Expression cloning and cDNA sequencing of the Na+/glucose co-transporter

Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.     

An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel

Two electroneutral, Na+-driven HCO3- transporters, the Na+-driven Cl-/HCO3- exchanger and the electroneutral Na+/HCO3- cotransporter, have crucial roles in regulating intracellular pH in a variety of cells, including cardiac myocytes, vascular smooth-muscle, neurons and fibroblasts; however, it is difficult to distinguish their Cl- dependence in mammalian cells. Here we report the cloning of three variants of an electroneutral Na+/HCO3- cotransporter, NBCn1, from rat smooth muscle. They are 89-92% identical to a human skeletal muscle clone, 55-57% identical to the electrogenic NBCs and 33-43% identical to the anion exchangers. When expressed in Xenopus oocytes, NBCn1-B (which encodes 1,218 amino acids) is electroneutral, Na+-dependent and HCO3(-)-dependent, but not Cl(-)-dependent. Oocytes injected with low levels of NBCn1-B complementary RNA exhibit a Na+ conductance that 4,4-diisothiocyanatostilbene-2,2'-disulphonate stimulates slowly and irreversibly.     

Cloning and expression of a functional serotonin transporter from rat brain

Selective antagonism of serotonin (5-hydroxytryptamine, 5HT) and noradrenaline transport by antidepressants is a key element in the 'amine' hypothesis of affective disorders. Uptake and/or transport sites of 5HT have been reported to be reduced in platelets of patients suffering from depression and in post-mortem brain samples of depressed patients and suicide victims. To date there has been little molecular information available on the structure and regulation of 5HT transporters. Using the polymerase chain reaction with degenerate oligonucleotides derived from two highly conserved regions of the transporters for noradrenaline and gamma-aminobutyric acid (GABA), we have identified a large family of related gene products expressed in rodent brain. One of these products hybridizes to a single 3.7-kilobase RNA restricted to rat midbrain and brainstem, where it is highly enriched within the serotonergic raphe complex. Transfection with a single 2.3-kilobase brainstem complementary DNA clone is sufficient to confer expression of a Na(+)-dependent 5HT transporter upon nonneural cells, with transport selectively and potently antagonized by 5HT uptake-specific antidepressants, including paroxetine, citalopram and fluoxetine.     

苦参中钠-葡萄糖协同转运蛋白2抑制活性成分的研究

对一个含400种中草药和民族药的组分库进行活性筛选,结果显示苦参的甲醇提取物具有良好的抑制钠-葡萄糖协同转运蛋白2（SGLT2）的活性.在生物活性导向的分离纯化策略下,运用中压柱色谱和半制备高效制备液相色谱对苦参甲醇提取物的活性成分进行分离纯化,根据理化性质和波谱数据鉴定所分离化合物的结构,并对所得到的化合物进行抑制SGLT2活性测试.从苦参的甲醇提取物中分离得到7个黄酮苷类化合物,鉴定为芒柄花素-7-O--D-吡喃木糖基-(16)--D-吡喃葡萄糖苷（1）、芒柄花素-7-O--D-呋喃芹糖基-(16)--D-吡喃葡萄糖苷（2）、三叶豆紫檀苷（3）、(-)-高丽槐素-3-O--D-吡喃半乳糖苷（4）、6-乙酰三叶豆紫檀苷（5）、罗思菌素（6）、芒柄花苷（7）.除化合物3外,其他化合物均为首次从苦参中分离得到.7个化合物均有抑制SGLT2的活性,其中具有高丽槐素苷元结构的苷类成分3～5具有较好的抑制SGLT2的活性,对SGLT2的抑制活性的IC50范围为2.61～4.47 mol/L.     

Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.     

肠道胆固醇转运关键蛋白NPC1L1的研究进展

Niemann-Pick C1Like1(NPC1L1)是肠道吸收胆固醇的关键转运蛋白.NPC1L1存在于小肠上皮细胞刷状缘膜上.影响着胆固醇吸收和血浆低密度脂蛋白水平,是维持体内胆固醇代谢平衡的重要因素.同时也是新型降脂药物依泽替米贝的作用靶点.     

Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger

Na+/Ca2+ exchange is electrogenic and moves one net positive charge per cycle. Although the cardiac exchanger has a three-to-one Na+/Ca2+ stoichiometry, details of the reaction cycle are not well defined. Here we associate Na+ translocation by the cardiac exchanger with positive charge movement in giant membrane patches from cardiac myocytes and oocytes expressing the cloned cardiac Na+/Ca2+ exchanger. The charge movements are initiated by step increments of the cytoplasmic Na+ concentration in the absence of Ca2+. Giant patches from control oocytes lack both steady-state Na+/Ca2+ exchange current (INaCa) and Na(+)-induced charge movements. Charge movements indicate about 400 exchangers per micron 2 in guinea-pig sarcolemma. Fully activated INaCa densities (20-30 microA cm-2) indicate maximum turnover rates of 5,000 s-1. As has been predicted for consecutive exchange models, the apparent ion affinities of steady state INaCa increase as the counterion concentrations are decreased. Consistent with an electroneutral Ca2+ translocation, we find that voltage dependence of INaCa in both directions is lost as Ca2+ concentration is decreased. The principal electrogenic step seems to be at the extracellular end of the Na+ translocation pathway.     

Role of Ca(2+)-dependent regulator protein in intestinal secretion

AFTER exposure to secretagogues the small intestine changes from a tissue that absorbs fluid and electrolyte from lumen to blood into a tissue that secretes electrolyte and fluid into the lumen(1-4). It has been shown that this secretion results from an increase in the passive Cl(-) permeability of the mucosal border, which permits Nad to leak passively from the lateral intercellular spaces, where it is present at hypertonic concentrations(5), into the mucosal bathing solution. Na(+) and water, electroosmotically coupled to Na(+) movement, leak through the tight junctions(1,2), and Cl(-) leaks through relatively anhydrous anion-selective channels, induced withira the mucosal border by secretagogues. The increased reflux of NaCl from the lateral intercellular space accounts for both the apparent decrease in electroneutral NaCl uptake across the mucosal border induced by secretagogues and the apparent increase in active CP secretion and short-circuit current(3,6,7). We have investigated the mechanism by which intestinal secretagogues increase passive Cl(-) permeability and thereby cause secretion. Cl(-) permeability is increased by several secretagogues, some of which, such as theophylline and choleragen, increase intracellular cyclic AMP concentration, and others, such as A23187, the Ca(2+) ionophore, or carbachol, do not(8). Thus there has been no known common mode of secretory induction. To investigate this problem we used two drugs that prevent intestinal secretion in vitro, RMI 12330A (Richardson Merrell), and the antipsychotic pheno-thiazine trifluoperazine (Stelazine, Smith, Kline and French). RMI 12330A prevents secretion by inhibiting choleragen-induced adenylyl cyclase activity(9). Stelazine inhibits phosphodiesterase in tissues(11,12) by preventing the activation of the enzyme by Ca(2+)-dependent regulator protein, CDR. We report here that it also inhibits Cl(-) secretion and binds to CDR.     

甘蔗条螟性外激素分泌腺研究

本文通过扫描电镜及光学显微镜观察了甘蔗条螟性外激素分泌腺的形态及组织的结构.甘蔗条螟性外激素腺十分发达,位于腹部第8—9节背面的节间膜上,是由该处节间膜部分细胞特化而成,属褶叠型腺体。外翻后,腺体的切面呈半球形.同时还观察描述了腺体各部份的形态结构.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

紫贻贝配子细胞表面糖基的定位研究

细胞表面糖蛋白中糖的特性对于细胞的相互识别起重要作用,配子细胞表面糖配基的差异恰恰体现不同物种间受精作用的特异性,本研究分别选用可以特异性结合半乳糖、葡萄糖、甘露糖、岩藻糖的FITC标记凝集素,对紫贻贝配子细胞中对应的糖进行研究。结果显示,4种糖在卵细胞边缘的标记特征不明显,仅半乳糖和葡萄糖在一些卵细胞边缘有区域性分布,但在细胞质区,这4种糖均有少量分布;半乳糖和岩藻糖在贻贝的精巢的精细胞和成熟精子区为较均匀的弱阳性标记,甘露糖仅在输精小管边缘有分布,葡萄糖的含量相对较多,但也只是在输精小管边缘以及成熟精子区有一定的标记。上述结果表明,紫贻贝配子细胞表面不同糖基的分布和含量存在一定的差异,造成配子间起到识别作用的糖蛋白组成和结构不同,决定了紫贻贝配子识别的特异性。     

转海蓬子Na+/H+逆向运输蛋白基因番茄的耐盐性研究

以转海蓬子Na /H 逆向运输蛋白基因(Nhap)的番茄(Lycopersicon esculentum Mill.)阳性植株和对照植株为材料,测定叶片相对含水量、质膜透性、K /Na 比值和叶绿素含量4项耐盐指标.结果表明:在盐胁迫条件下,转基因植株的相对含水量、 K /Na 比值和叶绿素含量明显高于对照植株,而叶片质膜透性明显低于对照植株,说明Nhap基因已经在番茄转化植株体内表达,提高了转基因植株的耐盐性.     

