Three-dimensional map of the plasma membrane H+-ATPase in the open conformation

The H+-ATPase from the plasma membrane of Neurospora crassa is an integral membrane protein of relative molecular mass 100K, which belongs to the P-type ATPase family that includes the plasma membrane Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase. The H+-ATPase pumps protons across the cell's plasma membrane using ATP as an energy source, generating a membrane potential in excess of 200mV. Despite the importance of P-type ATPases in controlling membrane potential and intracellular ion concentrations, little is known about the molecular mechanism they use for ion transport. This is largely due to the difficulty in growing well ordered crystals and the resulting lack of detail in the three-dimensional structure of these large membrane proteins. We have now obtained a three-dimensional map of the H+-ATPase by electron crystallography of two-dimensional crystals grown directly on electron microscope grids. At an in-plane resolution of 8 A, this map reveals ten membrane-spanning alpha-helices in the membrane domain, and four major cytoplasmic domains in the open conformation of the enzyme without bound ligands.     

Ca-ATPase and bone cell mineralization

The plasma membrane Ca-ATPases play an important role in the regulation of intracellular Ca2+ ion concentration by pumping Ca2+ out of the cell into the extracellular fluid at the expense of ATP. These pumps potentially play an important role in the delivery of Ca2+ during mineralization of hard tissues such as bone. The properties of the plasma membrane Ca2+ pump are compared with those of the sarco(endo)plasmic reticulum and the factors regulating pump function are presented. The different gene products for the plasma membrane Ca-ATPases are described as well as their known functional significance. Particular attention is paid on the plasma membrane Ca2+ pumps present in mineralizing tissues and evidence supporting a role for these pumps in the transcellular delivery of Ca2+ during the process of mineralization is also presented.     

Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.     

Endothelium-smooth muscle interactions in blood vessels

1. Blood vessel tone is determined both by smooth muscle and endothelial functions. In coronary arteries taken from rat (Fisher-Lewis) cardiac transplanted hearts, the inducible form of NOS (iNOS) in smooth muscle is more active, while acetylcholine-induced nitric oxide production in the endothelium is greatly diminished. This causes a greatly reduced myogenic constriction, in pressurized septal arteries taken from immunologically challenged transplanted hearts. 2. The sarcoplasmic reticulum (SR) of smooth muscle and the endoplasmic reticulum (ER) of endothelial cells sequester Ca2+ from the cytoplasm. This reduces the intracellular concentration of free Ca2+, which is necessary for the activation of cellular processes. The release of Ca2+ from internal stores occurs through ryanodine and IP3 recoptors located on the SR membrane. 3. The superficial SR/ER also interacts with ion exchangers and pumps in the plasma membrane. This allows for the superficial SR/ER to function in Ca2+ extrusion; for example, inhibition of the SR/ER Ca(2+)-ATPase (SERCA) partially inhibits the rate of loss Ca2+ from the cell. Recent data suggest that the SR Ca(2+)-ATPase and the Na(+)-Ca2+ exchanger of smooth muscle cells function in series; that is, Ca2+ uptake by the SR followed by release towards the exchanger to mediate extrusion. This interaction between the SERCA of the superficial SR and ion exchangers and pumps creates intracellular Ca2+ gradients. 4. The SERCA of the superficial, peripherally distributed SR/ER also serves to regulate Ca2+ entry from the extracellular space. This occurs in part by inhibition of the superficial buffer barrier function of the SR as well as by depletion of stimulated Ca2+ entry. 5. Ca2+ entry is also regulated in endothelial and smooth muscle cells by the membrane potential. Membrane hyperpolarization increases the driving force for Ca2+ entry into endothelial cells, which lack voltage-gated Ca2+ channels, and reduces open state probability of voltage-gated Ca2+ channels in vascular smooth muscle cells. The two cell types have electrical contact and interact in a dynamic manner to regulate blood vessel diameter.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] 相似文献   

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl

Debaryomyces hansenii showed an increased growth in the presence of either 1 M, KCl or 1 M NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86Rb+ and 22Na+. 86Rb+ transport was saturable, with K(m) and Vmax values higher for cells grown in 1 M NaCl. 22Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1 M KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+/H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth.     

