Permeability of reconstituted sarcoplasmic reticulum vesicles: Reconstitution of the K+, Na+ channel

Permeability properties of reconstituted rabbit skeletal muscle sarcoplasmic reticulum vesicles were characterized by measuring efflux rates of [³H]inulin, [³H]choline⁺, ⁸⁶Rb⁺, and ²²Na⁺, as well as membrane potential changes using the voltage-sensitive probe, 3,3′-dipentyl-2,2′-oxacarbocyanine. Native vesicles were dissociated with deoxycholate and were reconstituted by dialysis. Energized Ca²⁺ accumulation was partially restored. About $\text{1}\text{2}$ of the reconstituted vesicles were found to be ‘leaky’, i.e., permeable to choline⁺ or Tris⁺ but not to inulin. The remaining reconstituted vesicles were ‘sealed’, i.e., impermeable to choline⁺, Tris⁺ and inulin. Sealed reconstituted vesicles could be further subdivided according to their K⁺, Na⁺ permeability. About $\text{1}\text{2}$, previously designated Type I, were readily permeable to K⁺ and Na⁺, indicating the presence of the K⁺, Na⁺ channel of sarcoplasmic reticulum. The remaining sealed vesicles (Type II) formed a permeability barrier to K⁺ and Na⁺, suggesting that they lacked the K⁺, Na⁺ channel. These studies show that the K⁺, Na⁺ channel of sarcoplasmic reticulum can be solubilized with detergent and reconstituted with retention of activity. Furthermore, our results suggest that part or all of the decreased Ca²⁺-loading efficiency of reconstituted vesicles may be due to the presence of a significant fraction of leaky vesicles.     

Permeability of sarcoplasmic reticulum membrane. The effect of changed ionic environments on Ca2+ release

Summary Permeability properties and the effects of a changed membrane potential on Ca²⁺ release of sarcoplasmic reticulum vesicles of rabbit skeletal muscle were investigated by Millipore filtration. The relative permeability of sarcoplasmic reticulum to solutes determined under conditions of isotope exchange at equilibrium and/or under conditions of net flow of solute and water into the vesicles was as follows: sucrose, Ca²⁺, Mn²⁺–, choline⁺, Tris⁺–+, Na⁺, Li⁺, Cl^–. Transient membrane potentials were induced by rapidly changing the ionic environment of the vesicles. Knowledge of the relative permeation rates of the above ions allowed prediction of the direction and extent of membrane polarization. Osmotic effects in the polarization measurements due to the rapid influx of solute and water into the vesicles were minimized by using media containing a fast (K⁺ or Cl^–) and a relatively slow (gluconate^– or choline⁺) penetrating ion.⁴⁵Ca²⁺ efflux from vesicles derived from different parts of the sarcoplasmic reticulum structure was not appreciably changed when vesicles were made more positive inside (choline chloride potassium gluconate) or more negative inside (potassium gluconate choline chloride). These studies suggest that part or all of the ion-induced changes in sarcoplasmic reticulum membrane permeability, previously interpreted to indicate depolarization-induced Ca²⁺ release, may be due to osmotic effects.     

Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

Summary Ouabain-insensitive, furosemide-sensitive Rb⁺ influx (J _Rb) into HeLa cells was examined as functions of the extracellular Rb⁺, Na⁺ and Cl^– concentrations. Rate equations and kinetic parameters, including the apparent maximumJ _Rb, the apparent values ofK _m for the three ions and the apparentK _i for K⁺, were derived. Results suggested that one unit molecule of this transport system has one Na⁺, one K⁺ and two Cl^– sites with different affinities, one of the Cl^– sites related with binding of Na⁺, and the other with binding of K⁺(Rb⁺). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of²²Na⁺ and Rb⁺, and a 12 stoichiometry between those of Rb⁺ and³⁶Cl^–. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl^– inhibited ouabain-insensitive Rb⁺ influx, whereas sulfamate and probably also gluconate did not inhibitJ _Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na⁺ and one Cl^– bind concurrently to their sites and then one K⁺ (Rb⁺) and another Cl^– bind concurrently. 2) After completion of ion bindings Na⁺, K⁺(Rb^–) and Cl^– in a ratio of 112 show synchronous transmembrane movements.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

A non-specific Ca2+ (or Mg2+)-stimulated ATPase in rat heart sarcoplasmic reticulum

ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Increasing concentrations of Mg²⁺ produced an apparent inhibition of the Ca²⁺-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca²⁺ with a high affinity (K_0.5 Ca²⁺ = 0.41 M) suggesting the presence of a high affinity Ca²⁺-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations and failed to stimulate membrane phosphorylation and Ca²⁺ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca²⁺-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP adenosine 3,5-monophosphate - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - PLB phospholamban - SR sarcoplasmic reticulum - TFP trifluoperazine     

