Hyperpolarization-activated inward current associated with the frequency increase in ciliary beating of Paramecium

Summary In order to study the relationship between the inward Ca current activated by hyperpolarization and the frequency increase in ciliary beating, Paramecium cells were voltage clamped under conditions where K current was suppressed by use of CsCl electrodes and by extracellular tetraethyl ammonium. A 2-s pulse of hyperpolarization from the resting potential activated an inward current consisting of two components, an initial transient current peaking at 0.1–0.2 s (which had been identified as a Ca current) and a subsequent sustained current. The initial component was not associated with the frequency increase because the frequency increase was normally induced even when the peak current was almost completely inhibited by external addition of Ba²⁺. The second sustained current was closely correlated with the frequency increase. The frequency rose steeply with the sustained current and saturated at –0.6 nA. External addition of La³⁺ or replacement of Ca²⁺ by Mg²⁺ suppressed this current, and at the same time the frequency increase was inhibited. As the amplitude of the sustained current was not changed by deciliation, this current must pass through the somatic membrane. These results suggest that the frequency increase upon hyperpolarization is triggered by the voltage-activated inward current passing through the somatic membrane of the interciliary compartment.Abbreviations cAMP cyclic adenosine monophosphate - HEPES hydroxyethylpiperazine ethanesulfonate - TEA⁺ tetraethyl ammonium     

Ca2+ transport and chemoreception in Paramecium

Intracellular Ca²⁺ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca²⁺ pump and that a Ca²⁺-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca²⁺ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca²⁺ homeostasis and pump activity.Abbreviations EGTA ethyleneglycol tetra-acetate - ER endoplasmic reticulum - IBMX isobutyl methylxanthine - I _che index of chemokinesis - Mops 3-[N-morpholino] propanesulfonic acid - PEI phosphoenzyme intermediate - STEN sucrose, TRIS, EDTA, sodium chloride - TCA trichloroacetic acid - TRIS tris[hydroxymethyl] aminomethane     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Quantitative competition of calcium with sodium or magnesium for sorption sites on plasma membrane vesicles of melon (Cucumis melo L.) root cells

The presence of Ca²⁺ ions in solution is vital for root growth. The plasma membrane is one of the first sites where competition between Ca²⁺ and other ions occurs. We studied the competition between Ca²⁺ and Na⁺ or Mg²⁺ for sorption sites on the plasma membrane of melon root cells.Sorption of ⁴⁵Ca²⁺ to right-side-out PM vesicles of melon (Cucumis melo L.) roots (prepared by aqueous two-phase partitioning) was studied at various Ca²⁺ concentrations, in the presence of increasing concentrations of Na⁺ or Mg²⁺ chlorides. Experimentally determined amounts of Ca²⁺ sorbed to the plasma membrane vesicles agreed fairly well with those calculated from a competitive sorption model. The best fit of the model to the experimental data was obtained for an average surface area of 370 Ų per charge, and binding coefficients for Na⁺, Mg²⁺ and Ca²⁺ of 0.8, 9 and 50 m ^-1, respectively.Our results suggest that nonphospholipid components in the plasma membrane contribute significantly to Ca²⁺ binding. The high affinity of Ca²⁺ binding to the plasma membrane found in this study might explain the specific role of Ca²⁺ in relieving salt stress in plant roots.This research was supported by the GIFRID German-Israel fund for research and international development.     

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane

Summary Two-dimensional crystalline arrays of Ca²⁺-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na₃VO₄ in a Ca²⁺-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C₂(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca²⁺-ATPase, while negative potential disrupts preformed Ca²⁺-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca²⁺-ATPase.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Irreversible effects of calcium ions on the plasma membrane calcium pump

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mm Ca²⁺, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca²⁺ affinity at 37°C and is unaffected by serine-protease inhibitors. The activation caused by 1 mm Ca²⁺ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 m Ca²⁺ is not inhibited. Preincubations at 0°C also lead to activation, to a level up to half that seen at 37°C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca²⁺-containing solutions at 37°C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 m Ca²⁺. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca²⁺ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.We thank The Wellcome Trust for a research grant, the Medical Research Council for an equipment grant and the Regional Transfusion Service (Sheffield) for bank blood supplies.     

Functional reconstitution of ion channels from Paramecium cortex into artificial liposomes

Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless dior trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca²⁺ release, and the prospects of their purification are discussed.This work was supported by National Institutes of Health GM 26286 and GM 22714.     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca²⁺ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl₂ (0.1–2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

Spontaneous All-or-Nothing Action Potentials in the Ciliate Bursaridium difficile

The electrical membrane properties and the swimming behaviour of the freshwater ciliate Bursaridium difficile were studied by current clamp recordings and video analysis. The resting membrane potential was –45 ± 6 mV (mean ± SD, n = 80), and the input resistance and membrane capacitance were 109 ± 42 megaohms (MΩ) (n = 63) and 457 ± 150 picofarads (pF) (n = 42), respectively. Based on an estimated surface area of 6.8 × 10^-4 cm², the corresponding specific membrane resistance and capacitance are 7.4 × 10⁴Ω× cm² and 0.7 μF/cm². Bursaridium difficile generates spontaneous, all-or-nothing action potentials with a well-defined threshold in normal medium. The spontaneous firing frequency was 0.22 ± 0.06 Hz (n = 80). The maximum rate of rise of the action potentials was less than 1 V/s, and they displayed a prolonged plateau phase (0.5–1 s). The action potentials were abolished in nominal Ca²⁺-free solution and are thus Ca²⁺-spikes. The swimming pattern of Bursaridium in homogeneous surroundings is composed of forward swimming periods interrupted by regular, short periods of backward swimming followed by a change in the forward swimming direction. The turning frequency corresponded to the spontaneous firing frequency, and only forward swimming was observed in nominal Ca²⁺-free solution. The periods of backward swimming activity are thus linked to the spontaneous action potentials.     

Spontaneous inactivation of the Ca-sensitive K channels of human red cells at high intracellular Ca activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a (dCa²⁺/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about −65 mV was observed. At a membrane potential of about −70 mV and an intracellular concentration of about 2·10⁻⁴M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.