Bifurcation of periodic activity from periodic activity in a molluscan neurone

The response of a molluscan neurone to intra-somatic micro-iontophoresis of TEA⁺ is a periodic discharge of action potentials that bifurcates into doublet, triplet and bursting discharges, and then through regular, small amplitude oscillations to a depolarized steady state. This response pattern is explained by the presence of two TEA⁺-sensitive, K⁺-selective conductances: we conjecture that the maximal fast, transient K⁺-conductance acts as the bifurcation parameter.     

Do independent processes control the activation and inactivation of potassium contracture tension in rat skeletal muscle?

Potassium (K⁺) contracture tension, measured in small bundles of rat soleus muscle fibers during maintained depolarization, increases to a peak value and then decays either to the baseline or to a pedestal level. We have tested the hypothesis that the rise and fall of tension are determined by independent activation and inactivation processes. If the “Independence” hypothesis is correct, tension during the decay of K⁺ contractures should equal tension predicted from the product of the activation and inactivation parameters determined from the same K⁺ contractures. Both the measured and predicted tensions decayed to a pedestal level that was increased in amplitude in the presence of perchlorate ions. However, the measured tensions in normal solutions and in the presence of perchlorate were three to five times smaller than the predicted tensions. This result indicates that the activation and inactivation of processes controlling the rise and decay of K⁺ contracture tension are not independent.     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

The Roles of Sodium and Potassium Ions in the Generation of the Electro-Olfactogram

In order to clarify whether or not the electronegative olfactory mucosal potentials (EOG) are generator potentials, the effects of changed ionic enviroment were studied. The EOG decreased in amplitude and in some cases nearly or completely disappeared, when Na⁺ in the bathing Ringer solution was replaced by sucrose, Li⁺, choline⁺, tetraethylammonium⁺ (TEA), or hydrazine. In the K⁺-free Ringer solution, the negative EOG's initially increased and then decreased in amplitude. In Ringer's solution with increased K⁺, the negative EOG's increased in amplitude. When K⁺ was increased in exchange for Na⁺ in Ringer's solution, the negative EOG's decreased, disappeared, and then reversed their polarity (Fig. 6). Next, when the K⁺ was replaced by equimolar sucrose, Li⁺, choline⁺, TEA⁺, hydrazine, or Na⁺, the reversed potentials recovered completely only in Na⁺-Ringer's solution, but never in the other solutions. Thus, the essential role of Na⁺ and K⁺ in the negative EOG's was demonstrated. Ba⁺⁺ was found to depress selectively the electropositive EOG, but it hardly decreased and never increased the negative EOG. Hence, it is concluded that Ba⁺⁺ interferes only with Cl^- influx, and that the negative EOG's are elicited by an increase in permeability of the olfactory receptive membrane to Na⁺ and K⁺, but not to Cl^-. From the ionic mechanism it is inferred that the negative EOG's are in most cases composites of generator and positive potentials.     

Plasmalemmal voltage-activated K + currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Summary.  Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco ‘Bright Yellow-2’ cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K⁺ currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E_K when external [K⁺] was either 6 or 60 mM. Several channel blockers, including external Ba²⁺, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K⁺ current. Among the monovalent cations tested (NH₄ ⁺, Rb⁺, Li⁺, Na⁺), only Rb⁺ had appreciable permeation (P_Rb/P_K ⁼ 0.7). In addition, in 60 mM K⁺ solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K⁺ current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E_K, and other monovalent cations (NH₄ ⁺, Rb⁺, Li⁺, Na⁺) were not permeant. The inward current was blocked by external Ba²⁺ and niflumic acid. External Cs⁺ reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K⁺ current was generally small compared to the amplitude of the outward K⁺ current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K⁺ currents in tobacco ‘Bright Yellow-2’ cells. Received December 7, 2001; accepted April 15, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Pharmacologie et Physicochimie, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, 74 route du Rhin, BP 24, 67401 Illkirch, France. Abbreviations: TBY-2 Tobacco ‘Bright Yellow-2’; DHCB dihydrocytochalasin B; I_Kin inward rectifier K⁺ current; I_Kout outward K⁺ current; MFs microfilaments; MTs microtubules; NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid.     