K+ influx components in ascites cells: The effects of agents interacting with the (Na++K+)-pump

Summary Several agents known to interact with the (Na⁺+K⁺)-pump were tested for their effects on the components of steady-state K⁺ flux in ascites cells.⁸⁶Rb⁺ was used as a tracer for K⁺, and influx was differentiated into a ouabain-inhibitable pump component, a Cl^–-dependent and furosemide-sensitive exchange component, and a residual leak flux. All agents tested (ouabain, quercetin, oligomycin, phosphate) affected both the pump flux and the Cl^–-linked flux. These findings suggest a linkage between the activity of the Na/K ATPase and the Cl^–-dependent K⁺ exchange flux. In the discussion we point out that the mechanism of this linkage could be direct; e.g., Cl^–-dependent exchange may represent a mode of operation of the Na/K ATPase. However, data from this and other systems tend to suggest an indirect linkage between the Na⁺ pump and a KCl symporter, perhaps via a change in the level of intracellular ATP.     

Interdependence of H+ and K+ fluxes during the Ca2+-pumping activity of sarcoplasmic reticulum vesicles

The release of H⁺ during the oxalate-supported Ca²⁺ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca²⁺ accumulation. The Ca²⁺ uptake is increased and the H⁺ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H⁺-monovalent cation exchange. The functioning of the Ca²⁺-pump is disturbed by the presence of potassium gluconate and, to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca²⁺-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K⁺, and also Cl^–, appear to be involved in secondary fluxes during the active accumulation of Ca²⁺. The microsomal preparation seems homogeneous with respect to the K⁺-channel, showing an apparent rate constant for K⁺ release of approximately 25 s^–1 measured with the aid of⁸⁶Rb⁺ tracer under equilibrium conditions. A Rb⁺ efflux, sensitive to Ca²⁺-ionophore, can be also detected during the active accumulation of Ca²⁺. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca²⁺ uptake and H⁺ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

The comparative transport of K+ and Rb+ in normal and malignant rat tissuesin vivo and in liver slices,diaphragm, and tumor slicesin vitro

Summary The selectivity in the steady state uptakes of Rb⁺ and K⁺ has been studied in a number of normal and malignant rat tissues. The selectivity is minimal in erythrocytes and the two fastest-growing of four transplantable tumors, in which there is little discrimination between the two ions, and ranges upwards to a maximum Rb⁺ uptake in liver. In each tissue, the selectivity is independent of Rb⁺ concentration or of K⁺ deficiency (except in skeletal muscle). In liver slicesin vitro, reduction of energy metabolism by lowering the temperature or by the addition of metabolic inhibitors reduces the Rb⁺K⁺ discrimination proportionately much more than K⁺ transport. Diaphragm and slices of a transplantable tumor give similar results. With temperature reduction, there is a logarithmic relation between the Rb⁺K⁺ discrimination ratio and the respiration rate of liver slices. The results are quantitatively accounted for by simultaneous diffusion and metabolically coupled transport across a homogeneous membrane in which Rb⁺ transport is more closely coupled than that of K⁺ to a metabolic flux across the membrane. There is evidence that the tissue differences in Rb⁺K⁺ selectivity originate in the different levels of the coupling metabolic flux in different cell types and thus of the energy expenditure on ion transport. In contrast to the differences in steady state selectivity between Rb⁺ and K⁺, the initial ratio of uptakes of trace⁴³K and⁸⁶Rb, in otherwise steady state conditions, is close to unity in both liver and tumor slices, in agreement with theoretical calculations.     

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.     

Resistance adaptation of the freshwater crayfish and thermal inactivation of membrane-bound enzymes

Summary Heat death and resistance adaptation of freshwater crayfish are thought to be properties of its muscle membranes. The inactivation at high temperatures of a membrane-bound enzyme, the Ca⁺⁺-stimulated ATPase of crayfish abdominal muscle sarcoplasmic reticulum, and the effect of thermal acclimation of crayfish upon the inactivation kinetics have been investigated. In the absence of KCl, the Ca⁺⁺-stimulated ATPase is irreversibly inactivated with pseudo-first order kinetics at temperatures that cause heat death in the whole animal. 0.1–10.0 mM KCl resulted in slower inactivation, while 100 mM KCl activated the enzyme to 120–180% of its original activity. Enzyme activation by KCl and heat involved a shift in the enzyme concentration/activity curve. Thermal acclimation of crayfish had no significant effect upon the kinetics or Arrhenius activation energy for enzyme inactivation (100.6±10.5 and 92.3±14.6 kcal/mole for preparations from 4°C and 25°C acclimated crayfish).Ca⁺⁺-stimulated ATPase isolated from heat dead crayfish exhibited normal in vitro activity due presumably to the high intracellular K⁺ concentration. Nevertheless, the close correspondence between heat death temperatures and inactivation temperatures for several membrane-bound enzymes of muscle is thought to reflect some perturbation of muscle structure that occurs during heat death.Abbreviations ATP Ademosine 5-Triphosphate - EGTA Ethyleneglycol-bis [-amino-ethyl ether] - N N-tetraacetic acid - Hepes N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - FSR Fragmented sarcoplasmic reticulum - Tris Tris (hydroxymethyl)aminomethane     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Ionic permeability of sarcoplasmic reticulum vesicles measured by light scattering method

