Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Permeability changes in the cytoplasmic membrane of Escherichia coli K-12 early after infection with bacteriophage T1.

The nature of the bacteriophage T1-induced changes in the permeability of the cytoplasmic membrane of Escherichia coli K-12 was investigated. At 20 degrees C and with glucose as a substrate, the addition of one bacteriophage per cell induced a complete and irreversible loss of K+ ions (single-hit phenomenon). K+ loss was compensated by an uptake of Na+, Li+, or choline by the cell, depending on which of these ions was the major cation in the medium. T1 depolarized the cells and inhibited 86Rb+-K+ exchange across the cytoplasmic membrane. The loss of K+ occurred independently of the Mg2+ concentration in the medium. By contrast, at low but not at high Mg2+ concentrations, T1 caused efflux of Mg2+ which in turn caused inhibition of respiration and a decrease of delta pH.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Insights into the biochemistry of the ubiquitous NhaP family of cation/H+ antiporters

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

The isomorphism of univalent thallium and potassium in membrane transport processes]

Thallium ions (T1+) are able to isomorphous replacement of K+ in various minerals. The similarity between. T1+ and K+ is based upon the closeness of their crystal radii, hydration energy and mobility in aqueous solutions. Under certain conditions, the behaviour of T1+ can be substantially different from that of K+. As distinguished from K+, thallium ions tend to associate with different anions forming ion pairs and complexes. As a rule, the stability of these compounds is rather low, but in many cases the T(1+)-anion interactions appear to play an important role in discriminating between T1+ and K+ involved in transport processes. T1+/K(+)-selectivity characterizes K(+)-transport mechanisms operating in different kinds of cells membranes. In excitable membranes (muscles, nerves) the rates of passive transport of T1+ and K+ are similar. In non-excitable membranes (epithelial cells, red blood cells, mitochondrial membranes, bacteria) the T1+ passive permeability is about one or two orders of magnitude higher than that of K+. A moderate T1+/K(+)-selectivity was reported for various types of K+ active transport mechanisms.     

The effect of membrane-bound calcium on the activity of adenosine triphosphatase from erythrocytes and erythrocyte permeability for monovalent cations]

The activities of Ca2+, Mg2+-ATPase and Na+, K+-ATPase and the permeability of reconstituted human erythrocytes for Na and K ions were measured, using Ca2+-EGTA, Ca2+ATP and Ca2+-sodium citrate buffers. It was found that the increase in the Ca2+/chelate ratio caused stimulation of Ca2+, Mg2+- and Na+, K+-Atpases and an increase in the rate constants of ouabain--dependent 42K+ influx and 22Na+ efflux from the erythrocytes. The use of the Ca2+-sodium citrate system as a calcium buffer did not change the parameters of the functional state of erythrocyte membranes. The data obtained are discussed in terms of a possible role of calcium ions, which are bound to the inner surface of the erythrocyte membrane, in the regulation of the systems of active and passive transport of cations.     

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles.

We have characterized the asymmetric effect of Ca2+ on passive K+ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca2+, but not Mg2+, can induce an increase in K+ uptake in inside out vesicles. The half-maximal concentration of Ca2+ required to induce the K+ uptake is 0.2 mM, and the permeability increase is not specific for K+. Thus, the Ca2+- induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca2+ and is specific for K+. Removal of spectrin had no effect on the vesicle permeability increase due to Ca2+. Studies with N-ethylmaleimide show that the vesicle channel openings is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca2+-induced vesicle pathway is distinct from the normal channel for passive K+ leak in the absence of Ca2+. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca2+ -stimulated channel opening.     

Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.     

K+/H+ antiport in heart mitochondria

Heart mitochondria depleted of endogenous divalent cations by treatment with A23187 and EDTA swell in (a) K+ acetate or (b) K+ nitrate when an uncoupler is present. These mitochondria also exchange matrix 42K+ with external K+, Na+, or Li+ in a reaction that does not require respiration and is insensitive to uncouplers. Untreated control mitochondria do not swell in either medium nor do they show the passive cation exchange. Both the swelling and the exchange reactions are inhibited by Mg2+ and by quinine and other lipophilic amines. Swelling and exchange are both strongly activated at alkaline pH, and the exchange reaction is also increased markedly by hypotonic conditions. All of these properties correspond to those reported for a respiration-dependent extrusion of K+ from Mg2+-depleted mitochondria, a reaction attributed to a latent Mg2+- and H+-sensitive K+/H+ antiport. The swelling reactions are strongly inhibited by dicyclohexylcarbodiimide reacted under hypotonic conditions, but the exchange reaction is not sensitive to this reagent. Heart mitochondria depleted of Mg2+ show marked increases in their permeability to H+, to anions, and possibly to cations, and the permeability to each of these components is further increased at alkaline pH. This generalized increase in membrane permeability makes it likely that K+/H+ antiport is not the only pathway available for K+ movement in these mitochondria. It is concluded that the swelling, 42K+ exchange, and K+ extrusion data are all consistent with the presence of the putative K+/H+ antiport but that definitive evidence for the participation of such a component in these reactions is still lacking.     

