Resting potential of the mouse neuroblastoma cells: II. Significant contribution of the surface potential to the resting potential of the cells under physiological conditions

In the previous paper, we showed that the K⁺ channels of the mouse neuroblastoma cell (clone N-18) are closed at low concentration of external K⁺ ([K⁺]₀) including the physiological concentration for the cells. In the present study, the origin of the resting membrane potential of N-18 cells has been examined. (1) The resting membrane potential of N-18 cells was depolarized by increasing concentration of the polyvalent cations (La³⁺, Fe³⁺, Co²⁺, Ca²⁺, Sr²⁺, Mg²⁺) and by decreasing the pH of the medium. The input membrane resistance was slightly increased during the depolarization. The depolarization was not explained in terms of the diffusion of the cations across the membrane, since the trivalent cations of greater ionic size were effective at much lower concentrations than the divalent cations. The results obtained from the measurements of ⁸⁶Rb efflux suggested that the depolarization cannot be explained in terms of blocking of the K⁺ channels by the cations. (2) An increase in Ca²⁺ concentration from 0.3 to 1.8 mM induced depolarization of about 10 mV at low [K⁺]₀ where the K⁺ channels are closed, but did not induce any depolarization at high [K⁺]₀ where the channels are open. (3) In order to estimate the changes in the zeta-potential, the electrophoretic mobility of N-18 cells was measured under various conditions. There was a close correlation between the changes in the zeta-potential and those in the membrane potential in response to the polyvalent cations and proton. On the other hand, an increase in K⁺-concentration in the medium, which induced a large depolarization in the cells, did not affect the zeta-potential. (4) The results obtained were explained by an electrical circuit model for the membranes of N-18 cells. In this model, an electrical circuit for the membrane part carrying no selective ionic channels, in which changes in the surface potential directly affect the transmembrane potential, is connected in parallel to the usual circuit model representing selective ionic channel systems. It was concluded that the surface potential contributes significantly to the resting membrane potential of N-18 cells at low [K⁺]₀ where the K⁺ channels are closed.     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg²⁺ external to the isolated intact chloroplast interacts with stromal K⁺, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg²⁺ result in lower stromal K⁺, which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg²⁺ interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg²⁺ was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg²⁺ effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg²⁺ caused lower stromal K⁺ by restricting the rate of K⁺ influx; Mg²⁺ did not affect K⁺ efflux from the stroma. Mg²⁺ restriction of K⁺ influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg²⁺ with the chloroplast envelope likely mediated external Mg²⁺ effects on chloroplast metabolism.     

The concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.     

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

Summary Leakage of ions (Na⁺, K⁺) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by pore-forming agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu²⁺, Cd²⁺ or Zn²⁺. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca²⁺, Mn²⁺ or Ba²⁺; Mg²⁺, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca²⁺) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na⁺, K⁺, Rb⁺, NH ₄ ⁺ ) and divalent (Mg²⁺, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn²⁺ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

The effect of ionic strength on a Mg2+-ATPase and its relevance to the determination of (Na++K+)-ATPase

A ouabain-insensitive Mg²⁺-ATPase present in a microsomal fraction prepared from the dog submandibular gland was studied. This Mg²⁺-ATPase was inhibited by increasing concentrations of NaCl, KCl, RbCl and CsCl. The addition of an osmotically equal amount of sucrose was without effect. This inhibition was obtained over a pH range of from 6.3 to 8.8. The Mg²⁺-ATPase present in microsomes treated with NaI showed a similar inhibition. These results indicate that it is advisable to keep the ionic strength constant in solutions used to obtain (Na⁺+K⁺)-ATPase activities.     

Inorganic speciation of organotin(IV) cations in natural waters with particular reference to seawater

Abstract

This paper examines the inorganic complexing capacity of seawater, where chloride and sulfate ions are present in high concentration, towards mono- di- and tri-organotin(IV) cations which show a different trend of acidity, depending on cation charges, and a corresponding tendency to hydrolysis. By considering hydrolytic species and chloride and sulphate complex formation, a basic inorganic speciation model of organotins in synthetic seawater (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?, SO₄^2?) has been built up. The model has been extended to also consider interactions of organotins with carbonate and fluoride ions, which are other important components of seawater. Because of the strength of hydrolysis processes, the main complexes formed are in general mixed hydroxo-species. No species are formed by organotin cations and/or their hydroxo-species with fluoride owing to their very low concentration in fluoride, in comparison to the other components of seawater. In order to simplify calculations and to establish a cumulative inorganic binding capacity for seawater, we applied a chemical complexation model, according to which the major inorganic components of seawater are considered as a single salt BA.     

