胰岛素对蟾蜍卵母细胞膜电位及电解质的影响

应用普通玻璃微电极和离子选择性微电极,对正常及经过胰岛素处理的中华大蟾蜍卵母细胞膜电位、细胞内Na~+、K~+、Cl~-、H~+等活度及膜对Na~+、K~+的转运系数进行了测定。结果表明,胰岛素在促进蟾蜍卵母细胞发育成熟同时,具有使膜电位降低、细胞内Na~+、Cl~-活度增加、K~+、H~+活度减少及K~+转运系数降低等作用。胰岛素的上述作用可能与膜的通透性改变及膜上钠泵活性和Na~+/H~+交换的改变有关。     

沙枣幼苗根尖离子流对NaCl胁迫的响应

为了进一步从离子动态运输方面了解沙枣(Elaeagnus angustifolia)耐盐机制和揭示沙枣种源间的K~+/Na~+平衡调控差异,该研究利用非损伤微测技术(non-invasive micro-test technology,NMT)测定银川种源(盐敏感型)和阿拉尔种源(耐盐型)沙枣幼苗根系在3种不同NaCl处理方式下的离子流:1)在150 mmol·L–1 NaCl胁迫24 h后的Na~+和K~+离子流;2)NaCl瞬时处理后的K~+和H~+的动态离子流;3)先NaCl胁迫24 h,再用Na~+/H~+逆向转运体抑制剂阿米洛利(Amiloride)和K~+通道抑制剂氯化四乙胺(TEA)处理后的Na~+和K~+离子流。结果表明:NaCl胁迫24 h后,沙枣根系Na~+和K~+外排净流量显著增加,并且银川种源沙枣幼苗根系Na~+净流量显著低于阿拉尔种源,净流量分别为720和912 pmol·cm~(–2)·s~(–1),而K~+外流净流量显著高于阿拉尔种源。瞬时NaCl处理后,沙枣根系K~+的外流迅速增加,并且银川种源的K~+外排净流量始终高于阿拉尔种源,而H~+由内流转为外排,阿拉尔种源的H~+净外流量大于银川种源。NaCl和NaCl+Amiloride处理下,阿拉尔种源沙枣幼苗Na~+外流的净流量均大于银川种源,但K~+外流的净流量均小于银川种源,而在对照和NaCl+TEA处理下,Na~+和K~+的净流量在两个种源间无明显差异。研究证明NaCl胁迫造成根系Na~+积累和K~+外流,沙枣幼苗为减少Na~+积累,通过根系Na~+/H~+逆向转运体将Na~+从体内排出,并且耐盐型种源沙枣幼苗根系在NaCl胁迫时能更好地维持体内的K~+/Na~+平衡,其原因主要在于具有较强的Na~+外排能力和较弱的K~+流失。该研究可以为进一步发掘优良耐盐沙枣种质资源提供理论参考依据。     

细胞膜钠钾泵生理学

动物机体的细胞内外液中,Na~+、K~+浓度有显著差别。以神经细胞为例,静息状态下,膜内K~+浓度高于膜外约30倍,膜外Na~+浓度高于膜内约12倍。这个浓度差是产生静息电位的基础。采用微电极技术可测到各种细胞的静息电位,一般在—10———100毫伏之间。细胞靠什么机制产生并维持着如此巨大的电化学梯度呢?人们早就设想:这些细胞膜上普遍存在一种能逆着浓度差主动地将细胞外液的K~+移入膜内,同时把进入细胞内的Na~+移至膜外的机构,并称之为钠钾泵或简称钠泵。     

拟南芥内膜Na+,K+/H+反向转运体研究进展

拟南芥内膜Na,K~+/H~+反向转运体(Endosomal NHX)的亚细胞定位、离子转运特性及生物学功能阐释取得了重要进展。拟南芥内膜Na~+,K~+/H~+反向转运体包含AtNHX5和AtNHX6两个成员,它们的氨基酸序列相似性为78.7%。研究表明,AtNHX5和AtNHX6具有功能冗余,它们都定位在高尔基体(Golgi)、反面高尔基体管网状结构(TGN)、内质网(ER)和液胞前体(PVC),参与调控耐盐胁迫、pH平衡和K~+平衡等。有报道显示内膜NHXs跨膜结构域存在能够调控自身离子活性的酸性保守氨基酸残基,对其自身功能具有决定性作用。最新研究结果表明,拟南芥内膜NHXs影响囊泡运输和蛋白存贮,并参与生长素介导的植物生长和发育。文中主要对拟南芥内膜NHXs的亚细胞定位、离子转运、功能及应用进展进行了概述。     

