein. We have studied a novel human immunodeficiency disease in which LFA-1 and two other proteins which share the same beta subunit are lacking from the surface of leukocytes. The basis of the inherited defect in cell surface expression of both the alpha and beta subunits of LFA-1 was determined by somatic cell fusion of patient or normal human cells with an LFA-1+ mouse T cell line. Human LFA-1 alpha and beta subunits from normal cells could associate with mouse LFA-1 subunits to form interspecies hybrid alpha beta complexes. Surface expression of the alpha but not the beta subunit of patient cells was rescued by the formation of interspecies complexes. The findings show that the LFA-1 alpha subunit in genetically deficient cells is competent for surface expression in the presence of an appropriate beta subunit, and suggest that the genetic lesion affects the beta subunit. The human LFA-1 alpha and beta subunits were mapped to chromosomes 16 and 21, respectively. The genetic defect is inferred to be on chromosome 21.     

Molecular and biochemical basis of intermediate maple syrup urine disease. Occurrence of homozygous G245R and F364C mutations at the E1 alpha locus of Hispanic-Mexican patients.

Maple syrup urine disease (MSUD) is caused by a deficiency of the mitochondrial branched-chain alpha-keta acid dehydrogenase (BCKAD) complex. The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha 2 beta 2) structure. Four unrelated Hispanic-Mexican MSUD patients with the intermediate clinical phenotype were diagnosed 7 to 22 mo after birth during evaluation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The remaining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene product. Transfection studies in E1 alpha-deficient lymphoblasts indicate that both G245R and F364C mutant E1 alpha subunits were unable to significantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficiently rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chaperone-augmented bacterial overexpression system. The kinetics of initial assembly of the G245R E1 alpha subunit with the normal E1 beta was shown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins were produced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameric assembly and function of the BCKAD complex. Moreover, the results suggest that the G245R mutant E1 alpha allele may be important in the Hispanic-Mexican population.     

The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells

Fc epsilon RI is a tetrameric receptor, composed of a ligand recognition subunit, alpha, a beta chain, and dimeric gamma chains. Previous studies have indicated that the dimeric gamma chain is associated with Fc gamma RIIIA (CD16) on natural killer cells and macrophages as well as the clonotypic T cell receptor. Here we show that in mast cells, in addition to the dimeric gamma chains, the beta subunit is associated not only with Fc epsilon RI, but also with Fc gamma RIIIA. Functional reconstitution studies with a mastocytoma cell line indicate that Fc gamma RIIIA composed of alpha, beta, and gamma subunits has the capacity for signal transduction. These studies suggest that through the association of alternative ligand recognition subunits (alpha epsilon, alpha gamma), a common signal transduction complex (beta gamma 2) mediates similar biochemical and effector functions in response to immunoglobulin G (IgG) and IgE.     

人粒-巨噬细胞集落刺激因子受体在NIH3T3细胞中的功能表达

目的　探讨人类粒巨噬细胞集落刺激因子（ＧＭＣＳＦ） 受体（ＧＭＲ） 在ＮＩＨ３Ｔ３ 细胞中表达的功能特性。方法　将编码人ＧＭＲα和β亚单位的ｃＤＮＡ 转染到无人ＧＭＲ表达的小鼠ＮＩＨ３Ｔ３ 细胞中,并检测其阳性转染子在配体刺激后的增殖信号传导与酪氨酸磷酸化。结果　重建的功能性ＧＭＲα／β可以介导细胞增殖与细胞集落形成,并诱导βｃ、Ｊａｋ２、Ｓｈｃ 及Ｓｈｃ 相关蛋白Ｐ１４５（ＳＨＩＰ） 酪氨酸磷酸化,然而,人ＧＭＣＳＦ并不能激活仅含ＧＭＲα的ＮＩＨ３Ｔ３ 细胞出现有丝分裂信号表达。结论　人ＧＭＲα与β亚单位同时转染入ＮＩＨ３Ｔ３ 细胞后,可通过βｃ 磷酸化,激活Ｊａｋ２ 、Ｓｈｃ 和ＳＨＩＰ信号传导途径,而导致配体依赖性细胞生长与集落形成     

Molecular and pharmacological characterization of GABA(A) receptor alpha1 subunit knockout mice