Correlated effects of external magnesium on cation content and DNA synthesis in culture chicken embryo fibroblasts.

Depletion of Mg²⁺ in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by ³H-thymidine incorporation, and concomitant decreases in cellular K⁺ and Mg²⁺ and increases in Na⁺ and Ca²⁺. In cells grown in media containing 0.2 mM Ca²⁺, graded reduction of Mg²⁺ from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10% of control at 0.016 mM Mg²⁺. Concomitantly, cell cations showed graded changes, Na⁺ increasing to 227%, K⁺ decreasing to 52.5%, Mg²⁺ decreasing to 57.5% and Ca²⁺ increasing to 153.5% of control. The effects of Mg²⁺ depletion on DNA synthesis and cell cation content exhibited a dependence on Ca²⁺ concentration, the effects being larger at low Ca²⁺ concentration. Use of inorganic pyrophosphate in the growth medium as a selective complexor of Mg²⁺ caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg²⁺ depletion. The effects of Mg²⁺ depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg²⁺ concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg²⁺ in the coordinate control of growth and metabolism in animal cells.     

Cation-induced,inhibitor-resistant photosystem II reactions in cyanobacterial membranes

Ferricyanide-supported oxygen evolution in sonic vesicles from the cyanobacterium $\text{Spirulina}\text{platensis}$ is only partially sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and addition of cations to inhibited membranes stimulates the rate of oxygen evolution. The order of cation effectiveness (M³⁺ > M²⁺ > M⁺) suggests that this stimulation is due at least in part to surface charge screening effects which permit freer access of anionic ferricyanide to the vesicle membrane surface; La³⁺, Ca²⁺, and K⁺ are most effective in this regard. Ferricyanide photoreduction is completely sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and neither mono- nor divalent cations affect this inhibition. Addition of La³⁺, on the other hand, causes a nearly complete restoration of ferricyanide-supported oxygen evolution. We conclude that the membrane surfaces of these vesicles are uniquely different from those o higher plants; sites of ferricyanide reduction associated with the interphotosystem chain are surface localized, and the primary acceptor region of photosystem II is susceptible to a trivalent cation-specific reaction in which ferricyanide may directly oxidize the primary acceptor.     

Subcellular Distribution of Basal and Cation-sensitive ATPases in Roots of Phaseolus vulgaris

A smooth microsomal fraction isolated from homogenates of Pbaseolus vulgaris root tissue has been found to possesss a highly active basal ATPase (measured in the absence of added cations). The microsomal membranes also feature a cation-sensitive ATPase which responds to Mg²⁺, Na⁺ and K⁺, but in a manner that is highly variable with pH. In contrast, membrane fragments prepared by a technique designed to yield purified plasma membrane were capable of little or no hydrolysis of ATP either in the presence or absence of added cations. This suggests that the microsomal activity is a reflection of membrane-bound ATPase which has been derived from cytoplasmic membranes, possibly the tonoplast, rather than plasma membrane.     

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li⁺, Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K⁺, Mg²⁺, and Ca²⁺ significantly, decreased after exposure to Az, except that Na⁺ and NH₄ ⁺ remained constant. Li⁺ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.     

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current.On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca²⁺ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca²⁺, Mg²⁺, or Mn²⁺ are detected. A23187 produces fluorescent transients under conditions of Ca²⁺ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca²⁺ content. Changes in A23187 fluorescence due to mitochondrial Ca²⁺ accumulation are much smaller by comparison and fluorescence transients are not detected.Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca²⁺ and Mg²⁺, and less than 2 nsec in the presence of excess EDTA.The present results are consistent with a model of ionophore-mediated cation transport in which free M²⁺ binds with A23187 at the membrane surface to form the complex M(A23187)⁺. Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187)₂, which carries the divalent cation through the membrane.These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.     

pH gradient across the lysosomal membrane generated by selective cation permeability and donnan equilibrium

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [¹⁴C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H⁺ ? Cs⁺ > Rb⁺ > K⁺ Na⁺ > Li⁺ ? Mg²⁺, Ca²⁺. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondifussible anions and (2) a selective permeability of the lysosomal membrane to cations.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Osmotic swelling of heart mitochondria in acetate and chloride salts. Evidence for two pathways for cation uptake.

