Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

The ionic components of the current pulses generated by developing fucoid eggs.

Using a newly developed, extracellular vibrating electrode, we studied the ionic composition of the current pulses which traverse the developing Pelvetia embryo. External Na⁺, Mg²⁺, or SO₄^2?, are not needed for the first 20 min of pulsing. In fact, lowering external Na⁺ or Mg²⁺ (or K⁺) actually stimulates pulsing. Since tracer studies show that Ca²⁺ entry is speeded by Na⁺, Mg²⁺, or K⁺ reduction, these findings suggest that Ca²⁺ entry triggers pulsing. A sevenfold reduction in external Cl^? raises pulse amplitudes by 60%. Moreover, Cl^? is the only major ion with an equilibrium potential near the pulse reversal potential. These facts suggest that Cl^? efflux carries much of the “inward” current. We propose a model for pulsing in which increased Ca²⁺ within the growing tip opens Cl^? channels. The resulting Cl^? efflux slightly depolarizes the membrane and thus drives a balancing amount of K⁺ out. Thus, the pulses release KCl and serve to relieve excess turgor pressure. By letting Ca²⁺ into the growing tip, they should also strengthen the transcytoplasmic electrical field which is postulated to pull growth components toward this tip.     

The mechanism of vanadium action on selective K+-permeability in human erythrocytes

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO²⁺, +4 oxidation state) to vanadate (VO₃^?, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K⁺ permeability, which is very similar to the Gardos Ca²⁺-induced K⁺ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K⁺. In ultrapure media with very low Ca²⁺, however, vanadyl has no effect on K⁺ permeability. These experiments suggest that Ca²⁺ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K⁺ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca²⁺ from EDTA. Vanadate, an inhibitor of Ca²⁺-ATPase, causes a selective increase in K⁺ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca²⁺. Earlier observations of effects of vanadyl and vanadate on erythrocyte K⁺ permeability can thus be explained on the basis of inhibition of the Ca²⁺ pump by vanadium, leading to an increase in intracellular Ca²⁺ concentration.     

The cyclic nucleotide‐gated channel AtCNGC10 transports Ca2+ and Mg2+ in Arabidopsis

The suppression of the cyclic nucleotide‐gated channel (CNGC) AtCNGC10 alters K⁺ transport in Arabidopsis plants. Other CNGCs have been shown to transport Ca²⁺, K⁺, Li⁺, Cs⁺ and Rb⁺ across the plasma membrane when expressed in heterologous systems; however, the ability of the AtCNGC10 channel to transport nutrients other than K⁺ in plants has not been previously tested. The ion fluxes along different zones of the seedling roots, as estimated by the non‐invasive ion‐specific microelectrode technique, were significantly different in two AtCNGC10 antisense lines (A2 and A3) in comparison to the wild type (WT). Most notably, the influxes of H⁺, Ca²⁺ and Mg²⁺ in the meristem and distal elongation zones of the antisense A2 and A3 lines were significantly lower than in the WT. The lower Ca²⁺ influx from the external media corresponded to a lower intracellular Ca²⁺ activity, which was estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, the intracellular pH values in the meristem zone of the roots of A2 and A3 seedlings were significantly lower (more acidic) than that of the WT, which might indicate a feedback block of H⁺ influx into meristematic cells caused by low intracellular pH. Under the control conditions, mature plants from the A2 and A3 lines contained significantly higher K⁺ and lower Ca²⁺ and Mg²⁺ content in the shoots, indicating disturbed long‐distance ion transport of these cations, possibly because of changes in xylem loading/retrieval and/or phloem loading. Exposing the plants in the flowering stage to various K⁺, Ca²⁺ and Mg²⁺ concentrations in the solution led to altered K⁺, Ca²⁺ and Mg²⁺ content in the shoots of A2 and A3 plants in comparison with the WT, suggesting a primary role of AtCNGC10 in Ca²⁺ (and probably Mg²⁺) transport in plants, which in turn regulates K⁺ transporters' activities.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Calcium-Binding Properties of the Mitochondrial Channel-Forming Hydrophobic Component

A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa²⁺-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca²⁺ with a K_d of 8 × 10^–6 M, while at pH7.5 this K_d was decreased to 9 × 10^–5 M. B_max for the Ca²⁺-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca²⁺/g component were boundor one calcium ion to eight molecules of the component. The Ca²⁺ binding was stronglydecreased by 50–100 mM Na⁺, but not by K⁺. Treatment of mitochondria withCaCl₂ priorto ethanolic extraction resulted in a high level of Ca²⁺-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca²⁺-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

