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.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

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.     

Evidence for voltage-dependent Cl− permeability in mouse pancreatic β-cells

Microdissected -cell-rich pancreatic islets fromob/ob-mice were used in studies of transmembrane³⁶Cl^– efflux. The mean rate coefficient for³⁶Cl^– efflux was stable at 0.158 min^–1 during the initial 10 min. Depolarization of the -cell plasma membrane by acute increases in extracellular K⁺ (5–130mM) stimulated the³⁶Cl^– efflux in a concentration-dependent manner. Glucose-induced (20mM) and K⁺-induced increases in³⁶Cl^– efflux were largely overlapping, but even at 135.9 mM K⁺, glucose slightly further enhanced the³⁶Cl^– efflux rate. The data suggest (1) that pancreatic -cells are equipped with a voltage-dependent Cl^– permeability, (2) that glucose-induced increase in Cl^– permeability may, at least partly, be mediated by primary membrane depolarization, and (3) that glucose in addition may activate other mechanisms for -cell Cl^– transport.     

K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Summary Bidirectional transepithelial K⁺ flux measurements across high-resistance epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K⁺ flux.Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K⁺.K⁺ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain-and furosemide-insensitive component.The action of furosemide upon K⁺ influx is independent of (Na⁺–K⁺)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K⁺, Na⁺ and Cl^– content. Acetate and nitrate are ineffective substitutes for Cl^–, whereas Br^– is partially effective. Partial Cl^– replacement by NO₃ gives a roughly linear increase in the furosemide-sensitive component. Na⁺ replacement by choline abolishes the furosemide-sensitive component, whereas Li⁺ is a partially effective replacement. Partial Na⁺ replacement with choline gives an apparent affinity of 7mm Na, whereas variation of the external K⁺ content gives an affinity of the furosemide-sensitive component of 1.0mm.Furosemide inhibition is of high affinity (K _1/2=3 m). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K⁺ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective.Externally applied furosemide and Cl^– replacement by NO ₃ ^– inhibit K⁺ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of KK exchange.     

Modulation of maize roots H+-ATPase by sulfated polysaccharides

Vesicles derived from maize roots retain a membrane bound H⁺-ATPase that is able to pump H⁺ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H⁺-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent K_m for ATP from 0.28 to 0.95 mM and a decrease of the V_max from 14.5 to 7.1 mol P_i/mg · 30 min^–1. At pH 7.0 dextran 8000 promotes an increase in V_max from 6.7 to 11.7 mol Pi/mg · 30 min^–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K⁺ and Na⁺. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K⁺>Na⁺Li⁺).Abbreviations Mops 4-morpholinopropanesulfonic acid - EDTA ethylenediaminetetraacetic acid - ACMA 9-amino-6-chloro-2-methoxyacridine - FCCP carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone     

Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Summary The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from –23.7 to –46.7 and –4.3 to –5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G and 25m, respectively. The cells are electrically coupled via low resistance intercellular communications (58 M). Intracellular ion activities for Na⁺, K⁺ and Cl^– averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofP _K>P _Cl>P _Na. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na⁺–K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.     

Differential sensitivity of cardiac K(ATP) + channels to guanine nucleotides — evidence for a heterogeneous channel population

In cell-free patches from cultured neonatal rat cardiocytes, the cytosolic presence of GTP--S (100 µmol/l) or GDP-\-S (100 µmol/1) activated K_(ATP) ⁺ channels. GTP--S required cytosolic Mg⁺⁺, suggesting that an activated G-protein causes the increase in open probability. The great variations of the channel response to GTP--S and GDP-\-S indicates that cardiac K_(ATP) ⁺ channels represent a heterogeneous family. Correspondence to: M. Kohlhardt     

Exocytosis in Single Chromaffin Cells: Regulation by a Secretory Granule-Associated Go Protein

1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, Go has beenfound associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells.2. Microinjection of GTPS and mastoparan strongly inhibits the amperometric response to either nicotine or high K⁺.3. The presence of mastoparan in the cell incubation medium had no effect on K⁺-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane.4.Microinjection of anti-Go antibodies potentiates by more than 50% the K⁺-evoked secretion, whereas anti-Gi_1/2 antibodies have no effect.5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolicprotein GAP-43 stimulates Go in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin cells by stimulating a granule-associated Go protein.     

Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca²⁺-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with K_m for Ca ions of 0.4 M and V_max=12.5±2.4 mol P_i.mg^–1h^–1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive³H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with K_i=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >ZnCo>Ni. In contrast to low affinity (high K_m) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 M) and N-ethylmaleinimide (100 M). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.Abbreviations used BSA bovine serum albumin - HA Ca-ATPase high-affinity Ca²⁺-ATPase - K-pNPPase onabain-sensitive, K⁺-stimulated p-nitrophenyl phosphatase - NEM N-ethylmaleinimide - SIM 250 mM sucrose, 10 mM imidazole-HCl pH 7.4 - TRIS tris (hydroxymethyl) aminomethane     

Death of Cerebellar Granule Neurons Induced by Piperine Is Distinct from that Induced by Low Potassium Medium

We compared neurotoxicity of piperine and low K⁺on cultured cerebellar granule neurons. As considered from lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide reduction, both piperine and shifting from high K⁺(25 mM) to low K⁺(5.4 mM) were cytotoxic to cerebellar granule neurons. Protein synthesis inhibitors, cycloheximide and anisomycin, and an endonuclease inhibitor, aurintricarboxylic acid, were protective against low K⁺-induced neuronal death whereas they were ineffective against that induced by piperine. D--tocopherol, trolox, and a spin trap 3,3,5,5-tetramethyl-l-pyrroline-l-oxide were protective against piperine neurotoxicity whereas they had no effect on that induced by low K⁺. These results suggest that piperine and low K⁺may exert neurotoxic effects on cerebellar granule neurons through different mechanisms. Death of cerebellar granule neurons induced by piperine may be mediated by non-apoptotic mechanisms and may involve membrane lipid peroxidation and/or free radical generation.     

A K+-selective,three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle

Summary Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K⁺-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting (closed) state and two conducting states and . In 0.1m K⁺ the single-channel conductances are 50 and 150 pS for and , respectively. The probabilities of appearearance of the three states are voltage-dependent, and transitions between the closed and states proceed through the state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺>Cs⁺. Both open states are blocked by Cs⁺ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs⁺ block for the two states differ in a way suggesting that the Cs⁺ blocking site is located more deeply inside the membrane in the than in the state.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Na+ and K+ fluxes stimulated by Na+-coupled glucose transport: Evidence for a Ba2+-insensitive K+ efflux pathway in rabbit proximal tubules

Summary Addition of glucose or the nonmetabolizable analogue -methyl-d-glucoside to rabbit proximal tubules suspended in a glucoseand alanine-free buffer caused a sustained increase in intracellular Na⁺ content (+43±7 nmol · (mg protein)^–1) and a concomitant but larger decrease in K⁺ content (–72±11 nmol· (mg protein)^–1). A component of the net K⁺ efflux was Ba²⁺ insensitive, and was inhibited by high (1mm) but not low (10 m) concentrations of the diuretics, furosemide and bumetanide. The increase in intracellular Na⁺ content is consistent with the view that the increased rates of Na⁺ and water transport seen in the proximal tubule in the presence of glucose can be attributed (at least in part) to a stimulation of basolateral pump activity by an increased [Na⁺] _i .     

Reduction of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to chemically induced hyperhomocysteinemia

Hyperhomocysteinemia occurs in homocystinuria, an inherited metabolic disease clinically characterized by thromboembolic episodes and a variable degree of neurological dysfunction whose pathophysiology is poorly known. In this study, we induced elevated levels of homocysteine (Hcy) in blood (500 M), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 mol/g of body weight) twice a day at 8-hr intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the hippocampus of treated Hcy- and saline-treated rats. Chronic administration of Hcy significantly decreased (40%) Na⁺,K⁺-ATPase activity but did not alter Mg²⁺-ATPase activity. Considering that Na⁺,K⁺-ATPase plays a crucial role in the central nervous system, our results suggest that the brain dysfunction found in homocystinuria may be related to the reduction of brain Na⁺,K⁺-ATPase activity.