GABA(A) receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain alpha1 subunits. GABA(A) receptor alpha1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA(A) receptor alpha1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of alpha1 subunit-containing GABA(A) receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the alpha1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of alpha1 subunit peptide expression. Stable adaptations in the expression of several GABA(A) receptor subunits are observed in the fifth to seventh generations, including decreased expression of beta2/3 and gamma2 subunits and increased expression of alpha2 and alpha3 subunits. There was no change in alpha4, alpha5, or delta subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA(A) receptors measured by [(3)H]muscimol, [(3)H]2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([(3)H]SR-95531), and t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding. [(3)H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([(3)H]Ro15-4513) binding is reduced by variable amounts in different regions across brain. GABA(A) receptor alpha1(-/-) mice lose all high-affinity [(3)H]zolpidem binding and about half of [(3)H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes are reduced in alpha1(-/-) mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of alpha1 subunit expression and its involvement in the regulation of CNS excitability.     

Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.     

Identification of residues that confer alpha-conotoxin-PnIA sensitivity on the alpha 3 subunit of neuronal nicotinic acetylcholine receptors

Neuronal nicotinic receptors composed of the alpha3 and beta2 subunits are at least 1000-fold more sensitive to blockade by alpha-conotoxin-PnIA than are alpha2beta2 receptors. A series of chimeric subunits, formed from portions of alpha2 and alpha3, were coexpressed with beta2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of alpha3. Analysis of receptors formed by a series of mutant alpha3 subunits, in which residues that differ between alpha3 and alpha2 were changed from what occurs in alpha3 to what occurs in alpha2, allowed identification of three determinants of alpha-conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of alpha-conotoxin-MII and kappa-bungarotoxin sensitivity on the alpha3 subunit revealed overlapping, but distinct, arrays of determinants for each of these three toxins. When tested against an EC50 concentration of acetylcholine, the IC50 for alpha-conotoxin-PnIA blockade was 25 +/- 4 nM for alpha3beta2, 84 +/- 7 nM for alpha3P182Tbeta2, 700 +/- 92 nM for alpha3I188Kbeta2, and 870 +/- 61 nM for alpha3Q198Pbeta2. To examine the location of these residues within the receptor structure, we generated a homology model of the alpha3beta2 receptor extracellular domain using the structure of the acetylcholine binding protein as a template. All three residues are located on the C-loop of the alpha3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.     

CD8 beta chain influences CD8 alpha chain-associated Lck kinase activity

The CD8 molecule plays an important role in the differentiation of CD8+ T cells in the thymus and in their normal function in the periphery. CD8 exists on the cell surface in two forms, the alpha alpha homodimer and the alpha beta heterodimer. Recent studies indicate an important role for the CD8 beta chain in thymic development of CD8+ T cells and suggest that signaling via CD8 alpha beta may be distinct from CD8 alpha alpha. To better understand these differences, we introduced the CD8 beta gene into a T cell hybridoma which only expressed the CD8 alpha alpha homodimer. In the parent hybridoma, cross-linking of the CD8 alpha chain led to minimal enhancement of CD8-associated Lck tyrosine kinase activity. In the CD8 beta+ transfectants, several observations suggested that CD8 beta modifies CD8 alpha-associated Lck tyrosine kinase activity: (a) in in vitro kinase assays, antibody- mediated crosslinking of CD8 alone, or CD8 cross-linking with the TCR, resulted in 10-fold greater activation of Lck kinase activity, compared to cells expressing CD8 alpha alpha alone; (b) in vivo, markedly enhanced tyrosine phosphorylation of several intracellular proteins was observed upon CD8 cross-linking with the TCR in CD8 alpha beta- expressing cells, compared to cells expressing CD8 alpha alpha alone; and (c) Lck association with CD8 alpha was stabilized by the coexpression of CD8 beta. Thus, the differential Lck kinase activation and tyrosine phosphorylation seen with CD8 alpha alpha vs. CD8 alpha beta may reflect the unique signaling capabilities of the CD8 beta molecule. These differences in signaling may, in part, account for the diminished ability to generate CD8 single positive thymocytes in mice bearing a homozygous disruption of the CD8 beta gene.     

Detection of receptors for interleukin-6, interleukin-11, leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in bone marrow stromal/osteoblastic cells.

