Reconstitution and phosphorylation of chloride channels from airway epithelium membranes

Airway epithelial chloride secretion is controlled by the apical-membrane chloride permeability. Purified apical-membrane vesicles from bovine tracheal epithelium have now been shown to contain functional chloride channels by using the planar-bilayer technique. Three types of chloride channels were observed; a voltage-dependent, calcium-independent, 71-picoSiemen (in 150 mM NaCl) channel accounted for more than 80 percent of the vesicular chloride conductance and was under strict control of phosphorylation. The channel underwent a fast rundown in less than 2 to 3 minutes of recording, and reactivation required in situ exposure to a phosphorylating "cocktail" containing the catalytic subunit of the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Mean open time and open probability were increased after phosphorylation, whereas slope conductance remained unchanged. Thus, metabolic control of tracheal chloride single channels can now be studied in vitro.     

An apical-membrane chloride channel in human tracheal epithelium

The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.     

Activation of apical chloride channels in the gastric oxyntic cell

Oxyntic cells that retain distinct morphological polarity between apical and basolateral membranes were isolated from the gastric mucosa of the amphibian Necturus. Patch-clamp techniques were applied to these cells to identify apical membrane ion channels associated with hydrochloric acid secretion. A single class of voltage-dependent, inwardly rectifying chloride channels was observed in the apical membranes of both resting and stimulated (acid-secreting) oxyntic cells. Stimulation of the cells with dibutyryladenosine 3',5'-monophosphate and isobutylmethylxanthine increased channel open probability and simultaneously increased apical membrane surface area. This chloride channel is probably responsible for electrogenic chloride secretion by the gastric mucosa and may also participate in the fluid- and enzyme-secretory functions of the oxyntic cell, analogous to the chloride channels found in the apical membranes of other exocrine cells.     

The human laminin receptor is a member of the integrin family of cell adhesion receptors

A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.     

Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle

CLC proteins transport chloride (Cl(-)) ions across cell membranes to control the electrical potential of muscle cells, transfer electrolytes across epithelia, and control the pH and electrolyte composition of intracellular organelles. Some members of this protein family are Cl(-) ion channels, whereas others are secondary active transporters that exchange Cl(-) ions and protons (H(+)) with a 2:1 stoichiometry. We have determined the structure of a eukaryotic CLC transporter at 3.5 angstrom resolution. Cytoplasmic cystathionine beta-synthase (CBS) domains are strategically positioned to regulate the ion-transport pathway, and many disease-causing mutations in human CLCs reside on the CBS-transmembrane interface. Comparison with prokaryotic CLC shows that a gating glutamate residue changes conformation and suggests a basis for 2:1 Cl(-)/H(+) exchange and a simple mechanistic connection between CLC channels and transporters.     

TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.     

Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp--specific adhesion receptors

Adhesive interactions of the platelet surface with plasma proteins such as fibrinogen and fibronectin play an important role in thrombosis and hemostasis. The binding of both of these proteins to platelets is inhibited by synthetic peptides containing the sequence Arg-Gly-Asp, which corresponds to the cell adhesion site in fibronectin and is also present in the alpha chain of fibrinogen. An affinity matrix made of an insolubilized heptapeptide containing the Arg-Gly-Asp sequence selectively binds the platelet membrane glycoprotein IIb/IIIa from detergent extracts of platelets. When incorporated into liposome membranes, the isolated protein confers to the liposomes the ability to bind to surfaces coated with fibrinogen, fibronectin, and vitronectin but not to surfaces coated with thrombospondin or albumin. This platelet receptor is related to the previously identified fibronectin and vitronectin receptors in that it recognizes an Arg-Gly-Asp sequence but differs from the other receptors in its wider specificity toward various adhesive proteins. These results establish the existence of a family of adhesion receptors that recognize the sequence Arg-Gly-Asp.     

Gating the selectivity filter in ClC chloride channels

ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion.     

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.     

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.     