Altered permeability of the erythrocyte membrane for sodium and potassium ions in spontaneously hypertensive rats

Permeability of the erythrocyte membrane for sodium and potassium ions was studied in 8-10-week old spontaneously hypertensive rats (SHR, Kyoto Wistar strain), normotensive Wistar and Sprague-Dawley rats. The rate constnat of Na/Na exchange was considerably greater in the SHR than in the normotensive Wistar and Sprague-Dawley rats. This difference remained the same in the rats adrenalectomized 7 days prior to the experiment. The maximum difference in the constants was found when the sodium pump was blocked by ouabain. The accumulation of 42K in the erythrocytes of the SHR (the sodium pump being blocked) took place at a considerably slower rate, and the K+ washout into a potassium-free medium was faster than in the normotensive Wistar and Sprague-Dawley rats. These results seem to indicate a higher permeability of the SHR's erythrocyte membrane for Na+ and K+ ions, as compared to normotensive Wistar and Sprague-Dawley strains. It is suggested that the increased permeability of the erythrocyte membrane for Na+ and K+ in the SHR may reflect a more widespread cell membrane defect, which could serve as a general cause for activating the mechanisms maintaing high blood pressure.     

Energy transduction in the thermophilic anaerobic bacterium Clostridium fervidus is exclusively coupled to sodium ions

The thermophilic, peptidolytic, anaerobic bacterium Clostridium fervidus is unable to generate a pH gradient in the range of 5.5-8.0, which limits growth of the organism to a narrow pH range (6.3-7.7). A significant membrane potential (delta psi approximately -60 mV) and chemical gradient of Na+ (-Z delta pNa approximately -60 mV) are formed in the presence of metabolizable substrates. Energy-dependent Na+ efflux is inhibited by the Na+/H+ ionophore monensin but is stimulated by uncouplers, suggesting that the Na+ gradient is formed by a primary pumping mechanism rather than by secondary Na+/H+ antiport. This primary sodium pump was found to be an ATPase that has been characterized in inside-out membrane vesicles and in proteoliposomes in which solubilized ATPase was reconstituted. The enzyme is stimulated by Na+, resistant to vanadate, and sensitive to nitrate, which is indicative of an F/V-type Na(+)-ATPase. In the proteoliposomes Na+ accumulation depends on the presence of ATP, is inhibited by the ATPase inhibitor nitrate, and is completely prevented by the ionophore monensin but is stimulated by protonophores and valinomycin. These and previous observations, which indicated that secondary amino acid transport uses solely Na+ as coupling ion, demonstrate that energy transduction at the membrane in C. fervidus is exclusively dependent on a Na+ cycle.     

The structure of the potassium channel: molecular basis of K+ conduction and selectivity

The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.     

Dopamine restores lung ability to clear edema in rats exposed to hyperoxia

Exposure to hyperoxia causes lung injury, decreases active sodium transport and lung edema clearance in rats. Dopamine (DA) increases lung edema clearance by stimulating vectorial Na+ flux and Na, K-ATPase function in rat alveolar epithelium. This study was designed to test whether DA (10(-)5 M) would increase lung edema clearance in rats exposed to 100% O2 for 64 h. Active Na+ transport and lung edema clearance decreased by approximately 44% in rats exposed to acute hyperoxia (p < 0.001). DA increased lung edema clearance in room air breathing rats (from 0.50 +/- 0.02 to 0.75 +/- 0.06 ml/h) and in rats exposed to 100% O2 (from 0.28 +/- 0.03 to 0. 67 +/- 0.03 ml/h). Disruption of cell microtubular transport system by colchicine blocked the stimulatory effect of DA on active Na+ transport in control and hyperoxic rats, whereas the isomer beta-lumicolchicine, which does not affect cell microtubular transport, did not inhibit the stimulatory effects of dopamine. The Na,K-ATPase alpha1-subunit protein abundance increased in the basolateral membranes of alveolar type II (ATII) cells incubated with 10(-)5 M DA for 15 min, probably by recruiting Na+ pumps from intracellular pools. Colchicine, but not beta-lumicolchicine, prevented the recruitment of alpha1 subunits to the plasma membrane by DA. Accordingly, DA restored lung ability to clear edema in hyperoxic-injured rat lungs. Conceivably, dopamine induces recruitment of Na+ pumps from intracellular pools to the plasma membrane of alveolar epithelial cells and thus increases lung edema clearance.     

