Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.     

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.     

External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts

Coupled active transport of Na+ and K+ across cellular plasma membranes is mediated by (Na+ + K+)-stimulated Mg2+-dependent ATPase. Active cation transport by this Na pump involves a cyclic Na-dependent phosphorylation of the enzyme by intracellular ATP and hydrolytic dephosphorylation of the phosphoenzyme, stimulated by K+ (ref. 1). In human red blood cells, skeletal muscle and squid axons, replacement of extracellular K by Na results in a ouabain-sensitive efflux of Na coupled to an influx of extracellular Na. There is apparently no net Na movement nor net hydrolysis of ATP. The rate of Na:Na exchange is stimulated by increased levels of ADP and exchange transport is not observed in cells totally depleted of intracellular ATP. These characteristics suggest that the biochemical mechanism underlying the Na exchange mode of the Na pump involves phosphorylation of the enzyme by ATP (which requires intracellular Na) followed by its dephosphorylation by ADP. Such a reaction has been observed in partially purified (Na+ + K+) ATPase from a variety of sources and its dependence on Na concentration has been described (although not previously for the red cell enzyme). In the present work, intracellular ATP:ADP exchange reaction was initiated by photoreleased ATP following brief irradiation at 350 nm of ghosts containing caged-ATP. The ouabain-sensitive component of the ensuing ATP:ADP exchange reaction shows a biphasic response to extracellular Na. External Na in the range 0--10 mM has an inhibitory effect whilst increasing concentrations beyond this range stimulate the rate of exchange in a roughly linear fashion up to 100 mM Na. These results represent the first direct demonstration of the sidedness of the effects of Na on this partial sequence in the overall enzyme cycle and bear a qualitative resemblance to the Na effects on the Na-ATPase which occur in the absence of intracellular ADP in human red blood cells.     

Substituted benzimidazoles inhibit gastric acid secretion by blocking (H+ + K+)ATPase

Studies both in vivo and in vitro have shown that substituted benzimidazoles inhibit the stimulation of acid secretion produced by dibutyryl cyclic AMP and histamine. Furthermore, the results differ from those produced by H2 antagonists and anticholinergic agents in that the inhibition is not competitive, and the site of action is intracellular and peripheral to that of dibutyryl cyclic AMP. To investigate the biochemical mechanism of action of substituted benzimidazoles, one such compound, H 149/94 (2-([2-(3-methyl)pyridyl-methyl]-sulphinyl)-5-methoxycarbonyl-6-methylbenzimidazol), has been tested either directly on an (H+ + K+)ATPase isolated from pig and human gastric mucosa or on the function of this enzyme in gastric glands isolated from rabbit and human gastric mucosa. (H+ + K+)ATPase, which has only been found at the secretory surface of the parietal cell, catalyses a one-to-one exchange of protons and potassium ions. It is possibly the proton pump within the gastric mucosa, and may thus be the terminal or one of the terminal steps of the acid secretory process. We show here that H 149/94 inhibits (H+ + K+)ATPase, which may explain its inhibitory action on acid secretion in vitro and in vivo. Because of the unique distribution and properties of the (H+ + K+)ATPase, the inhibitory action of H 149/94 on this enzyme may be a highly selective clinical means of suppressing the acid secretory process.     

Combined effects of adrenaline and insulin on active electrogenic Na+-K+ transport in rat soleus muscle.