Summary The volume change of sarcoplasmic reticulum vesicles was followed by measuring the light scattering intensity. When the salt concentration of the suspension of sarcoplasmic reticulum vesicles was increased by using a stopped flow apparatus, the light scattering intensity rapidly increased at the beginning and then decreased. The fast increase in the light scattering intensity is caused by the decrease of the volume of sarcoplasmic reticulum vesicles due to the outflow of water. The following decrease in the light scattering intensity is caused by the increase of the volume due to the inflow of the solutes and water. From the former and the latter rates, the permeation times of water and the solutes could be calculated, respectively. According to the same method, permeation times of various salts were determined. The rate of the inflow of the salts was dependent on the movement of the slower ions, that is, ions move as a pair.In the case of potassium salts, an increase in the permeation rate of the salts was observed when valinomycin was added to the membrane suspensions. From these experiments, as a measure of permeability, half permeation times of various ions and molecules were determined. The following are typical results: water 0.1, Li⁺ 36, Na⁺ 26, K⁺ 20, Rb⁺ 16, Cl^– 0.4, methanesulfonate 20, phosphate 10.5, oxalate 40 in seconds at room temperature. As a whole, sarcoplasmic reticulum was found to be an anion permeable membrane.     

Modulation of HERG channel inactivation by external cations

We investigated the effect of external cations on the permeability characteristics and gating kinetics of the human ether-à-go-go-related gene (HERG) current using the whole-cell patch-clamp technique. Inward HERG currents were recorded on hyperpolarization in 140 mM external Cs⁺ and Rb⁺, as well as K⁺. The permeability ratios of Rb⁺ and Cs⁺ relative to K⁺ were 1.25 and 0.56, respectively. Biphasic outward currents were recorded on depolarization in 140 mM Cs⁺ and in Rb⁺ with much smaller amplitude. The voltage dependence of inactivation was affected by external cations, such that the half-inactivation voltage shifted from –69.4±3.7 mV in K⁺ to –30.7±1.6 mV in Cs⁺ and to –35.8±1.9 mV in Rb⁺ (n=5). The time constants of inactivation were also changed significantly by external cations; of inactivation at +40 mV was 16.4±2.2 ms in 140 mM K⁺, 181±20.3 ms in Cs⁺, and 94.1±7.6 ms in Rb⁺ (n=5). Voltage dependence of activation was not altered significantly. The inhibition of the rapid inactivation mechanism by large cations may suggest that the foot-in-the-door model of gating is involved in HERG channel inactivation.     

Voltage-dependent inhibition of the sodium pump by external sodium: Species differences and possible role of the N-terminus of the α-subunit

Currents generated by the Na⁺/K⁺ ATPase were measured under voltage clamp in oocytes of Xenopus laevis. The dependence of pump current on external [Na⁺] was investigated for the endogenous Xenopus pump as well as for wild-type and mutated pumps of electroplax of Torpedo californica expressed in the oocytes. The mutants had -subunits truncated before position Lys²⁸ (K28) or Thr²⁹ (T29) of the N-terminus. The currents generated by all variants of pump molecules in the presence of 5 mM K⁺ show voltage-dependent inhibition by external [Na⁺]. The apparent K₁ values increase with membrane depolarisation, and the potential dependence can be described by the movement of effective charges in the electrical potential gradient across the membrane. Taking into account Na⁺-K⁺ competition for external binding to the E₂P form, apparent K₁ values and effective charges for the interaction of the Na⁺ ions with the E₂P form can be estimated. For the Xenopus pump the effective charge amounts to 1.1 of an elementary charge and the K₁ value at 0 mV to 44 mM. For the wild-type Torpedo pump, the analysis yields values of 0.73 of an elementary charge and 133 mM, respectively. Truncation at the N-terminus removing a lysinerich cluster of the a-subunit of the Torpedo pump leads to an increase of the effective charge and decrease of the K₁ value. For K28, values of 0.83 of an elementary charge and 117 mM are obtained, respectively. If LyS²⁸ is included in the truncation (·T29), the effective charge increases to 1.5 of an elementary charge and the apparent K₁ value is reduced to 107 mM. The K, values for pump inhibition by external Na⁺, calculated by taking into account Na⁺-K⁺ competition, are smaller than the K_/12 values determined in the presence of 5 mM [K⁺]. The difference is more pronounced for those pump variants that have higher K_m, values. The variations of the parameters describing inhibition by external [Na⁺] are qualitatively similar to those described for the stimulation of the pumps by external [K⁺] in the absence of extracellular [Na⁺]. The observations may be explained by an acess channel within the membrane dielectric that has to be passed by the external Na⁺ and K⁺ ions to reach or leave their binding sites. The potential-dependent access and/or the interaction with the binding sites shows species differences and is affected by cytoplasmic lysine residues in the N-terminus.     