The functional receptor complexes assembled in response to interleukin-6 and -11 (IL-6 and IL-11), leukemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), all involve the signal transducer gp130: IL-6 and IL-11 induce homodimerization of gp130, while the rest heterodimerize gp130 with other gp130-related beta subunits. Some of these cytokines (IL-6, IL-11, and CNTF) also require a specificity-determining alpha subunit not directly involved in signaling. We have searched for functional receptor complexes for these cytokines in cells of the bone marrow stromal/osteoblastic lineage, using tyrosine phosphorylation of the beta subunits as a detection assay. Collectively, murine calvaria cells, bone marrow-derived murine cell lines (+/+LDA11 and MBA13.2), as well as murine (MC3T3-E1) and human (MG-63) osteoblast-like cell lines displayed all the previously recognized alpha and beta subunits of this family of receptors. However, individual cell types had different constellations of alpha and beta subunits. In addition and in difference to the other cell types examined, MC3T3-E1 cells expressed a heretofore unrecognized form of gp130; and MG-63 displayed an alternative form (type II) of the OSM receptor. These findings establish that stromal/osteoblastic cells are targets for the actions of all the members of the cytokine subfamily that shares the gp130 signal transducer; and suggest that different receptor repertoires may be expressed at different stages of differentiation of this lineage.     

Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle.

Integrins are a class of cell adhesion molecules that participate in cell-cell and cell-substratum interactions and are present on essentially all human cells. The distribution of nine different alpha and beta integrin subunits in human endometrial tissue at different stages of the menstrual cycle was determined using immunoperoxidase staining. Glandular epithelial cells expressed primarily alpha 2, alpha 3, and alpha 6 (collagen/laminin receptors), while stromal cells expressed predominantly alpha 5 (fibronectin receptor). The presence of alpha 1 on glandular epithelial cells was cycle specific, found only during the secretory phase. Expression of both subunits of the vitronectin receptor, alpha v beta 3, also underwent cycle specific changes on endometrial epithelial cells. Immunostaining for alpha v increased throughout the menstrual cycle, while the beta 3 subunit appeared abruptly on cycle day 20 on luminal as well as glandular epithelial cells. Discordant luteal phase biopsies (greater than or equal to 3 d "out of phase") from infertility patients exhibited delayed epithelial beta 3 immunostaining. These results demonstrate similarities, as well as specific differences, between endometrium and other epithelial tissues. Certain integrin moieties appear to be regulated within the cycling endometrium and disruption of integrin expression may be associated with decreased uterine receptivity and infertility.     

Sex steroid hormone regulation of follicle-stimulating hormone subunit messenger ribonucleic acid (mRNA) levels in the rat.

Follicle-stimulating hormone (FSH) beta, luteinizing hormone (LH) beta, and alpha subunit messenger RNA (mRNA) levels were examined in rats after castration and sex-steroid replacement. Subunit mRNAs were determined by blot hybridization using rat FSH beta genomic DNA, and alpha and LH beta complementary DNA (cDNA). Rat FSH beta mRNA is 1.7 kilobase in size. After ovariectomy, female FSH beta mRNA levels increased fourfold, whereas those of LH beta and alpha increased twenty- and eightfold, respectively. With estradiol, all subunits returned toward normal levels. Male LH beta and alpha mRNA levels rose eight- and fourfold, respectively, 40 d postcastration, but FSH beta mRNA levels increased minimally. After 7 d of testosterone propionate, LH beta and alpha mRNAs declined to normal levels, whereas FSH beta mRNA increased slightly. We conclude that in female rats FSH beta is negatively regulated by gonadal steroids, but to a lesser extent than LH beta or alpha mRNAs, and there is a differential regulation of FSH beta mRNA levels in males as compared with females at the time points examined.     

The delta subunit of gamma-aminobutyric acid type A receptors does not confer sensitivity to low concentrations of ethanol