外源甜菜碱对两种玉米耐盐性影响的研究

以玉米沈单和农大108为实验材料,研究了外源甜菜碱对盐分胁迫条件下玉米(Zea may)幼苗水势、叶绿素含量、丙二醛(MDA)含量、细胞膜透性、cNa /cK 等的影响.结果表明,外源甜菜碱能够提高玉米幼苗叶片水势、叶绿素含量,降低膜透性、丙二醛含量,参与多种代谢调节,影响作物的耐盐性.另外,外源甜菜碱可以增强根系对Na ,K 的选择吸收能力,促进质膜主动排Na 过程,降低盐胁迫下叶片中Na 含量.     

Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ～10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30?? away, in a manner that allows the Na(+) gradient to power the transport mechanism.     

单引物PCR扩增DNA指纹图谱的稳定性研究

以人胶原蛋白基因下游一段ＤＮＡ的互补序列设计引物，对人染色体ＤＮＡ进行单引物ＰＣＲ扩增，在低温退火的条件下，这种引物与ＤＮＡ模板的一处完全配对，并且可以随机地结合在其他一些位置上。由此进行单引物ＰＣＲ扩增，它们的扩增产物形成了稳定的引物－模板特异的ＤＮＡ指纹图谱。本文所建立的单引物ＰＣＲ扩增技术在类似的研究中均可获得稳定的实验结果，具有一定的应用价值。     

Arginine vasopressin enhances pHi regulation in the presence of HCO3- by stimulating three acid-base transport systems

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.     

Amplification of P-glycoprotein genes in multidrug-resistant mammalian cell lines

The multidrug-resistance phenotype expressed in mammalian cell lines is complex. Cells selected with a single agent can acquire cross-resistance to a remarkably wide range of compounds which have no obvious structural or functional similarities. The basis for cross-resistance seems to be a decreased net cellular accumulation of the drug involved, and has been attributed to alterations in the plasma membrane. An over-expressed plasma membrane glycoprotein of relative molecular mass (Mr) 170,000 (P-glycoprotein) is consistently found in different multidrug-resistant human and animal cell lines, and in transplantable tumours. Consequently, it has been postulated that P-glycoprotein directly or indirectly mediates multidrug resistance. Here we report the cloning of a complementary DNA encoding P-glycoprotein. Southern blot analysis of hamster, mouse and human DNA using this cDNA as a probe showed that P-glycoprotein is conserved and is probably encoded by a gene family, and that members of this putative family are amplified in multidrug-resistant cells.     

alpha-Actinin-containing branched microvilli isolated from an ascites adenocarcinoma

Microvilli, slender projections approximately 0.1 micrometer in diameter which occur on the surfaces of many cell types, are bounded by plasma membrane except at the site of attachment to the cell body and contain microfilament bundle cores. The presence of both microfilaments and plasma membrane suggests the use of microbilli for investigations of membrane cytoskeleton interactions. Immunofluorescence studies with anti-alpha-actinin have suggested that alpha-actinin is concentrated at the tips of intestinal brush border microvilli and might link actin microfilaments and the plasma membrane. However, this idea was disputed by later immunofluorescence and electrophoresis studies. To investigate the components and organization of microvilli from a less highly differentiated cell type, we have used an ascites sub-line (MAT-Cl) of a rat mammary tumour, the 13762 mammary adenocarcinoma, whose microvilli are high branched. Becaused such unusual structures may provide an understanding of cell-surface assemblies important in determining cell morphology, we have developed a procedure for isolating the branched microvilli and have shown that they contain significant quantities of alpha-actinin.     

Sodium-dependent uptake of inorganic phosphate by the intracellular malaria parasite

As the malaria parasite, Plasmodium falciparum, grows within its host erythrocyte it induces an increase in the permeability of the erythrocyte membrane to a range of low-molecular-mass solutes, including Na+ and K+ (ref. 1). This results in a progressive increase in the concentration of Na+ in the erythrocyte cytosol. The parasite cytosol has a relatively low Na+ concentration and there is therefore a large inward Na+ gradient across the parasite plasma membrane. Here we show that the parasite exploits the Na+ electrochemical gradient to energize the uptake of inorganic phosphate (P(i)), an essential nutrient. P(i) was taken up into the intracellular parasite by a Na+-dependent transporter, with a stoichiometry of 2Na+:1P(i) and with an apparent preference for the monovalent over the divalent form of P(i). A P(i) transporter (PfPiT) belonging to the PiT family was cloned from the parasite and localized to the parasite surface. Expression of PfPiT in Xenopus oocytes resulted in Na+-dependent P(i) uptake with characteristics similar to those observed for P(i) uptake in the parasite. This study provides new insight into the significance of the malaria-parasite-induced alteration of the ionic composition of its host cell.