Heart sarcolemmal (Na+ + K+)-ATPase has an essential amino group in the potassium binding site on the enzyme molecule

Selective modification of primary amino groups of (Na+ + K+)-ATPase by trinitrobenzene sulfonic acid (TNBS) resulted in a considerable inhibition of the specific activity of the enzyme. Investigation by means of enzyme and sorption kinetics of activation of heart sarcolemmal (Na+ + K+)-ATPase by its monovalent cationic ligands added simultaneously with TNBS revealed: a considerable competition between K+-ions and TNBS for the potassium binding site on the enzyme molecule; a non-competitive type of inhibition of Na+-induced activation of the enzyme. Both, potassium and sodium ions depressed, and magnesium ions enhanced the initial rate of TNBS-sorption; however, none of the above cations influenced the equilibrium value of TNBS sorption onto isolated sarcolemmal membranes. Ouabain, on the other hand, did not inhibit the initial rate and decreased the equilibrium value of TNBS sorption onto the membranes. The results obtained enabled the identification of an essential amino group in the potassium binding site of the (Na+ + K+)-ATPase molecule.     

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

Temperature dependence of action of polymyxin B on Escherichia coli

The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes. At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s. The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C. The uptake of TPP+ also increased after polymyxin addition. The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously. In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C. This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes. The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E. coli. Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability. The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.     

The effects of protons on 3'',5''-cGMP-activated currents in photoreceptor patches.

Macroscopic 3',5'-guanine cyclic monophosphate (cGMP)-activated currents from photoreceptor outer segment membranes were examined as the pH on the cytoplasmic face of inside/out patches was reduced. In the absence of divalent cations, protons reduced the current in both directions without affecting the shape of the current-voltage relation consistent with a voltage-independent block. When Ca2+ was added to the bath, increasing the [H+] relieved the Ca2+ block and eliminated the Ca(2+)-induced reversal potential shifts seen at pH 7.4. These results suggest that protons alter Na+/Ca2+ permeability of the channel and relieve Ca2+ block of the sodium transport.     

Activity and properties of fructose bisphosphatase of turbellarian Phagocata sibirica

Activity and properties of fructose bisphosphatase (FBPase) was studied in the free-living turbellarian Phagocata sibirica. All subcellular fractions of P. sibirica (12 000 g cytosol, 105 000 g cytosol, mitochondria, and microsomes) have the FBPase activity. There was studied dependence of the FBPase reaction rate on the substrate concentration. For realization of the enzyme activity, the high affinity to substrate and presence of bivalent cations (Mg2+ or Mn2+) are necessary. The was studied the effect of various effectors as well as of monovalent (Na+, K+, Li+, and NH4+) and bivalent (Zn2+ and Cu2+) cations.     

Role of bivalent and monovalent cations in the functioning of myosin ATPase

The effects of bivalent (Mg2+, Ca2+, Sr2+) and monovalent (K+, Na+, NH4+) cations on the ATPase activity of subfragment 1 of myosin (SI) with a decreased Mg2+ content (EDTA-SI) were studied. Mg2+ activate the EDTA-SI ATPase, but only in the absence of other activating cations. K+, NH4+, a2+ and Sr2+ have a much stronger activating effect on EDTA-SI ATPase than on Mg-SI (SI enriched with Mg2+) ATPase. Monovalent cations inhibit Mg2+-ATPase and Ca2+-ATPase of EDTA-SI, while K+ and NH4+ activate Sr2+-ATPase of EDTA-SI. Based on experimental results and literary data, a hypothesis on the participation of the cations in the functioning of myosin ATPase was postulated. This hypothesis entails the existence of two closely interconnected cation-binding sites in the vicinity of the myosin active center (one for bivalent and one for monovalent cations); the ATPase activity of myosin is at any moment dependent on the nature of cations present in these two sites. An attempt to explain the role of the cations in the accomplishment of the ATPase reaction by myosin was made.