钙对盐胁迫下冰叶日中花不同器官离子含量和根部K~+、Na~+吸收的影响

以冰叶日中花(Mesembryanthemum crystallinum L.)实生苗为材料,经NaCl、NaCl+ CaCl_2、NaCl+LaCl_3处理后,利用电感耦合等离子发射光谱仪检测叶、茎、根中Na~+、K~+、Ca~(2+)、Mg~(2+)含量,计算K~+/Na~+、Ca~(2+)/Na~+和Mg~(2+)/Na~+比值,利用非损伤微测技术测定根尖Na~+流和K~+流,研究盐胁迫下钙在维持离子平衡中的作用。结果显示,NaCl处理后,冰叶日中花各器官中Na~+含量增加,K~+、Ca~(2+)、Mg~(2+)含量降低,离子比值降低;CaCl_2处理降低了Na~+含量,提高了K~+、Ca~(2+)、Mg~(2+)含量,离子比值升高,而LaCl_3处理后的结果相反。经NaCl处理24 h后,冰叶日中花根尖Na~+和K~+明显外流,加入CaCl_2后,Na~+外流速度显著增加,K~+外流速度受到抑制,而加入LaCl_3后则降低了Na~+的外流速度,促进了K~+的外流。研究结果表明冰叶日中花受到盐胁迫后,钙参与了促进根部Na~+外排、抑制K~+外流的过程,进而保持各器官中较低的Na~+含量,表明钙在维持和调控离子平衡中起到重要作用。     

日本血吸虫转氨酶的研究

用纸上层析法测定了日本血吸虫匀浆对22种氨基酸与α-酮戊二酸的转氨作用,当用丙氨酸、精氨酸及天门冬氨酸作底物时,可明显地测出谷氨酸的生成。日本血吸虫的谷氨酸-两酮酸转氨酶(谷丙酶)及谷氨酸-草酰乙酸转氨酶(谷草酶)的活力经用比色法多次测定,所得结果波动不大,雌雄合抱虫活力的平均值(微克分子/毫克氮量/60分)谷丙酶为20.1,谷草酶为16.9。在雌雄虫分别测定中,雌虫的酶活力较高。二种酶的最适pH均为7.2—7.5,底物最适浓度α-酮戊二酸为0.02M,DL-丙氨酸及DL-天门冬氨酸同为0.2-0.4M。吐酒石、Sb-58及(月弟)芬对日本血吸虫的谷丙酶有明显的抑制作用,当吐酒石的浓度为10~(-4)M时正逆二个方向的反应均被抑制约5O%,这种抑制作用能被二巯基丁二酸钠所解除。锑剂对小白鼠肝脏的谷丙酶无作用,但对肝吸虫的作用与血吸虫相似。血吸虫的谷草酶亦能被Sb-58所抑制,但不受其他二种锑剂的影响。     

缬氨霉素对紫膜碎片双层平板脂膜(BLM)光电响应的影响

本实验中测量了缬氨霉素对双层平板膜系统的电性质和紫膜碎片BLM上光电响应的作用.实验结果表明:缬氨霉素对K~+很灵敏,它可使大豆磷脂BLM的膜电阻下降近四个量级,膜的稳定性也下降.它对Na~+也有作用,但其作用不如K~+那么明显.在缬氨霉素作用下,K~+对紫膜碎片BLM光电响应的影响十分明显,它可以使光电压完全消失.Na~+对紫膜碎片BLM的作用则比K~+的作用小得多.     

杜氏盐藻质膜ATPase在渗透调节中的可能作用

杜氏盐藻是一种以甘油为渗透调节物质的单细胞海藻,能够在0.08～5.0mol/L NaGl的培养液中生长。当外界NaGl浓度从0.5mol/L上升到4.0mol/L时,藻细胞内的Na~+和K~+含量变化不大,甘油含量则从6.20Pg/cell上升到51.50pg/cell。当藻细胞承受2.0mol/L到3.0mol/L NaCl的高渗胁迫时,能通过增加细胞内甘油含量来恢复原有形态;同时,藻细胞的H~+分泌增加,ATP含量下降;20μmol/L Na_3VO_4抑制了这些变化。KGN处理虽降低藻细胞内的ATP含量,却增加K~+外流和Na~+内渗。     