Both beta 2-adrenoreceptor stimulants (such as adrenaline and salbutamol) and insulin can increase active Na+-K+ transport and hyperpolarise skeletal cells. Thus, adrenaline and insulin, which are otherwise antagonistic regulators of several metabolic processes, have one action in common, namely, stimulation of active ion translocation. This is especially interesting as cyclic AMP stimulates Na+-K+ transport, whereas a lowering of the cytoplasmic concentration of cyclic AMP has been proposed as an early signal in the action of insulin. Here we report the results of experiments in which the active Na+-K+ transport and membrane potential (EM) of rat soleus muscles were studied during the action of supramaximal doses of insulin and beta 2-adrenoreceptor stimulants, alone and in combination. We conclude that the stimulant action of insulin on active electrogenic Na+-K+ transport is unlikely to be evoked by a lowering of the intracellular concentration of cyclic AMP.     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Ion permeation through the Na+,K+-ATPase

P-type ATPase pumps generate concentration gradients of cations across membranes in nearly all cells. They provide a polar transmembrane pathway, to which access is strictly controlled by coupled gates that are constrained to open alternately, thereby enabling thermodynamically uphill ion transport (for example, see ref. 1). Here we examine the ion pathway through the Na+,K+-ATPase, a representative P-type pump, after uncoupling its extra- and intracellular gates with the marine toxin palytoxin. We use small hydrophilic thiol-specific reagents as extracellular probes and we monitor their reactions, and the consequences, with cysteine residues introduced along the anticipated cation pathway through the pump. The distinct effects of differently charged reagents indicate that a wide outer vestibule penetrates deep into the Na+,K+-ATPase, where the pathway narrows and leads to a charge-selectivity filter. Acidic residues in this region, which are conserved to coordinate pumped ions, allow the approach of cations but exclude anions. Reversing the charge at just one of those positions converts the pathway from cation selective to anion selective. Close structural homology among the catalytic subunits of Ca2+-, Na+,K+- and H+,K+-ATPases argues that their extracytosolic cation exchange pathways all share these physical characteristics.     

同温度及曲率NaK-BASE管内Na+和K+迁移数值模拟

本文以NaK-AMTEC的BASE管内的Na+和K+迁移为研究对象,建立了NaK-BASE管显微结构的分形模型,采用微观Poisson-Nernst-Planck多离子运移模型模拟了Na+和K+在BASE管中的迁移,考察了不同温度下NaK BASE管内离子的迁移过程。研究结果表明,NaK-BASE管内的阳离子迁移浓度和表面电荷密度与BASE管的温度直接相关;温度的升高会使BASE管内阳离子浓度峰值有所减小,可通过增加BASE管曲率来提高该峰值。BASE管内的表面电荷密度随着温度的升高逐渐增大,且不同温度表面电荷密度之差随着曲率的增加逐渐增大。     

Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase

The Na+/K+ pump, a P-type ion-motive ATPase, exports three sodium ions and then imports two potassium ions in each transport cycle. Ions on one side of the membrane bind to sites within the protein and become temporarily occluded (trapped within the protein) before being released to the other side, but details of these occlusion and de-occlusion transitions remain obscure for all P-type ATPases. If it is deprived of potassium ions, the Na+/K+ pump is restricted to sodium translocation steps, at least one involving charge movement through the membrane's electric fields. Changes in membrane potential alter the rate of such electrogenic reactions and so shift the distribution of enzyme conformations. Here we use high-speed voltage jumps to initiate this redistribution and show that the resulting pre-steady-state charge movements relax in three identifiable phases, apparently reflecting de-occlusion and release of the three sodium ions. Reciprocal relationships among the sizes of these three charge components show that the three sodium ions are de-occluded and released to the extracellular solution one at a time, in a strict order.     

(Na/ + K+)ATPase has one functioning phosphorylation site per alpha subunit

(Na+ + K+)ATPase contains two different subunits, a catalytic subunit (alpha) and a subunit with uncertain function (beta). The enzyme binds ATP, ouabain and vanadate, and can be phosphorylated by ATP as well as by inorganic phosphate. From the previously reported maximal binding and phosphorylation capacities of 3.5--4.3 nmol P per mg protein (based on Lowry protein determination) and the earlier molecular weight value of approximately 250,000, a molar binding and phosphorylation capacity of 0.87--1.07 mol per mol enzyme was derived. As it is generally agreed that the enzyme molecule contains two alpha subunits or even a multiple of two, it has been suggested that the enzyme operates by means of a so-called "half-of-the-sites mechanism" whereby only of the two alpha subunits can be phosphorylated at any one time. We now present evidence that every alpha subunit can be phosphorylated simultaneously, which rules out the operation of such a mechanism.     