ROLE OF INORGANIC IONS IN CONTROLLING SEDIMENTATION RATE OF A MARINE CENTRIC DIATOM DITYLUM BRIGHTWELLI1,2

The settling rates and intracellular levels of K⁺, Na⁺, Cl^-, Mg²⁺ and Ca²⁺ were measured in Ditylum bright-welli (West) Grunow, grown axenically in an enriched seawater medium at 20 C at 4,000 lx on an 8:16 LD schedule. Cells at the end of the dark period have high Na⁺ (118 mM), low K⁺ (64 mM) and low Cl^- (117 mM) relative to levels at the end of the light period when K⁺ (126 mM) and Cl^- (154 mM) are high and Na⁺ (101 mM) is low. There is no significant change in Mg²⁺ (16–18 mM) or Ca²⁺ (3–4 mM) with time. The net result of the ion changes during the light period is to increase cell density by about 3.4 mg ml^-1. This change can account for the increase in settling rate of ca. 0.3 day^-1 during the same interval. The density of the cell contents, calculated from observed ion concentrations, is 15–18 mg.ml^-1 less than that of the seawater medium. The ion and settling rate changes are light-dependent and do not persist in the dark or under constant light (ca. 850 lx), but cells do exhibit a free-running circadian rhythm in cell division under continuous dim illumination. The cell vacuole expands during the light period and contracts during the dark, apparently in response to the net ion fluxes. D. brightwelli appears to regulate its density by active ion selectivity accompanied by trans-vacuolar water movement.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.

     

Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture

We have addressed the possibility that Ca²⁺, Mg²⁺ and K⁺ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca²⁺, Mg²⁺ or K⁺ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their respective intracellular levels. From several lines of evidence, the deprivation of K⁺ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells. Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes. Removal of Mg²⁺ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death. The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features. These results highlighted the importance of utilizing several assays for the determination of apoptosis. The absence of Ca²⁺ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death. Hybridoma cells overexpressing the apoptotic suppresser gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg²⁺ ion deprivation and apoptosis inducing effects of Ca²⁺ ion deprivation. However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K⁺ free medium. The inclusion of bcl-2 activity in the mechanisms of Ca²⁺ Mg²⁺ or K⁺ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

Calcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledons

In developing seeds of bean (Phaseolus vulgaris L.), phloem‐imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K⁺ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole‐cell currents across their plasma membranes were measured with the patch‐clamp technique. A weakly rectified cation current displaying a voltage‐dependent blockade by external Ca²⁺ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal‐cell pH and Calcium‐dependent Cation Conductance (Pv‐CD‐pHCaCC) was highly selective for K⁺ over Ca²⁺ and Cl^–. For K⁺ current through Pv‐CD‐pHCaCC a sigmoid shaped current–voltage (I–V) curve was observed with negative conductance at voltages between ?200 and ?140 mV. This negative K⁺ conductance was Ca²⁺ dependent. With other univalent cations (Na⁺, Rb⁺, NH₄⁺) the currents were smaller and were not Ca²⁺ dependent. Reversal potentials remained constant when external K⁺ was substituted with these cations, suggesting that Pv‐CD‐pHCaCC channels were non‐selective. The Pv‐CD‐pHCaCC would provide a pathway for K⁺ and other univalent cation influx into developing cotyledons. These cation influxes could be co‐ordinated with sucrose influx via pH and Ca²⁺dependence.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

A Mathematical Model of Action Potential in Cells of Vascular Plants

A mathematical model of action potential (AP) in vascular plants cells has been worked out. The model takes into account actions of plasmalemma ion transport systems (K⁺, Cl^? and Ca²⁺ channels; H⁺- and Ca²⁺-ATPases; 2H⁺/Cl^? symporter; and H⁺/K⁺ antiporter), changes of ion concentrations in the cell and in the extracellular space, cytoplasmic and apoplastic buffer capacities and the temperature dependence of active transport systems. The model of AP simulates a stationary level of the membrane potential and ion concentrations, generation of AP induced by electrical stimulation and gradual cooling and the impact of external Ca²⁺ for AP development. The model supports a hypothesis about participation of H⁺-ATPase in AP generation.     

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux ‘signatures’ during these stress conditions are poorly characterized. We investigated the kinetics of K⁺, Ca²⁺ and H⁺ ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non‐invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K⁺ efflux was observed following acute ER stress with peak K⁺ efflux being ?30·6 and ?138·7 nmolm^?2 s^?1 for 10 and 50 µg ml^?1, respectively (p < 0·01). TM‐dependent Ca²⁺ uptake was more prolonged with peak values of 0·85 and 2·68 nmol m^?2 s^?1 for 10 and 50 µg ml^?1 TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K⁺ efflux and Ca²⁺ uptake. While K⁺ changes were significantly higher at each time point tested, Ca²⁺ uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K⁺ efflux and Ca²⁺ uptake. Functional assays to investigate the effect of inhibiting K⁺ efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K⁺, Ca²⁺ and H⁺ ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Differential effects of heavy metal ions on Ca2+-dependent K+ channels