Allosteric and Non-Allosteric Regulation of Rubidium Influx in Barley Roots

Uptake of Rb⁺ was investigated in 6–8-day-old intact barley plants (Hordeum vulgare cv. Kristina), which had been cultivated or pretreated in nutrient solutions with various K⁺ concentrations. The relationship between Rb⁺ influx and the K⁺ concentration of roots appeared to be sigmoidal for plants grown in solutions containing K⁺, indicating regulation of Rb⁺ uptake by allosteric inhibition of the uptake mechanism. Pretreatment of the roots in K⁺-free solutions changed the pattern of uptake and caused the Rb⁺ influx to become linearly related to the chemical Rb⁺ potential of the uptake solution. Pretreatment in K⁺-free solutions probably abolishes the allosteric inhibition of a carrier system.     

Ca2+‐Inactivated K+ Current is Modulated by Endothelin‐1 in B‐16 Murine Melanoma Cells

It is well established that endothelin‐1 (ET‐1) plays a role in differentiation and proliferation in a variety of cells such as fibroblasts and human melanoma cells via a receptor‐mediated mechanism. However, whether ET‐1 modulates ion channel activity in these cell types is still unknown. In this report, we recorded the voltage‐dependent outward K⁺ current in cultured B16 melanoma cells using the patch‐clamp technique. Biophysical and pharmacological properties of the K⁺ current, and the effect of ET‐1 on the K⁺ current were investigated. When cells were loaded with a Ca²⁺‐chelating agent (EGTA or BAPTA), the K⁺ current amplitude gradually increased with time after establishment of the whole cell configuration. Replacement of Ca²⁺ with Co²⁺ in the extracellular medium caused no significant modulation of the K⁺ current amplitude. Addition of BaCl₂ or quinidine to the extracellular solution reduced the K⁺ current amplitude, whereas the K⁺ current was insensitive to tetraethylammonium. ET‐1 (10 nM) reversibly decreased the K⁺ current amplitude and accelerated the decay of the K⁺ current. The ET‐1‐induced inhibitory effect displayed no desensitization following repeated ET‐1 application. Pretreatment with pertussis toxin (PTX) or perfusion of cells with the protein kinase C (PKC) inhibitor H‐7 abolished the inhibitory effect of ET‐1 on the K⁺ current. We conclude that the outward K⁺ current recorded in murine B‐16 melanoma cells represents a Ca²⁺‐inactivated K⁺ current, and that the inhibitory effect of ET‐1 on the K⁺ current may reveal a novel mechanism to control the differentiation and proliferation of melanoma cells.     

Mesurement of longitudinal ion profiles in single roots of Hordeum and Atriplex by use of flameless atomic absorption spectroscopy

Summary A method is described by which the Na⁺ and K⁺ content in 0.5 mm sections of single roots of Hordeum distichon L. and Atriplex hortensis L. can be determined by use of flameless atomic absorption spectroscopy. By this method the longitudinal profiles of K⁺ and Na⁺ along low salt roots and roots which had been equilibrated with or grown in K⁺-free 1 mM Na⁺-solution were determined. The profiles reveal that high K⁺/Na⁺ ratios in the cytoplasm are maintained also in K⁺-free solutions. In solutions containing 1 mM Na⁺ a high K⁺/Na⁺ selectivity was found to be dependent on sufficient aeration. From the ion profiles the cytoplasmic (110 mM) and vacuolar (20 mM) K⁺ concentration in low salt barley roots—values which are unobtainable by compartmental analysis—could be estimated.     

Potassium-dependent changes in the expression of membrane-associated proteins in barley roots : I. Correlations with k(rb) influx and root k concentration

Barley (Hordeum vulgare L. cv Halcyon) seedlings which had been grown in full strength complete inorganic nutrient media (containing 6 millimolar K⁺) had high internal K⁺ concentrations and low values of K⁺ (⁸⁶Rb⁺) influx when influx was measured from solutions containing 100 micromolar K⁺. Transfer of these plants to solutions lacking K⁺ resulted in significant reductions of root and shoot K⁺ concentrations and values of K⁺ (⁸⁶Rb⁺) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K⁺, the changes induced by K⁺ deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K⁺ (⁸⁶Rb⁺) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K⁺ status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washed in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar.     