Swelling of nonenergized heart mitochondria suspended in acetate salts appears to depend on the activity of an endogenous cation/H⁺ exchanger. Passive swelling in acetate shows a characteristic cation selectivity sequence of Na⁺ >Li⁺ >K⁺, Rb⁺, Cs⁺, or tetramethylammonium, a sharp optimum at pH 7.2–7.3, activation by Ca²⁺, and loss of activity on aging which can be related to loss of endogenous K⁺. The reaction is nearly insensitive to either addition of exogenous Mg²⁺ or removal of membrane Mg²⁺ with EDTA. Each of these characteristics of passive swelling in acetate salts is duplicated in chloride media when tripropyltin is added to induce Cl^?/OH^? exchange. In contrast to nonenergized mitochondria, swelling of respiring mitochondria has been postulated to depend on electrophoretic uptake of cations in response to an interior negative membrane potential. Respiration-dependent swelling in acetate shows an indistinct cation selectivity sequence with Li⁺ and Na⁺ supporting higher rates of swelling at higher efficiency than K⁺, Rb⁺, and Cs⁺. The high rates of respiration-dependent swelling in Li⁺ and Na⁺ are inhibited by low levels of exogenous Mg²⁺ (K_i of 5–10 μm), but a significant swelling with almost no cation selectivity persists in the presences of 2 mm Mg²⁺. Removal of membrane Mg²⁺ by addition of EDTA strongly activates the rate of respiration-dependent swelling and converts a sigmoid dependency of swelling rate on Li⁺ concentration to a hyperbolic one with a Km of about 14 mm Li⁺. The cation selectivity and Mg²⁺ dependence of the reaction induced in chloride salts by tripropyltin are identical to these properties in acetate. Energy-dependent swelling in acetate shows optimum activity at pH 6.5 which appears related to the availability of free acetic acid, since the corresponding reaction induced in chloride shows a broad optimum at about pH 7.5. These studies support the concept that monovalent cations enter nonenergized mitochondria by electroneutral exchange with protons but penetrate respiring mitochondria by electrophoretic movement through one or more uniport pathways. They further suggest that both a Mg²⁺-sensitive uniport with high activity for Na⁺ and Li⁺ and a Mg²⁺-insensitive pathway with little cation discrimination are available in the membrane.     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

ATP sensitive K+ conductance in pancreatic zymogen granules: Block by glyburide and activation by diazoxide

Summary The properties of transporters (or channels) for monovalent cations in the membrane of isolated pancreatic zymogen granules were characterized with an assay measuring bulk cation influx driven by a proton diffusion potential. The proton diffusion potential was generated by suspending granules in an isotonic monovalent cation/acetate solution and increasing the proton conductance of the membrane with a protonophore. Monovalent cation conductance had the sequence Rb⁺ > K⁺ > Na⁺ > Cs⁺ > Li⁺ > N-methyl glucamine⁺. The conductance could be inhibited by Ca²⁺, Mg²⁺, Ba²⁺, and pharmacological agents such as quinine, quinidine, glyburide and tolbutamide, but not by 5 mm tetra-ethyl ammonium or 5mm 4-aminopyridine, when applied to the cytosolic surface of the granule membrane. Over 50% of K⁺ conductance could be inhibited by millimolar concentrations of ATP or MgATP. The inhibition by MgATP, but not by ATP itself, was reversed by the K⁺ channel opener diazoxide. The inhibitory effect is probably by a noncovalent interaction since it could be mimicked by nonhydrolyzable analogs of ATP and by ADP. The reversal of MgATP inhibition by diazoxide may be mediated by phosphorylation since it was not affected by dilution, and was blocked by the protein kinase inhibitor H7. The properties of the K⁺ conductance of pancreatic zymogen granule membranes are similar to those of ATP-sensitive K⁺ channels found in the plasma membrane of insulin-secreting islet cells, neurons, muscle, and renal cells.This research was supported by grants from the Cystic Fibrosis Foundation (ZO298) and NIH (DK-39658). F.T. is recipient of a Fellowship from the American Cystic Fibrosis Foundation. K.C.V. is a participant of a summer research program for undergraduate students from Knox College, Galesburg, IL.     

Properties of (Na+ + K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124–132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na⁺ + K⁺)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na⁺ + K⁺)-activated ATPase activity was documented by demonstrating specific cation requirements for Na⁺ and K⁺, while the divalent cation, Ca²⁺, and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na⁺ + K⁺)-activated ATPase activity averaged 10.07 ± 2.80 μmol P_i/mg protei per h compared to 50.03 ± 11.41 for Mg²⁺-activated ATPase and 58.66 ± 10.07 for 5′-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na⁺ + K⁺)-activated ATPase without any effect on Mg²⁺-activated ATPase. Both (Na⁺ + K⁺)-activated ATPase and Mg²⁺-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na⁺ + K⁺)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na⁺ + K⁺)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.