钒酸钠、叠氮钠对小麦根细胞K~+吸收和质膜K~+-Mg~(2+)-ATPase的作用

Neurospora细胞膜质子泵(H~+-ATPase)专一性抑制剂钒酸钠,抑制小麦离体根K~+的吸收与H~+分泌,并抑制小麦根细胞膜-K~+-Mg~(2+)-ATPase活力。它对K~+吸收的抑制效应,可能是抑制质膜K~+-Mg~(2+)-ATPase活力的结果。而且在起抑制作用的时间上有明显地不同,表明钒酸钠对K~+、H~+在细胞膜中的通道影响不同。叠氮钠解链小麦根的呼吸,降低根细胞的ATP水平,但从实验开始就完全抑制小麦根K~+的吸收,对质膜K~+-Mg~(2+)-ATP-ase的活力没有影响。可能叠氮钠只阻止“载体”对K~+接受的过程。应用~86R_b+示踪的K~+吸收试验表明,钒酸钠对小麦根K~+吸收的抑制%,不为增加外部溶液K~+浓度而减低。增加底物ATP浓度,也不能减低钒酸钠对质膜-ATPase的抑制%。钒酸钠的抑制作用是非竞争性抑制。~3H-亮氨酸渗入试验表明钒酸钠对“载体”的合成没有干扰作用。VO_4~(3-)离子明显促进小麦根的呼吸,并提高根细胞的ATP水平,这种ATP水平的提高,可能是质膜-ATPase受到抑制,主动运输过程减弱的结果。     

星形神经胶质细胞摄取谷氨酸与环境因子和离子运输的关系

本文以星形神经胶质细胞为对象,用同位素示踪技术较详细地研究了介质中Na、、K~+和CL~-、不同浓度的卡因酸以及几种抑制剂对L-谷氨酸摄取的影响;并观察了L-谷氨酸对星形神经胶质细胞膜运输Na~+、K~+、Cl~-和Ca~(2+)等的作用.结果表明:L-谷氨酸的摄取依赖于介质中是否存在Na~+ ,在缺Na~+介质中对Cl~-的依赖性也较明显,但在正常Na~+浓度下,含Cl~_和缺Cl~_没有明显差别.当增加介质中K~+浓度引起膜的去极化时,则能降低L~_谷氨酸的摄取.反过来,L-谷氨酸的摄取也对Na~+、K~+、Cl~-等的运输起刺激作用.此外,卡因酸及所用的几种抑制剂对谷氨酸的摄取办有明显抑制作用.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Absence of effect of aldosterone on sodium efflux catalyzed by the human erythrocyte Na+, K+-ATPase in vitro

The effect of physiological and pharmacological concentrations of aldosterone on Na+ efflux catalyzed by the human erythrocyte Na+,K+-ATPase in vitro were studied. Aldosterone had no significant effect on ouabain-sensitive Na+ efflux from fresh erythrocytes. In addition, aldosterone did not alter Na+ transport activity of stimulated Na+,K+-ATPase of Na+ loaded erythrocytes. Finally, Na+ efflux from Na+ loaded erythrocytes was not changed by preincubation of the cells with aldosterone. It is concluded that aldosterone in vitro does not modify pump activity of the human erythrocyte Na+, K+-ATPase.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

The Na+,K+,2Cl- -cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway

We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl- -cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+,K+,2Cl- -cotransport system. LK1 cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide-sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1 cells (increase in Vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)-bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)-bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1 cells present, 1.44 pmol/mg protein, specifically bound (3H)-bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)-ouabain binding sites as well as an increase in the activity of the Na+,K+-ATPase, expressed as a function of ouabain-sensitive 86Rb+ uptake. Furthermore, LK1 cells were different in the concentrations of intracellular Na+ (increases) and K+ (decreases) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.     

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

The cyclic nucleotide-gated channel, AtCNGC10, influences salt tolerance in Arabidopsis

Cyclic nucleotide-gated channels (CNGCs) in the plasma membrane transport K+ and other cations; however, their roles in the response and adaptation of plants to environmental salinity are unclear. Growth, cation contents, salt tolerance and K+ fluxes were assessed in wild-type and two AtCNGC10 antisense lines (A2 and A3) of Arabidopsis thaliana (L.) Heynh. Compared with the wild-type, mature plants of both antisense lines had altered K+ and Na+ concentrations in shoots and were more sensitive to salt stress, as assessed by biomass and Chl fluorescence. The shoots of A2 and A3 plants contained higher Na+ concentrations and significantly higher Na+/K+ ratios compared with wild-type, whereas roots contained higher K+ concentrations and lower Na+/K+ ratios. Four-day-old seedlings of both antisense lines exposed to salt stress had smaller Na+/K+ ratios and longer roots than the wild-type. Under sudden salt treatment, the Na+ efflux was higher and the K+ efflux was smaller in the antisense lines, indicating that AtCNGC10 might function as a channel providing Na+ influx and K+ efflux at the root/soil interface. We conclude that the AtCNGC10 channel is involved in Na+ and K+ transport during cation uptake in roots and in long-distance transport, such as phloem loading and/or xylem retrieval. Mature A2 and A3 plants became more salt sensitive than wild-type plants because of impaired photosynthesis induced by a higher Na+ concentration in the leaves.     

The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1-independent fashion.

We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ari?o, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.     

A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.