Inhibition by dopamine of (Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism

The (Na(+)+K+)ATPase, an integral membrane protein located in virtually all animal cells, couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane. In neurons, a large portion of cellular energy is expended by this enzyme to maintain the ionic gradients that underlie resting and action potentials. Although neurotransmitter regulation of the enzyme in brain has been reported, such regulation has been characterized either as a nonspecific phenomenon or as an indirect effect of neurotransmitter-induced changes in ionic gradients. We report here that the neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the (Na(+)+K+)ATPase activity of isolated striatal neurons. Our data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They also demonstrate that synergism between D1 and D2 receptors, which underlies many of the electrophysical and behavioural effects of dopamine in the mammalian brain, can occur on the same neuron. In addition, the results support the possibility that dopamine and other neurotransmitters can regulate neuronal excitability through the novel mechanism of pump inhibition.     

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

Vanadate inhibits uncoupled Ca efflux but not Na--Ca exchange in squid axons.

Nerve cells can maintain a very low intracellular calcium concentration ([Ca2+]i) against large Ca2+ electrochemical gradients (see ref. 1 for review). The properties of the calcium efflux from these cells depend on [Ca2+]i (ref. 2), and within the physiological range, most Ca efflux depends on ATP (which stimulates with high affinity) and is insensitive to Na1, Na0 and Ca0 (uncoupled Ca efflux). When the [Ca2+]i is well above the physiological range, Ca efflux becomes only partially dependent on ATP (acting now with low affinity), is inhibited by Nai and is stimulated by Na0 and Ca0 (Na--Ca exchange). Orthovanadate, a powerful inhibitor of the (Na+ + K+)ATPase and the Na pump, also inhibits the Ca-stimulated ATPase activity, which is the enzymatic basis for the uncoupled Ca pump, in human red cells. The experiments reported here show that in squid axons the ATP-dependent uncoupled Ca efflux can be fully and reversibly inhibited by vanadate, whereas concentrations of vanadate 10 times higher have no effect on the Na--Ca exchange. This is another indication that the uncoupled Ca efflux represents an ATP-driven Ca pump, and supports the suggestion that the uncoupled Ca efflux and Na--Ca exchange are mediated by different mechanisms.     

Crystal structure of the plasma membrane proton pump

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.     

长角血蜱蜕皮激素含量与卵巢微粒体Na~+-K~+-ATP酶活力的关系(英文)

为阐明蜕皮激素在长角血蜱生殖中的作用机制，测定了雌蜱不同生殖时期体内（卵巢和血淋巴）蜕皮激素含量和卵巢微粒体Na＋－K＋－ATP酶活力的变化．结果表明，产卵前，蜕皮甾类含量和酶活力变化不明显；产卵时，则达到最高；产卵后，蜕皮甾类含量和酶活力又下降．外源燃皮激素（20－羟基蜕皮酮）处理饱血3d雌蜱有激活酶活力作用，但不表现剂量依赖关系．     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