Ion permeability of maize root membrane vesicles: Studies with light scattering

A possible modulation of permeabilities of membrane vesicles to anions and cations was explored by light scattering techniques, evaluated by measuring the capacity of the vesicles to shrink and swell in response to changes of the osmolarity of the incubation medium. Membrane fractions were obtained by phase partition. Purity was evaluated by detection and quantification of membrane enzyme markers: vanadate-sensitive ATPase for the plasma membrane, nitrate-sensitive ATPase for the tonoplast and azide-sensitive ATPase for mitochondria. Membrane vesicles (250 g protein) were exposed to hypertonic solutions of salts (0.6 osmolar). Kinetics of the changes in apparent absorbance at 546 nm were observed by the addition of potassium, nitrate and chloride salts. The diffusion of ions into vesicles was induced by an osmotic gradient across the membrane and brought about volume changes of vesicles. Upon addition of vesicles to the different solutions the following ion permselectivity sequences were observed: PNO ₃ ^– >PCl ^–>PSO ₄ ^2– and PK ⁺PNa ⁺>PNH ₄ ⁺.Abbreviations ATP adenosine 5-triphosphate - EDTA ethylene diaminetetraacetic acid - Tris-Mes (Tris[hydroxymethyl]aminomethane, Mes-(2-[N-Morpholino]ethanesulfonic acid) - PEG polyethylene glycol     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Voltage-gated cation conductance channel from fragmented sarcoplasmic reticulum: Steady-state electrical properties

Summary The interaction of fragmented sarcoplasmic reticulum (SR) with an artificial planar phospholipid membrane under conditions known to induce fusion of phospholipid vesicles raises the conductance of the planar bilayer by several orders of magnitude. Measurements of steady-state electrical properties of bilayers thus modified by SR show that two types of conductance pathways are present. One is a voltage-independent pathway which may be somewhat anion-selective. The other is a voltagegated ionophore showing selectivity to small monovalent cations. This latter ionophore is fully oriented within the artificial bilayer and is inhibited asymmetrically by divalent cations. It is also inhibited below pH 6. The ionophore displays single-channel conductance fluctuations between two states, open and closed, with an open-state conductance of 1.4×10^–10 mho in 0.1m K⁺. The physiological function of this ionophore is unknown.     

Variability of the K+−Na+ discrimination of beauvericin in mitochondrial membranes

In intact mitochondria supplemented with succinate or -hydroxybutyrate, the rates of oxygen consumption induced by beauvericin followed the ionic selectivity pattern: Na⁺>Rb⁺, Cs⁺, K⁺, Li⁺.When the respiratory substrate is glutamate plus malate in the absence of phosphate, the selectivity pattern is: K⁺>Rb⁺>Cs⁺>Li⁺>Na⁺.When the media are supplemented with phosphate, the Na⁺/K⁺ discrimination of beauvericin is considerably modified with all the respiratory substrates, being K⁺>Na⁺ with succinate and Na⁺>K⁺ with glutamate plus malate, whereas no significant ionic selectivity differences were obtained with -hydroxybutyrate.The respiratory control induced by oligomycin in submitochondrial particles is released by beauvericin only in the presence of a nigericin-like carboxylic antibiotic and an alkali metal cation, being far more effective in K⁺ than in Na⁺.This selectivity is maintained regardless of whether NADH or succinate is used as a respiratory substrate.Release of respiratory control can also be obtained with a combination of beauvericin and NH₄Cl.This information indicates that the ionic selectivity pattern obtained with beauvericin in mitochondrial membranes is an intrinsic property of the antibiotic which, however, can be significantly modified by factors such as the nature of the translocatable substrate anion or other anionic species, as well as the possible operation of a Na⁺/H⁺ antiporter existent in the membrane.