GABA(A) receptors (GABA(A)Rs) are usually formed by alpha, beta, and gamma or delta subunits. Recently, delta-containing GABA(A)Rs expressed in Xenopus oocytes were found to be sensitive to low concentrations of ethanol (1-3 mM). Our objective was to replicate and extend the study of the effect of ethanol on the function of alpha4beta3delta GABA(A)Rs. We independently conducted three studies in two systems: rat and human GABA(A)Rs expressed in Xenopus oocytes, studied through two-electrode voltage clamp; and human GABA(A)Rs stably expressed in the fibroblast L(tk-) cell line, studied through patch-clamp electrophysiology. In all cases, alpha4beta3delta GABA(A)Rs were only sensitive to high concentrations of ethanol (100 mM in oocytes, 300 mM in the cell line). Expression of the delta subunit in oocytes was assessed through the magnitude of the maximal GABA currents and sensitivity to zinc. Of the three rat combinations studied, alpha4beta3 was the most sensitive to ethanol, isoflurane, and 5alpha-pregnan-3alpha,21-diol-20-one (THDOC); alpha4beta3delta and alpha4beta3gamma(2S) were very similar in most aspects, but alpha4beta3delta was more sensitive to GABA, THDOC, and lanthanum than alpha4beta3gamma(2S) GABA(A)Rs. Ethanol at 30 mM did not affect tonic GABA-mediated currents in dentate gyrus reported to be mediated by GABA(A)Rs incorporating alpha4 and delta subunits. We have not been able to replicate the sensitivity of alpha4beta3delta GABA(A)Rs to low concentrations of ethanol in four different laboratories in independent studies. This suggests that as yet unidentified factors may play a critical role in the ethanol effects on delta-containing GABA(A)Rs.     

Alpha subunit of glycoprotein hormone: presence in peripheral serum after LHRH.

A method is described for the selective measurement of human luteinizing hormone (hLH) and alpha subunit. The assays employ highly purified tracers of hLH and of subunit of human chorionic gonadotropin (hCGalpha) and a "mixed population" of antibody: Population I (directed against determinants on beta subunit of hCG) and Population II (directed against determinants on alpha subunit of hCG). The former is present in greater concentration than the latter. When the mixed antibody is used at higher dilution (1:1.2x10(6)), Population II is effectively diluted out, and using 125I-labelled hLH as tracer, the assay recognize hLH, hCG and their beta subunits 20-50 times more sensitively than hCGalpha. When the mixed antibody is used at fivefold higher concentration, Population I is present in relative excess and acts as a "sink" for hCG, hLH and their beta subunits. Under these conditions, using 125I-hCGalpha as tracer, the assay recognize the alpha subunit 20 to 50-fold more sensitively than hLH and hCG. These assays have been employed in the study of sera which have been filtered over Sephadex G-100. Alpha subunit was detected in serum within minutes after intravenous injection of luteinizing hormone releasing hormone (LHRH) in four subjects tested.     

Mutation causing congenital myasthenia reveals acetylcholine receptor beta/delta subunit interaction essential for assembly

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the beta subunit gene. One mutation causes skipping of exon 8, truncating the beta subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (beta426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing beta426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that beta426delEQE impairs AChR assembly by disrupting a specific interaction between beta and delta subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the beta subunit is crucial for interaction with the delta subunit. The findings imply that the mutated residues are positioned at the interface between beta and delta subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.     

Deletion of the alpha1 or beta2 subunit of GABAA receptors reduces actions of alcohol and other drugs

Enhancement of the activation of GABAA receptors is a common feature of many sedative and hypnotic drugs, and it is probable that the GABAA receptor complex is a molecular target for these drugs in the mammalian central nervous system. We set out to elucidate the role of the two predominant (alpha1 and beta2) subunits of GABAA receptor in sedative drug action by studying mice lacking these two subunits. Both alpha1 (-/-) and beta2 (-/-) null mutant mice showed markedly decreased sleep time induced by nonselective benzodiazepine, flurazepam, and GABAA agonist, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol. The sleep time induced by the beta-selective drug etomidate was decreased only in beta2 (-/-) knockout mice. In contrast, alpha1 (-/-) mice were more resistant to the alpha1-selective drug zolpidem than beta2 (-/-) or wild-type animals. Knockout mice of both strains were similar to wild-type mice in their responses to pentobarbital. The duration of loss of the righting reflex produced by ethanol was decreased in male mice for both null alleles compared with wild-type mice, but there were no differences in ethanol-induced sleep time in mutant females. Deletion of either the alpha1 or beta2 subunits reduced the muscimol-stimulated 36Cl36 influx in cortical microsacs suggesting that these mutant mice have reduced number of functional brain GABAA receptors. Our results show that removal of either alpha1 or beta2 subunits of GABAA receptors produce strong and selective decreases in hypnotic effects of different drugs. Overall, these data confirm the crucial role of the GABAA receptor in mechanisms mediating sedative/hypnotic effects.