Monensin-mediated transports of H+, Na+, K+ and Li+ ions across vesicular membranes: T-jump studies.

Theoretical expression for the rate of decay of delta pH across vesicular membrane due to carrier-mediated ion transports, 1/tau, has been modified taking note of carrier states (such as mon- and mon-H-M+) for which the translocation rate constants in the membrane are small. The rates of delta pH decay due to monensin-mediated H+ and M+ transports (M+ = Na+, K+, Li+) observed in our experiments in the pH range 6-8, and [M+] range 50-250 mM at 25 degrees C have been analysed with the help of this expression. delta pH across soybean phospholipid vesicular membranes were created by temperature jump in our experiments. The following could be inferred from our studies. (a) At low pH (approximately 6) 1/tau in a medium of Na+ is greater than that in a medium of K+. In contrast with this, at higher pH (approximately 7.5) 1/tau is greater in a medium of K+. Such contradictory observations could be understood with the help of our equation and the parameters determined in this work. The relative concentrations of the rate-limiting species (mon-H, mon-K, and mon-Li at Ph approximately 7 in vesicle solutions having Na+, K+ and Li+, respectively) can explain such behaviours. (b) The proton dissociation constant KH for mon-H in the lipid medium (pKH approximately 6.55) is larger than the reported KH in methanol. (c) The concentrations of mon- and mon-H-Na+ are not negligible under the conditions of our experiments. The latter species cause a [Na+]-dependent inhibition of ion transports. (d) The relative magnitudes of metal ion dissociation constants KHM (approximately 0.05 M) for mon-H-Na+ and KM (approximately 0.03 M) for mon-Na suggest that the carboxyl group involved in the protonation may not be dominantly involved in the metal ion complexation. (e) The estimates of KM (approximately 0.03 M for Na+, 0.5 M for K+ and 2.2 M for Li+) follow the ionophore selectivity order. (f) The rate constants k1 and k2 for the translocations of mon-H and mon-M (M+ = Na+, K+ and Li+) are similar in magnitude (approximately 9 x 10(3) s-1) and are higher than that for nig-H and nig-M (approximately 6 x 10(3) s-1) which can be expected from the relative molecular sizes of the ion carriers.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Transport of H+, K+, Na+ and Ca++ in Streptococcus

Summary The streptococci differ from other bacteria in that cation translocations (with the possible exception of one of the K⁺ uptake systems) occur by primary transport systems, i.e., by cation pumps which use directly the free energy released during hydrolysis of chemical bonds to power transport. Transport systems in other bacteria, especially for Na⁺ and Ca⁺⁺, are often secondary, using the free energy of another ion gradient to drive cation transport. In streptococci H⁺ efflux occurs via the F₁F₀-ATPase. This enzyme is composed of eight distinct subunits. Three of the subunits are embedded in the membrane and form a H⁺ channel; this is called the F₀ portion of the enzyme. The other five subunits form the catalytic part of the enzyme, called F₁, which faces the cytoplasm and can easily be stripped from the membrane. Physiologically, this enzyme functions as a H⁺-ATPase, pumping protons out of the cell to form an electrochemical proton gradient, . The F₁F₀-ATPase, however, is fully reversible and if supplied with P_i, ADP and a + of sufficient magnitude (ca –200 mv) catalyzes the synthesis of ATP. Streptococcus faecalis can accumulate K⁺ and establish a gradient of 50 000:1 (in>out) under some conditions. Uptake occurs by two transport systems. The dominant, constitutive system requires both an electrochemical proton gradient and ATP to operate. The minor, inducible K⁺ transport system, which has many similarities to the K⁺-ATPase of the Kdp transport system found in Escherichia coli, requires only ATP to power K⁺ uptake.Sodium extrusion occurs by a Na⁺/H⁺-ATPase. Exchange is electroneutral and there is no requirement for a . The possibility that the Na⁺/H⁺-ATPase may consist of two parts, a catalytic subunit and a Na⁺/H⁺ antiport subunit, is suggested by the finding that damage to the Na⁺ transport system either through mutation or protease action leads to the appearance of -requiring Na⁺/H⁺ antiporter activity.Ca⁺⁺ like Na⁺ is extruded from metabolizing, intact cells. Transport requires no but does require ATP. Reconstitution of Ca⁺⁺ transport activity with accompanying Ca⁺⁺-stimulated ATPase activity into proteoliposomes suggests that Ca⁺⁺ is transported by a Ca⁺⁺-translocating ATPase.Where respiring organelles and bacteria use secondary transport systems the streptococci have developed cation pumps. The streptococci, which are predominantly glycolyzing bacteria, generate a much inferior to that of respiring organisms and organelles. The cation pumps may have developed simply in response to an inadequate .Abbreviations electrochemical potential of protons - membrane potential - pH pH gradient - p proton-motive force - DCCD N,Na¹-dicyclohexlcarbodiimide - TCS tetrachlorosalicylanilide - FCCP carbonylcyanide-p-trifluoromethylphenylhydrazone - CCCP carbonylcyanie-m-chlorophenylhydrazone - TPMP⁺ triphenylmethyl phosphonium ion - DDA⁺ dibenzyldimethylammonium ion - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA ethyleneglycol-bis (amino-ethyl-ether)-N,N-tetraacetic acid     

A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.     

K+,H+ and Cl- fluxes across erythrocyte membrane irradiated with radio frequency electromagnetic fields

The effect of 460 MHz microwave radiation on the ion-transporting properties of the isolated rat erythrocytes was studied with the use of K+, H+ and Cl(-)-selective electrodes. In comparison with the control cells kept at 0 degree C the most significant changes were observed in the K+ transport system. Particularly, microwave radiation (specific absorbed rate 280 W/kg) caused an increased loss of K+ during treatment and 2-fold decrease in the rate of K+ efflux from the irradiated erythrocytes, when the latter were incubated in the isoosmotic, unbuffered sucrose. The same changes were observed when the erythrocytes were conventionally heated up to 39 degrees C for 20 minutes. It is concluded that high levels of microwave radiation cause temperature-induced changes of the membrane structure resulting in alterations in potassium transport across the membrane.     

Adenovirus-dependent changes in cell membrane permeability: role of Na+, K+-ATPase.

Adenovirus-dependent release of choline phosphate from KB cells at pH 6.0 was partially blocked by ouabain. In K+-containing medium, maximum inhibition of release was obtained by 10(-5) M ouabain and half-maximal inhibition was achieved by about 0.5 X 10(-6)M ouabain. Ouabain did not block either the binding or the uptake of adenovirus by KB cells. Without K+, about 25% of cell-associated choline phosphate was released by adenovirus, whereas with 1 mM K+ about 50% was released. This activation by K+ was blocked by 0.1 mM ouabain. HeLa cells behaved like KB cells, but a mutant of HeLa cells resistant to ouabain (D98-OR) released much lower amounts of choline phosphate in response to human adenovirus type 2 (Ad2). Wild-type D98-OR cells bound nearly the same amount of adenovirus as did normal HeLa cells. Ad2 also increased the activity of Na+,K+-ATPase in KB cells, with maximum activation at 50 micrograms of Ad2 per ml. In D98-OR cells, Ad2 failed to activate Na+,K+-ATPase activity. Ad2-dependent lysis of endocytic vesicles (receptosomes) was assayed by measuring Ad2-dependent enhancement of epidermal growth factor-Pseudomonas exotoxin toxicity. This action of adenovirus was increased when K+ was present in the medium. Under the conditions used, K+ had no effect on the amount of Ad2 or epidermal growth factor taken up by the cells. On the basis of these results, it is suggested that Ad2-dependent cellular efflux of choline phosphate and adenovirus-dependent lysis of receptosomes may require Na+,K+-ATPase activity.     

Ion pumps in polarized cells: sorting and regulation of the Na+, K+- and H+, K+-ATPases

The physiologic function of an ion transport protein is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na(+),K(+)-ATPase and the H(+),K(+)-ATPases are closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells, and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intramolecular interactions govern trafficking, distribution, and catalytic capacities of these proteins.     

Structural studies on H+,K+-ATPase: determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase

The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase.     

On the Na+,K+ pump in fluctuating membrane potentials

The present work investigates the usefulness of noise in the activity of the Na+,K+ pump. Random gating activity of the neighboring ion channels causes local fluctuations of the electric potential. They are modeled by a Markovian symmetric dichotomic noise, added to the membrane potential. The noise-averaged pump current is calculated for a general rectangular voltage signal and the model parameters of the effective two-state enzyme cycle are tuned to fit experimental results. Then, using these parameters, the amount of transported charge is calculated, and studied as a function of noise intensity. Signal and noise characteristics are identified at which fluctuations enhance pump activity. The biological impact of this phenomenon seems to be absent in physiological conditions for it occurs at noise amplitudes over 50 mV, which are unlikely to appear due to ion channels. However, under some conditions, externally applied dichotomic noise of intensity about 150 mV may sensibly increase the quantity of transported charge.     

Muscle K+, Na+, and Cl disturbances and Na+-K+ pump inactivation: implications for fatigue.

Membrane excitability is a critical regulatory step in skeletal muscle contraction and is modulated by local ionic concentrations, conductances, ion transporter activities, temperature, and humoral factors. Intense fatiguing contractions induce cellular K(+) efflux and Na(+) and Cl(-) influx, causing pronounced perturbations in extracellular (interstitial) and intracellular K(+) and Na(+) concentrations. Muscle interstitial K(+) concentration may increase 1- to 2-fold to 11-13 mM and intracellular K(+) concentration fall by 1.3- to 1.7-fold; interstitial Na(+) concentration may decline by 10 mM and intracellular Na(+) concentration rise by 1.5- to 2.0-fold. Muscle Cl(-) concentration changes reported with muscle contractions are less consistent, with reports of both unchanged and increased intracellular Cl(-) concentrations, depending on contraction type and the muscles studied. When considered together, these ionic changes depolarize sarcolemmal and t-tubular membranes to depress tetanic force and are thus likely to contribute to fatigue. Interestingly, less severe local ionic changes can also augment subtetanic force, suggesting that they may potentiate muscle contractility early in exercise. Increased Na(+)-K(+)-ATPase activity during exercise stabilizes Na(+) and K(+) concentration gradients and membrane excitability and thus protects against fatigue. However, during intense contraction some Na(+)-K(+) pumps are inactivated and together with further ionic disturbances, likely precipitate muscle fatigue.     

Phospholipid N-methylation in diabetic erythrocytes: effects on membrane Na+, K+ ATPase activity.

Phospholipid methylation was quantified in non-diabetic and streptozotocin diabetic rat erythrocytes. While the total mass of methylated lipids remained the same in both groups, the relative abundance of individual methylated lipid species differed significantly in diabetic erythrocytes. Moreover, incubation of erythrocytes membranes with S-adenosyl methionine, a substrate for methyl transferases, not only increased membrane lipid methylation but also decreased Na+, K+ ATPase activity significantly. These results suggest that phospholipid methylation may cause the observed depression of erythrocyte Na+, K+ ATPase activity in diabetes and could contribute to the altered rheology of erythrocytes in diabetes.     

Competitive membrane adsorption of Na+, K+, and Ca2+ in smooth muscle cells

Summary A theory for Na⁺, K⁺ and Ca²⁺ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na⁺, K⁺ and Ca²⁺ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10^–8 m, respectively. Furthermore, the effect of a Ca²⁺ pump that maintains free surface Ca²⁺ concentration constant is investigated. A decrease in intracellular Na⁺ content results in an increased Ca²⁺ uptake; part of this uptake is due to an increase in surface-bound Ca²⁺ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca²⁺ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.     

Dynamic lipid interactions in the plasma membrane Na+,K+-ATPase

The function of ion-transporting Na⁺,K⁺-ATPases depends on the surrounding lipid environment in biological membranes. Two established lipid-interaction sites A and B within the transmembrane domain have been observed to induce protein activation and stabilization, respectively. In addition, lipid-mediated inhibition has been assigned to a site C, but with the exact location not experimentally confirmed. Also, possible effects on lipid interactions by disease mutants dwelling in the membrane-protein interface remain relatively uncharacterized. We simulated human Na⁺,K⁺-ATPase α₁β₁FXYD homology models in E1 and E2 states in an asymmetric, multicomponent plasma membrane to determine both wild-type and disease mutant lipid-protein interactions. The simulated wild-type lipid interactions at the established sites A and B were in agreement with experimental results thereby confirming the membrane-protein model system. The less well-characterized, proposed inhibitory site C was dominated by lipids lacking inhibitory properties. Instead, two sites hosting inhibitory lipids were identified at the extracellular side and also a cytoplasmic CHL-binding site that provide putative alternative locations of Na⁺,K⁺-ATPase inhibition. Three disease mutations, Leu302Arg, Glu840Arg and Met859Arg resided in the lipid-protein interface and caused drastic changes in the lipid interactions. The simulation results show that lipid interactions to the human Na⁺,K⁺-ATPase α₁β₁FXYD protein in the plasma membrane are highly state-dependent and can be disturbed by disease mutations located in the lipid interface, which can open up for new venues to understand genetic disorders.     