Membrane function and vascular reactivity

The resting potential and monovalent ions in the marginal cells and scala media were measured before and 20 min after the onset of anoxia using ion-sensitive microelectrodes. The resting potential of the marginal cells decreased from 62.7 to -2.4 mV. The K+ activity decreased from 77.7 to 53.2 mEq/l, while the Na+ activity increased from 2.6 to 24.7 mEq/l. The Cl- activity did not change significantly. In the scala media, the endocochlear potential decreased rapidly from 80.9 to -28.0 mV after the onset of anoxia. The K+ activity decreased from 119.0 to 96.5 mEq/l, the Na+ activity increased from 1.3 to 9.5 mEq/l and that of Cl- decreased from 127.0 to 115.1 mEq/l. The electrochemical gradients determined for each ion based on the ionic changes in the scala media and marginal cells, suggested the existence of an Na/K pump and Na-K-2Cl cotransport at the basolateral membrane of the marginal cells, and a rheogenic K pump and Na-K-2Cl transport at the luminal membrane of the marginal cells. The Na+ and K+ must be recycled at the basolateral membrane and luminal membrane of marginal cells, respectively.     

Chromo-domain proteins: linking chromatin structure to epigenetic regulation

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200//trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na(+)-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about-18 mV, which is close to the predicated value of a perfect Nernst K+ or Na+ electrode. In 50//10 mM CaCl2 solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about -20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction.     

Na+ pump low and high ouabain affinity alpha subunit isoforms are differently distributed in cells

Three isoforms (alpha1, alpha2, and alpha3) of the catalytic (alpha) subunit of the plasma membrane (PM) Na+ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na+ pump inhibitor, ouabain. In the rat, alpha1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (alpha1) and high (alpha2 or alpha3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize alpha subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform alpha1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (alpha2 in astrocytes, alpha3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that alpha1 may regulate bulk cytosolic Na+, whereas alpha2 and alpha3 may regulate Na+ and, indirectly, Ca2+ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na+ pumps may thereby modulate reticulum Ca2+ content and Ca2+ signaling.     

Two types of stretch-activated channels coexist in the rabbit corneal epithelial cell

Ion channels contribute to the regulation of cellular function through control of the membrane potential and intracellular concentration of various ions. We examined stretch-activated channels in the corneal epithelial cell. Patch clamping was applied to enzymatically dissociated corneal epithelial cells to characterize their stretch-activated ion channels. The plasma membrane was stretched by applying suction to the patch pipette in cell-attached or inside-out patch configuration. The ion selectivity, voltage-dependence, and stretch-dependence were examined. Two kinds of stretch-activated channel events were observed; the previously-reported large conductance (L) channel and a novel small conductance (S) channel. The probability of recording L vs. S channels in the cell-attached configuration was about 2:1. The L channel was potassium selective with single channel conductance (gamma) of about 160 pS under the symmetrical (150 mm K+) solution. The S channel was permeable to Na+ and K+ with gamma of about 20 pS under the same conditions. Both L and S channels showed little activity in the absence of suction applied to the recording pipette. Channel activity was evoked by suction (negative pressure) stronger than -20 mmHg in both channels. The open probability (Po) and the mean current increased in proportion to further applied stretch and did not saturate for applied suction as strong as -80 mmHg, the pressure at which the gigaseal started to break. Thus, two types of stretch-activated channels coexist in corneal epithelial cells; a potassium-selective L channel and non-selective S channel. The contribution of these channels to the membrane potential is discussed.     