碱胁迫对宁夏枸杞叶中Na＋，K＋，Ca2＋动态变化的影响

摘要：为探讨宁夏枸杞叶中离子平衡与盐碱胁迫的关系,研究不同浓度的NaHCO3溶液胁迫下,枸杞叶中Na^＋,K^＋,Ca^2＋的浓度变化,同时采用非损伤微测技术研究了枸杞叶中Na^＋,K^＋,Ca^2＋的流速变化．结果表明,在同一时间内（7,14,21d）,Na^＋的浓度随NaHCO。浓度的升高总体呈升高趋势,K^＋和Ca^2＋的浓度总体呈下降趋势,c（Na^＋）／c（Ca^2＋）随NaHCO3浓度的升高而升高;随着时间的变化,各个处理下枸杞叶中Na^＋的浓度总体呈现先降后升的趋势,K^＋的浓度总体呈现下降趋势,Ca^2＋的浓度总体呈现先升后降的趋势,c（Na^＋）／c（K^＋）总体呈现升高趋势,c（Na^2＋）／c（Ca^2＋）总体呈现先降后升的趋势;NaHCO。溶液胁迫7d时,诱导了枸杞叶肉细胞中净Na^＋,K^＋,Ca^2＋外排的增加．碱胁迫下造成c（Na^＋）／c（K^＋）和f（Na^＋）／c（Ca^2＋）升高的原因为,叶片中K^＋和Ca^2＋外排和Na^＋大量积累,这也是枸杞不耐碱的原因之一．可为种植枸杞改良盐碱地提供参考．     

Role of intracellular Ca2+ sequestration in beta-adrenergic relaxation of a smooth muscle.

Various mechanisms have been proposed for beta-adrenergically mediated relaxation of smooth muscle. All theories suggest the involvement of cyclic AMP as a second messenger: beta-agonists stimulate adenylate cyclase which converts ATP to cyclic AMP and protein kinase, activated by cyclic AMP, is then thought to catalyse a protein phosphorylation that leads to a reduction in free Ca2+, thus effecting relaxation. How this last step is accomplished is much debated, but the following possibilities are currently considered as the mechanisms responsible for cyclic AMP-induced reduction of cytoplasmic Ca2+: activation of a Ca2+-ATPase in the plasma and/or sarcoplasmic reticulum membranes which lowers cytoplasmic [Ca2+] in a direct manner or stimulation of (Na+-K+)ATPase in the cell membrane which may indirectly effect Ca2+ extrusion. Among the hypotheses suggested, those of Ca2+ sequestration by the sarcoplasmic reticulum and of Ca2+ extrusion across the cell membrane are consistent with each other if it is assumed that both processes are effected by a cyclic AMP-sensitive Ca2+-ATPase. However, quite a different mechanism is implied by involving the Na+-K+ pump and Na+-Ca2+ exchange carrier. In this report, we present evidence that suggests intracellular Ca2+ sequestration is the mechanism involved.     

Effects of ATP and vanadate on calcium efflux from barnacle muscle fibres

Calcium ions carry the inward current during depolarization of barnacle muscle fibres and are involved in the contraction process. Intracellular ionized calcium ([Ca2+]i) in barnacle muscle, as in other cells, is kept at a very low concentration, against a large electrochemical gradient. This large gradient is maintained by Ca2+ extrusion mechanisms. When [Ca2+]i is below the contraction threshold, Ca2+ efflux from giant barnacle muscle fibres is, largely, both ATP dependent and external Na+ (Na+0) dependent (see also refs 5,6). When [Ca2+]i is raised to the level expected during muscle contraction (2-5 muM), most of the Ca2+ efflux from perfused fibres is Na0 dependent; as in squid axons, this Na+0-dependent Ca2+ efflux is ATP independent. Orthovanadate is an inhibitor of (Na+ + K+) ATPase and the red cell Ca2+-ATpase. We report here that vanadate inhibits ATP-promoted, Na+0-dependent Ca2+ efflux from barnacle muscle fibres perfused with low [Ca2+]i (0.2-0.5 microM), but has little effect on the Na+0-dependent, ATP-independent Ca2+ efflux from fibres with a high [Ca]i (2-5 microM). Nevertheless, ATP depletion or vanadate treatment of high [Ca2+]i fibres causes an approximately 50-fold increase of Ca2+ efflux into Ca2+-containing lithium seawater. These results demonstrate that both vanadate and ATP affect Ca2+ extrusion, including the Na+0-dependent Ca2+ efflux (Na-Ca exchange), in barnacle muscle.     

Functional analysis of an archaebacterial voltage-dependent K+ channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal K(V) channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels.