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).     

Intracellular Na+, K+ and Cl- activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (Vm) and intracellular Na+, K+ and Cl- activities were measured under steady-state conditions in normal saline medium (Na+ = 154, K+ = 6, Cl- 150 mequiv./l). Membrane potential was estimated to be -23.3 +/- 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of Vm and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na+, K+ and Cl- activities are 4.6 +/- 0.5; 68.3 +/- 8.0; and 43.6 +/- 2.1 mequiv./l, respectively. The apparent activity coefficients for Na+ and K+ are 0.18 +/- 0.02 and 0.41 +/- 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl- (0.67 +/- 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl- is not different from zero. The results establish that the energy available from the Na+ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Measurement of intracellular chloride activity in mouse liver slices with microelectrodes

Steady-state membrane potential (V_m) and intracellular Cl⁻ activity (a_Clⁱ) were measured with double-barreled Cl⁻-selective microelectrodes in mouse liver slices. In bathing solutions (33.8° C) containing pyruvate, glutamate, fumarate, and glucose, V_m and a_Clⁱ were −27.6 ± 1.0 mV and 32.6 ± 1.5 mM, respectively. This apparent value of a_Clⁱ exceeded the level required for passive distribution of this ion (a_Cl^eq = 26.4 ± 1.3 mM) by 6.2 ± 1.0 mM. This difference was essentially unchanged in experiments where (i) Na⁺ was replaced by choline, (ii) HCO₃⁻ was removed, and (iii) Cl⁻ was replaced by gluconate. These data argue against the presence of Na⁺- or HCO₃⁻-coupled Cl⁻ transport mechanisms in the plasma membrane of mouse liver cells. This implies that a_Clⁱ is in fact at equilibrium and interference with the response of Cl⁻-selective microelectrodes by intracellular anions is responsible for the apparent difference between a_Clⁱ and a_Cl^eq. We found that Cl⁻-selective microelectrodes containing Corning 477315 ligand are sensitive to taurocholate, a representative bile salt. Their selectivity to taurocholate is about 60-times their selectivity towards Cl⁻. This suggests that interference of bile acids at concentrations normally present in hepatocytes with determinations of a_Clⁱ can account for the apparent difference a_Clⁱ − a_Cl^eq.     

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.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Kinetics of net H+, Ca2+, K+, Na+, NH+4, and Cl- fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues

Although temperature-induced changes in membrane structure and activity seem to be central to chilling stress perception, the specific details of temperature effects on plant nutrient acquisition remain obscure. In this study, we have undertaken a comparative study of low temperature effects on the activity of plasma membrane transporters of different ions in corn ( Zea mays L.) leaf and root tissues by non-invasive measurements of net ion fluxes using ion-selective microelectrode (the MIFE) technique. Kinetics of net H⁺, Ca²⁺, K⁺, Na⁺,     and Cl^– fluxes were measured as plant tissues recovered after short-term (3 h) chilling stress. The major findings can be summarized as follows: (1) The critical temperatures, under which the recovery of the activity of plasma membrane transporters took place, were found to be the same for all ions measured and are likely to be associated with the phase transition of membrane lipids. (2) The most pronounced was the reduction in net uptake of K⁺ and     (3) Chilling treatment caused a significant net influx of Cl^– and Na⁺ in the leaf tissue. (4) For the same species, the critical temperatures for membrane-transport processes in roots were 2–2.5°C lower than in leaves. Possible physiological significance of these findings is discussed.     

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.     

The Na+,K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.     

Changes in Na+, K+-adenosinetriphosphatase and 5'-nucleotidase activities in cell membrane isolated from rat liver after acute white phosphorus poisoning

Na+, K+-ATPase and 5'-Nucleotidase activities in rat liver plasmamembranes after "in vivo" intoxication with a single dose of white phosphorus (10 mg/kg b.w. "per os") are investigated. Na+, K+-ATPase activity is significantly increased 1 hour and inhibited 12 hours after intoxication. 5'-Nucleotidase is strongly increased at 1, 2 and 4 hours after poisoning and is significantly decreased at 12 hours. The enhancement of both the enzymatic activities is evident prior to triglyceride accumulation in rat liver. Our results suggest that lipid fluidity of cell membrane is early and mildly affected during white phosphorus poisoning.     

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K+, Na+ or H+ concentrations

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3’-dipropylthiadicarbocyanine, diS-C₃(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559–569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K⁺, Na⁺ and H⁺ concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ?tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K⁺, particularly at lower extracellular potassium concentration [K⁺]_out. In the absence of external energy source plasma membrane depolarization by K⁺ was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K⁺]_out. The plasma membrane depolarization by Na⁺ was independent from the presence of Trk1,2 transporters. Contrary to K⁺, Na⁺ depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pH_o of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pH_o values.     

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     

Changes in membrane potential, membrane resistance, and intracellular H, K, Na, and Cl activities during the progesterone-induced maturation of urodele amphibian oocytes

Changes in intracellular activities of H⁺, K⁺, Na⁺, and Cl⁻ ions were recorded with ion-selective microelectrodes during progesterone-induced maturation of full-grown oocytes of the urodele amphibians Ambystoma mexicanum and Pleurodeles waltlii. The membrane potential (E_m) and electrical resistance (R_m) were also determined. During the first hours after initiation of maturation, the oocytes slowly depolarized and R_m gradually increased. By the end of maturation of Pleurodeles oocytes E_m had stabilized at about −10 mV and R_m had increased from 410 to 1760 kΩ. The same initial pattern was observed for Ambystoma, but in most oocytes a rapid transition occurred at about the time of germinal vesicle breakdown (GVBD): E_m spontaneously shifted from about −15 to about +30 mV; simultaneously R_m dropped from 1230 down to 100 kΩ (i.e., less than the initial 270 kΩ resistance). The internal K⁺ activity did not show any important variation during maturation of Ambystoma and Pleurodeles oocytes. Na⁺ activity increased slightly at the onset of GVBD in Ambystoma; a further marked increase of Na⁺, accompanied by an increase in Cl⁻ activity, was observed as soon as E_m shifted to a positive value. In Pleurodeles sodium activity was also more elevated in matured than in immature oocytes. The average pH of Ambystoma immature oocytes was 7.48 ± 0.05 (external pH 7.5). A transient alkalinization to 7.64 ± 0.04 took place during the first 4–6 hr postprogesterone. Cytoplasmic pH was restored to 7.50 ± 0.07 between 10 and 12 hr postprogesterone, before the onset of GVBD and the shift of E_m. The difference between the measured oocyte pH and the calculated equilibrium pH decreases during the course of maturation, due partly to the depolarization of E_m.     

Nigericin-mediated H+, K+ and Na+ transports across vesicular membrane: T-jump studies.

The decay of delta pH across vesicular membranes by nigericin-mediated H+ and metal ion (M+) transports has been studied at 25 degrees C after creating delta pH by temperature jump (T-jump). In these experiments K+ or Na+ were chosen as M+ for the compensating flux. Theoretical expressions derived to analyse these data suggest a method for estimating the intrinsic rate constants for the translocation of nig-H (k1) and for the translocation of nig-M (k2) across membrane, from the pH dependence of the delta pH decay. The following could be inferred from the analysis of data. (a) At pH approximately 7.5 and 250 mM ion concentrations, nigericin-mediated H+ and M+ transport rates are lower in a medium of K+ than in a medium of Na+, although ionophore selectivity of nigericin towards K+ is 25-45-times higher than that towards Na+. However, at lower [M+] (approximately 50 mM) the transport rates are higher in a medium of K+ than in a medium of Na+. Such behaviours can be understood with the help of parameters determined in this work. (b) The intrinsic rate constants k1 and k2 associated with the translocations of nig-H and nig-K or nig-Na across membrane are similar in magnitude. (c) At pH approximately 7.5 translocation of nig-H is the dominant rate-limiting step in a medium containing K+. In contrast with this, at this pH, translocation of nig-M is the dominant rate-limiting step when metal ion is Na+. (d)k1 approximately k2 approximately 6.10(3) s-1 could be estimated at 25 degrees C in vesicles prepared from soyabean phospholipid, and lipid mixtures of 80% phosphatidylcholine (PC) + 20% phosphatidylethanolamine and 92% PC + 8% phosphatidic acid. (e) The apparent dissociation constants of nig-M in vesicles were estimated to be approximately 1.5.10(-3) M for K+ and 6.4.10(-2) M for Na+ (at 50 mM ion concentrations) using approximately 10(-8.45) M for the apparent dissociation constant of nig-H.     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

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.