Accumulation of potassium and sodium by barley roots in a k-na replacement series

Excised roots of barley (Hordeum vulgare, var. Campana) were incubated for 24 hr in solutions containing constant total concentrations of KCl and NaCl but in which the mole fractions of K and Na were varied in replacement series. In solutions containing 1, 10, or 50 mm concentrations of K⁺ plus Na⁺, total cation accumulation was dependent upon the total salt concentration but was relatively independent of the mole fractions of K⁺ and Na⁺. These results imply that accumulation of K⁺ and Na⁺ was limited by a common factor. In solutions containing 0.01 mm K⁺ plus Na⁺ there was a strong preference for K⁺ over Na⁺ and the sum of K⁺ and Na⁺ accumulation increased with increasing K⁺ concentration.     

Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

The Influence of the Intracellular Potential on Potassium Uptake by Beetroot Tissue

Intracellular potentials were measured in beetroot tissue during the steady-state uptake of K⁺ from various solutions. In solutions containing bicarbonate, the membrane potential becomes up to 70 mv more negative than the estimated equilibrium potential for K⁺. The uptake of K⁺ from such solutions is correlated with variations in the potential, both when the bicarbonate concentration is changed and also when the metabolic activity of the tissue is changed by washing in water for various periods. However, the estimated permeability to K⁺ varies from 0.4 x 10^-7 to 1.5 x 10^-7 cm·sec^-1. It is postulated that the change of potential arises from the metabolic transport of HCO₃^- into the cell or H⁺ outwards, and that the associated uptake of K⁺ is partly or entirely by passive diffusion across the cell membrane. In contrast, K⁺ uptake from KCl solutions is not accompanied by any significant change in the membrane potential, which remains relatively close to the K⁺ equilibrium potential. In solutions containing both KHCO₃ and KCl, it appears that an amount of K⁺ equal to the influx of Cl^- is taken up independently of the potential, while the component of K⁺ uptake which is not balanced by Cl^- uptake is related to the potential in the manner described. These results suggest that K⁺ uptake is linked to Cl^- uptake in an electrically neutral active transport process.     

A mutant of Paramecium with increased relative resting potassium permeability

Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K⁺ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K⁺. Membrane potential was plotted against the concentration of K⁺ (0.5 to 16 mM) in solutions of fixed Na⁺ or Ca⁺⁺ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K⁺. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K⁺ relative to the permeabilities to Na⁺ and Ca⁺⁺ has been increased by the mutation.     

Bioelectric effects of ions microinjected into the giant axon of Loligo

1. A technique is described for recording the bioelectric activity of the squid giant axon during and following alteration of the internal axonal composition with respect to ions or other substances. 2. Experimental evidence indicates that the technique as described is capable of measuring changes in local bioelectric activity with an accuracy of 10 to 15 per cent or higher. 3. Alterations of the internal K⁺ or Cl^- concentrations do not cause the change in resting potential expected on the basis of a Donnan mechanism. 4. The general effect of microinjection of K⁺ Rb⁺, Na⁺, Li⁺, Ba⁺⁺, Ca⁺⁺, Mg⁺⁺, or Sr⁺⁺ is to cause decrease in spike amplitude, followed by propagation block. 5. The resting potential decreases when the amplitude of the spike becomes low and block is incipient. 6. The decrease in resting potential and spike amplitude may be confined to the immediate vicinity of the injection. 7. At block, the resting potential decreases up to 50 per cent, but injection of small quantities of divalent cations may cause much larger localized depolarization. 8. The blocking effectiveness of K⁺, Na⁺, and Ca⁺⁺ expressed as reciprocals of the relative amounts needed to cause block is approximately 1:5:100. Rb⁺ has the same low effectiveness as does K⁺. Li⁺ resembles Na⁺. Ba⁺⁺ and Mg⁺⁺ are approximately as effective as Ca⁺⁺. 9. Microinjection of Na⁺ may cause marked prolongation of the spike at the injection site as well as decrease in its amplitude. 10. The anions used (Cl^-, HCO₃^-, NO₃^-, SO₄^-, aspartate, and glutamate) do not seem to exert specific effects. 11. A tentative explanation is offered for the insensitivity of the resting potential to changes in the axonal ionic composition. 12. New data are presented on the range of variation, in a large sample, of the magnitude of the resting potential and spike amplitude.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na⁺ + K⁺)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na⁺ + K⁺)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na⁺ + K⁺)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.     