Potassium channels in epithelial cells

The broad array of K- channels in epithelial cells includes voltage-dependent (mainly outward) and Ca(2+)-activated channels, and K+ channels modulated by adenosine triphosphate (ATP). Voltage-dependent K+ channels mediate Na+/K+ absorption and secretion; typically, they are found in the basolateral membrane and exhibit burst activity. Ca(2+)-activated K+ (Ca2+/K+) channels regulate activity by decreasing Ca2+ influx via voltage-gated Ca2+ channels. Ca2+/K+ channels exhibit conductances of 4-300 pS, and have a low open probability (< 10(-7)) at the level of the resting membrane potential. ATP-sensitive channels have been observed mainly in insulin-secreting pancreatic beta-cells and in the urinary tract, where the open state is rapidly closed by ATP. The channels are voltage-dependent, exhibit burst activity, and, in the case of the urinary-tract cells, are Ca2+ dependent. Chemical compounds that selectively open or block K+ channels have been exploited to characterize channels found in different cells, but no opener or blocker has been found that specifically affects only one type of K+ channel. Specialized model systems and recombinant techniques have led to a general understanding of the structure of K+ channels, but many important details remain to be determined.     

Myotonic dystrophy protein kinase is involved in the modulation of the Ca2+ homeostasis in skeletal muscle cells

Myotonic dystrophy (DM), the most prevalent muscular disorder in adults, is caused by (CTG)n-repeat expansion in a gene encoding a protein kinase (DM protein kinase; DMPK) and involves changes in cytoarchitecture and ion homeostasis. To obtain clues to the normal biological role of DMPK in cellular ion homeostasis, we have compared the resting [Ca2+]i, the amplitude and shape of depolarization-induced Ca2+ transients, and the content of ATP-driven ion pumps in cultured skeletal muscle cells of wild-type and DMPK[-/-] knockout mice. In vitro-differentiated DMPK[-/-] myotubes exhibit a higher resting [Ca2+]i than do wild-type myotubes because of an altered open probability of voltage-dependent l-type Ca2+ and Na+ channels. The mutant myotubes exhibit smaller and slower Ca2+ responses upon triggering by acetylcholine or high external K+. In addition, we observed that these Ca2+ transients partially result from an influx of extracellular Ca2+ through the l-type Ca2+ channel. Neither the content nor the activity of Na+/K+ ATPase and sarcoplasmic reticulum Ca2+-ATPase are affected by DMPK absence. In conclusion, our data suggest that DMPK is involved in modulating the initial events of excitation-contraction coupling in skeletal muscle.     

Two functionally different Na/K pumps in cardiac ventricular myocytes

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half-saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half-maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.     

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.     

Na, K-ATPase--its structure, function and intracellular transport

Na, K-ATPase is an integral plasma membrane protein and plays essential roles such as maintaining sodium and potassium ion gradients across the plasma membrane. The enzyme consists of the alpha and the beta subunits with the stoichiometry of one to one. Three alpha subunit and two beta subunit isoforms have been detected in animal cells with the tissue-specific expression of both subunits. Recent advances in molecular biological studies on the Na, K-ATPase enable us to understand the structure-function relationships and mechanisms of intracellular transport of the enzyme. In this article we review the findings deduced from these studies, especially on the assembly and transport to the plasma membrane of the alpha and beta subunits.     

A case of lung adenocarcinoma associated with remarkably high levels of CA 19-9 and lymphangitis carcinomatosa

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation orate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (-223 mV and -230 mV respectively). The Mg2+-dependent ATPase activity of the 10(5) x g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na+-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.