Structure and function of voltage-sensitive ion channels

Voltage-sensitive ion channels mediate action potentials in electrically excitable cells and play important roles in signal transduction in other cell types. In the past several years, their protein components have been identified, isolated, and restored to functional form in the purified state. Na+ and Ca2+ channels consist of a principal transmembrane subunit, which forms the ion-conducting pore and is expressed with a variable number of associated subunits in different cell types. The principal subunits of voltage-sensitive Na+, Ca2+, and K+ channels are homologous members of a gene family. Models relating the primary structures of these principal subunits to their functional properties have been proposed, and experimental results have begun to define a functional map of these proteins. Coordinated application of biochemical, biophysical, and molecular genetic methods should lead to a clear understanding of the molecular basis of electrical excitability.     

Methylation increases sodium transport into A6 apical membrane vesicles: possible mode of aldosterone action

When isolated apical membrane vesicles prepared from cultured A6 epithelia were incubated in vitro with the methyl donor S-adenosylmethionine, the control rate of amiloride-inhibitable sodium transport was doubled. The methylation inhibitors 3-deazaadenosine and S-adenosyl homocysteine returned the S-adenosyl-methionine-stimulated sodium transport to control levels. Neither these agents nor adenosine affected sodium transport into control vesicles. In vesicles incubated with S-adenosyl-[3H-methyl]methionine, both membrane phospholipids and proteins were labeled, and this labeling was inhibited by deazaadenosine. In vesicles prepared from A6 cells treated with aldosterone, sodium transport was twice the control value and S-adenosylmethionine did not cause any further stimulation of transport. In those vesicles, both lipid and protein methylation were increased. These results suggest that methylation, which increases the rate of amiloride-sensitive sodium transport is involved in the action of aldosterone at the apical membrane level in epithelia.     

Single subunits of the GABAA receptor form ion channels with properties of the native receptor

The alpha and beta subunits of the gamma-aminobutyric acidA (GABAA) receptor were expressed individually in Xenopus oocytes by injection of RNA synthesized from their cloned DNAs. GABA-sensitive chloride channels were detected several days after injection with any one of three different alpha RNAs (alpha 1, alpha 2, and alpha 3) or with beta RNA. The channels induced by each of the alpha-subunit RNAs were indistinguishable, they had multiple conductance levels (10, 19, 28, and 42 picosiemens), and their activity was potentiated by pentobarbital and inhibited by picrotoxin. The beta channels usually expressed poorly but showed similar single channel conductance levels (10, 18, 27, and 40 picosiemens), potentiation by pentobarbital and inhibition by picrotoxin. The finding that both alpha and beta subunits, examined separately, form GABA-sensitive ion channels with permeation properties and regulatory sites characteristic of the native receptor suggests that the amino acid sequences that confer these properties are within the homologous domains shared by the subunits.     

Altered regulation of airway epithelial cell chloride channels in cystic fibrosis

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.     

Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits

Human gamma-aminobutyric acid A (GABAA) receptor subunits were expressed transiently in cultured mammalian cells. This expression system allows the simultaneous characterization of ligand-gated ion channels by electrophysiology and by pharmacology. Thus, coexpression of the alpha and beta subunits of the GABAA receptor generated GABA-gated chloride channels and binding sites for GABAA receptor ligands. Channels consisting of only alpha or beta subunits could also be detected. These homomeric channels formed with reduced efficiencies compared to the heteromeric receptors. Both of these homomeric GABA-responsive channels were potentiated by barbiturate, indicating that sites for both ligand-gating and allosteric potentiation are present on receptors assembled from either subunit.     

Absence of TGF-beta receptors and growth inhibitory responses in retinoblastoma cells

The responses of retinoblastoma tumor cells and normal retinal cells to various growth inhibitory factors were examined. Whereas fetal retinal cells were highly sensitive to the antimitogenic effects of transforming growth factor beta 1 (TGF-beta 1), retinoblastoma tumor cell lines were all resistant to this factor. Binding assays and affinity labeling of these cells with radioiodinated TGF-beta 1 revealed that the cells did not have TGF-beta receptors. The retinoblastoma cells lacked the three affinity-labeled proteins of 65, 95, and 300 kilodaltons typically seen in human cell lines and thus differed from normal retinal cells and from other types of neuroectodermal tumors that display the normal pattern of receptors. Loss of TGF-beta receptors, which is a rare event among tumor cells, may represent one mechanism through which these cells escape from negative control and form retinoblastomas.     

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.     

pH对大菱鲆幼鱼平均日增重及鳃显微结构的影响

研究不同pH对大菱鲆（Scophthalmusmaximus）幼鱼平均日增重及鳃显微结构的影响。结果显示：大菱鲆平均日增重随pH升高而降低,pH8．3和8．8组平均日增重显著低于其他处理组（P〈0．05）。pH6．3时鳃小片上皮细胞角质化较为严重;pH6．8和7．8时鳃上皮细胞未见明显变化;pH8.3时上皮细胞出现脱离,部分出现角质化;pH8．8时上皮细胞角质化较为严重。结果表明,水环境pH在6．8～7．8范围内比较适宜于大菱鲆幼鱼的健康生长。     

雾灵山物种多样性及景观格局多样性的研究

选取雾灵山10个主要森林景观类型,通过比较这些森林景观类型的物种多样性和相关的环境因子,揭示了这些景观类型物种多样性的差异,与环境因子的关系,及其空间分布规律。结果显示:各森林景观类型乔木层物种丰富度均较低,灌木层和草本层物种丰富度较高,大部分景观类型的物种丰富度均表现出:草本层>灌木层>乔木层的特点;各森林景观类型的Simpson指数和Shannon指数同物种丰富度具有相似的规律。根据平均相似度值,对这些森林景观类型进行了排序。运用亲和度分析方法测度景观格局多样性,根据平均亲和度值,可将这些森林景观类型划分为中心点、中间点和外点三部分。     

假禾谷镰孢细胞凋亡基因FpTatD的鉴定与表达分析

【目的】鉴定和克隆假禾谷镰孢(Fusarium pseudograminearum)细胞凋亡相关基因FpTatD,分析FpTatD在假禾谷镰孢菌丝、分生孢子和侵染不同时期的表达,为探索细胞凋亡在假禾谷镰孢侵染过程中的功能提供理论依据。【方法】从GenBank获得模式物种已知的TatD氨基酸序列,利用BLASTP的方法在镰孢中鉴定TatD同源蛋白,并构建进化树;分别以假禾谷镰孢的基因组DNA和cDNA为模板,通过PCR方法克隆假禾谷镰孢FpTatD的基因序列和开放阅读框（ORF）序列;利用实时荧光定量PCR方法分析FpTatD在假禾谷镰孢生长、产孢及侵染不同时期的表达;利用转录组测序方法分析FpTatD在假禾谷镰孢与感病小麦和抗病小麦互作中的表达差异。【结果】镰刀菌中有4个保守的TatD同源蛋白,与其他物种的TatD蛋白构建系统进化树,发现TatD在进化上分成两个大的分支,第一个分支的TatD在所有物种中都非常保守,包括镰刀菌的TatD1和TatD2,第二个分支的TatD蛋白属于植物和真菌特有的,包括镰刀菌的TatD3和TatD4;克隆假禾谷镰孢的FpTatD1、FpTatD2、FpTatD3、FpTatD4基因长度分别为993、1 331、1 227、1 176 bp,ORF区长度为993、1 182、1 227、1 176 bp。FpTatD2 5′端包含两段长度为94和55 bp的非编码序列,FpTatD1、FpTatD3和FpTatD4均不含非编码序列。4个FpTatD编码蛋白质的分子量大小分别为36.37、43.13、45.59、44.25 kD。蛋白结构和序列分析显示FpTatD蛋白均具有明显的DNase结构域以及大部分保守的氨基酸位点;实时荧光定量PCR分析显示,FpTatD1和FpTatD2在假禾谷镰孢中的表达量高,且在侵染初期阶段明显诱导表达,尤其是FpTatD1在侵染36 h和3 d时分别上调表达8.8倍和7.6倍;而FpTatD3和FpTatD4表达量非常低,且在不同阶段的表达量无明显变化,说明FpTatD1和FpTatD2在假禾谷镰孢中起主要作用;通过分析假禾谷镰孢与感病小麦国麦301和抗病小麦周麦24互作的转录组数据,与实时荧光定量PCR结果一致,FpTatD1和FpTatD2表达量高,并在侵染阶段上调表达,而且相对于假禾谷镰孢与感病小麦的亲和互作中,其与抗病小麦的非亲和互作中,FpTatD诱导表达倍数更高。【结论】假禾谷镰孢细胞凋亡相关基因FpTatD可能参与病原菌与寄主的互作过程。