Summary 1. The ability of various divalent metal ions to substitute for Ca²⁺ in activating distinct types of Ca²⁺-dependent K⁺ [K⁺(Ca²⁺] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca²⁺.2. At concentrations between 1 and 100 µM Pb²⁺, Cd²⁺ and Co²⁺ activate intermediate conductance K⁺(Ca²⁺) channels in erythrocytes and large conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Pb²⁺ and Co²⁺, but not Cd²⁺, activate small conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Mg²⁺ and Fe²⁺ do not activate any of the K⁺(Ca²⁺) channels.3. Rank orders of the potencies for K⁺(Ca²⁺) activation are Pb²⁺, Cd²⁺>Ca²⁺, Co²⁺>>Mg²⁺, Fe²⁺ for the intermediate erythrocyte K⁺(Ca²⁺) channel, and Pb²⁺, Cd²⁺>Ca²⁺>Co²⁺>>Mg²⁺, Fe²⁺ for the small, and Pb²⁺>Ca²⁺>Co²⁺>>Cd²⁺, Mg²⁺, Fe²⁺ for the large K⁺(Ca²⁺) channel in neuroblastoma cells.4. At high concentrations Pb²⁺, Cd²⁺, and Co²⁺ block K⁺(Ca²⁺) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb²⁺>Cd²⁺, Co²⁺>>Ca²⁺, and Cd²⁺>Pb²⁺, Co²⁺>>Ca²⁺, respectively, and are distinct from the potency orders for activation.5. It is concluded that the different subtypes of K⁺(Ca²⁺) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K⁺(Ca²⁺) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Effects of transition metal ions on spontaneous electrical activity and chemical synaptic transmission of neurons in the medicinal leech

Leech neurons exposed to salines containing inorganic Ca²⁺-channel blockers generate rhythmic bursts of impulses. According to an earlier model, these blockers unmask persistent Na⁺ currents that generate plateau-like depolarizations, each triggering a burst of impulses. The resulting increase in intracellular Na⁺ activates an outward Na⁺/K⁺ pump current that contributes to burst termination. We tested this model by examining systematically the effects of six transition metal ions (Co²⁺, Ni²⁺, Mn²⁺, Cd²⁺, La³⁺, and Zn²⁺) on the electrical activity of neurons in isolated leech ganglia. Each ion induced bursting activity, but the amplitude, form, and persistence of bursting differed with the ion used and its concentration relative to Ca²⁺. All ions tested suppressed chemical synaptic transmission between identified motor neurons, consistent with block of voltage-dependent Ca²⁺ currents in these cells. In addition, a strong correlation between suppression of synaptic transmission and burst amplitudes was obtained. Finally, burst duration was increased and the rate of repolarization decreased in reduced K⁺ saline, as expected for pump-dependent repolarization. These results provide further support for the hypothesis that a novel form of oscillatory electrical activity driven by persistent Na⁺ currents and the Na⁺/K⁺ pump occurs in leech ganglia exposed to Ca²⁺-channel blockers. Accepted: 15 May 1997     

Significance of Ca2+ and K+ in Micrasterias Growth and Morphogenesis

Significance of Ca²⁺ and K⁺ for the complex morphogenesis ofMicrasterias, which takes place through multipolar tip growth,was investigated. Studies with different external Ca²⁺ concentrationsand Ca²⁺ channel inhibitors LaCl₃ and verapamil indicate thatCa²⁺ and Ca²⁺ channels are essential in the development, whiletreatments with different K⁺ concentrations and K⁺ channel inhibitorTEA demonstrate that potassium or K⁺ channels are not neededin the process, albeit the existence of K⁺ channels. K⁺ is notneeded even for the regulation of turgor pressure, which wasfound to decrease clearly during cell development. The plasmamembrane ATPase inhibitors diethylstilbesterol (DES) and Na-orthovanadatestop morphogenesis and indicate the importance of ion pumpsin the developmental process. Both supraoptimal, external K⁺and Ca²⁺ cause abundant Ca²⁺ precipitate formation in chloroplasts,which shows that chloroplasts are important in regulation ofcytoplasmic Ca²⁺ metabolism and that K⁺ activates the uptakeof Ca²⁺ through Ca²⁺ channels. (Received June 13, 1995; Accepted September 13, 1996)     

Standard free energy maps for the hydrolysis of ATP as a function of pH and metal ion concentration: comparison of metal ions

The observed equilibrium constant K_obs for the hydrolysis of ATP to ADP and inorganic phosphate has been calculated as a function of pH and metal ion concentration pM (- log [M]) at 25 °C and μ = 0.2 with the use of literature values of the acid dissociation and complex dissociation constants for the phosphates.The resulting standard free energy changes ΔG °′ are presented by means of contour diagrams for the range pH 4–10 and pM 1–7. These maps summarize the results of some 1900 calculations per diagram, and clearly simulate a differential effect of the metal ions of interest, including Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Li⁺, Na⁺ and K⁺, on the equilibrium hydrolysis of ATP.