Chloride dependence of the K+-stimulated release of taurine from synaptosomes

Exposure of a crude synaptosomal fraction to K⁺ concentrations ranging from 25 to 100 mM evokes the release of [³H]taurine and [³H]GABA. These high concentrations of K⁺ induce, besides depolarization, a marked synaptosomal swelling, which is prevented by replacing chloride in the solutions with the largely impermeant anion gluconate. The depolarizing effect of K⁺ is unaffected by omission of chloride. The K⁺-evoked release of taurine seems related to K⁺-induced changes in synaptosomal volume rather than to a depolarizing effect, since it is totally calcium-independent but is abolished by reducing chloride and by making solutions hypertonic with mannitol. The release of [³H]GABA, in contrast is unaffected in chloride-free or hypertonic solutions.     

Genetic and physiological evidence concerning the development of chemically sensitive voltage-dependent ionophores in L6 cells

The electrophysiological properties of a tissue culture muscle line, L₆, and a K⁺ resistant mutant (MK₁) derived from L₆ were determined to elucidate certain aspects of membrane differentiation and function. MK₁ was selected as a clone of myoblasts resistant to the toxic effects of 55 mM K⁺. The resting potentials of L₆ and MK₁ myoblasts and myotubes were K⁺ dependent and equal. The amplitudes of the action potentials were equal in normal medium, but 27.7 mM K⁺ interfered with or eliminated the ability of L₆ myotubes to produce action potentials. MK₁ myotubes produced nearly normal action potentials under these conditions. Thus, the K⁺ resistant myoblasts differentiate into myotubes which have an action potential generating mechanism much less sensitive to K⁺ than the normal mechanism. Also, both d-tubocurarine and α-bungarotoxin enhance the amplitude of the action potentials produced by L₆ myotubes in the presence of 27.7 mM K⁺; these compounds do not enhance the amplitude of the action potentials produced by MK₁ myotubes under the same conditions. It is proposed that as a consequence of differentiation a type of ionophore present in myoblasts becomes a voltage-dependent ionophore in myotubes. Furthermore, these voltage-dependent ionophores can be chemically sensitive.     

THE ADENOSINE DIPHOSPHATE-ADENOSINE TRIPHOSPHATE EXCHANGE REACTION OF EXTRACTED CEREBRAL MICROSOMES

—Microsomal fractions prepared from guinea pig cerebral cortex manifested ADP-ATP exchange activity, 40–99 per cent of which was extractable by dilute salt solutions. All of the (Na⁺, K⁺)-ATPase activity remained in the particulate material. The unextracted ADP-ATP exchange activity was stimulated six to seven fold by a non-ionic detergent (Lubrol W). When pre-extracted microsomes were sedimented in a sucrose density gradient, the ADP-ATP exchange activity was more widely distributed than (Na⁺, K⁺)-ATPase or adenylate kinase activities. The ADP-ATP exchange activity of microsomes extracted with NaI was stimulated by Na⁺ ions when the Mg²⁺ concentration in the reaction mixture was low (0·2 mm ). The Na⁺ stimulation of exchange activity was more variable than was the stimulation of phosphate formation by Na⁺ plus K⁺. The Na⁺-stimulated ADP-ATP exchange reaction of extracted microsomes may be a component of the (Na⁺, K⁺)-ATPase system, which has not been freed from adenylate kinase or possibly other contributing enzyme systems.