Na+, K+, H+ and Cl- permeability properties of rabbit skeletal muscle sarcolemmal vesicles

The ion permeability properties of rabbit skeletal muscle sarcolemmal vesicles were investigated by means of radioisotope flux, membrane potential, and light-scattering measurements. An enriched sarcolemmal fraction was obtained from the 22-27% region of sucrose gradients after isopycnic centrifugation. The presence of contaminating sarcoplasmic reticulum was assessed with the use of a purified sarcoplasmic reticulum vesicle fraction. 22Na+, 86Rb+, 36Cl-, and [3H]sucrose flux measurements indicated that the sarcolemmal fraction possessed isotope spaces ranging between 1.5 and 4 microliters/mg protein. Membrane potential measurements using the voltage-sensitive fluorescent probe 3,3'-dipentyl-2,2'-oxadicarbocyanine iodide (diO-C5-(3)) indicated that sarcolemmal vesicles were impermeable to H+ and Na+ but that 10-15% of the vesicles were permeable to K+. Light-scattering measurements indicated a small fraction of sarcolemmal vesicles were permeable to both K+ and Cl-. Whether the low permeability of sarcolemmal vesicles to Na+, K+, and Cl- is the result of a low concentration of ion channels or the inactivation of these channels during isolation is at present uncertain.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Reaction of (Na+ + K+)-dependent adenosine triphosphatase with inorganic phosphate. Regulation by Na+, K+, and nucleotides

Effects of Na+, K+, and nucleotides on Mg2+-dependent phosphorylation of (Na+ + K+)-dependent adenosine triphosphatase by Pi were studied under equilibrium conditions. Na+ was a linear competitive inhibitor with respect to Mg2+ and a mixed inhibitor with respect to Pi. K+ was a partial inhibitor; it interacted with positive cooperativity and induced negative cooperativities in the interactions of Mg2+ and Pi with the enzyme. Adenyl-5'-yl (beta, gamma-methylene)diphosphonate, a nonhydrolyzable analog of ATP, interacted with negative cooperativity to inhibit phosphorylation in competition with Pi. ATP was also a competitive inhibitor. Na+ and K+ acted antagonistically, Na+ and nucleotides inhibited synergistically, and K+ and nucleotides were mutually exclusive. In the presence of ouabain, when nucleotides were excluded from the site inhibiting phosphorylation, a low affinity regulatory site for nucleotides became apparent, the occupation of which reduced the rate of dephosphorylation and the initial rate of phosphorylation of the enzyme without affecting the equilibrium constant of the reaction of Pi with the ouabain-complexed enzyme. The regulatory site was also detected in the absence of ouabain. The data suggest that catalytic and transport functions of the oligomeric enzyme may be regulated by homotropic and heterotropic site-site interactions, ligand-induced slow isomerizations, and distinct catalytic and regulatory sites for ATP.     

Intracellular K+, Na+ and Cl- concentrations and membrane potential in human monocytes

The relationship between the resting membrane potential and the intracellular ionic concentrations in human monocytes was investigated. Cell volume, cell water content, and amount of intracellular K+, Na+, and Cl- were measured to determine the intracellular concentrations of K+ (Ki), Na+ (Nai) and Cl- (Cli) of monocytes, and of lymphocytes and neutrophils. Values found for monocytes were similar to those for neutrophils, i.e., cell volumes were 346 and 345 micron3, respectively, cell water content 78%, and Ki, 128 and 125, Nai, 24 and 26, and Cli, 102 and 103 mmol/l cell water, respectively. Lymphocytes, however, had different values: 181 micron3 cell volume, 77% cell water content, and for Ki, Nai, and Cli, 165, 37, and 91 mmol/l cell water, respectively. The resting membrane potential of cultured human monocytes (range -30 to -40 mV), determined by measurement of the peak potential occurring within the first milliseconds after microelectrode entry, was most dependent on extracellular K+, followed by Cl-, and Na+. The membrane permeability ratio of Cl- to K+ was estimated by use of the constant field equation to be 0.23 (range 0.22 to 0.30).