Identification of ATP-sensitive Potassium Channel in Frog Ventricular Myocytes

Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80 μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with little yolk. Neither shortening τ_e (spin echo time) to 9 msec (from 18 msec) nor lengthening τ_r (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T₁ (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular medium (T₁= 3.0 sec), cytoplasm (T₁= 0.8 sec) and nucleoplasm (T₁= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass) of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference between the nuclear and cytoplasmic compartments. Received: 18 January 1996/Revised: 29 April 1996     

Membrane potential responses controlling chemodispersal of Paramecium caudatum from quinine

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca²⁺ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.     

Altered drug translocation mediated by the MDR protein: Direct,indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ltpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential () and/or intracellular pH (pH_i) and therebyindirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pH_i, or -dependent manner. A third model proposes that the protein alternates between drug pump and Cl^– channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pH_i, , and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.     

Parental effort of kestrels (Falco tinnunculus) in nest defense: effects of laying time, brood size, and varying survival prospects of offspring

We studied the nest defense behavior of Eurasian kestrels (Falcotinnunculus) towards a stuffed pine marten (Martes martes) througha 3-year vole cycle (1990–92) in western Finland. Survivalprobability of offspring decreases with a later start of breeding,and, therefore, early breeders should protect their offspringmore than late ones. We found this true for males during theincubation period, but not for females. In addition, we expectedthe nest defense intensity to increase with offspring number.During the incubation period, this was true for females, butnot for males. During the nestling phase, parents did not adjusttheir defense effort to natural or manipulated (by one to twoyoung) brood size. Survival prospects of kestrel offspring werehighest in the increasing vole year 1991 and lowest in the decreasingvole year 1992, and, therefore, we expected the defense activityof kestrels to follow the same trend. However, the oppositeresult appeared true for females with a similar tendency formales. Most hypotheses predicting avian nest defense behaviorwere not supported by our data. Temporally heterogeneous environmentand low degree of nest-site tenacity of migratory kestrels maymake them unfamiliar with environmental variation and survivalprospects of their offspring. Therefore, fitness benefits ofparental care are not predictable, and kestrels may thus adjusttheir parental effort to their own future reproductive potential(i.e., number of future breeding attempts), rather than to somecurrent investment indicator, like offspring age and number.     

The Hawk—Dove game revisited: Effects of continuous variation in resource-holding potential on the frequency of escalation

Summary The classic Hawk—Dove game is extended to deal with continuous variation in resource-holding potential or RHP, when RHP is observable (via any sensory modality) but RHP difference is less than perfectly reliable as a predictor of the outcome of an escalated contest. The relationship between sensory and physical magnitudes of RHP is assumed to be governed by Fechner's psychophysical law, whose effect is that contestants interact as if they had perfect information about their relative RHP (as opposed to RHP difference). Thus, an animal is aggressive if its RHP exceeds a certain fraction, called its threshold, of its opponent's RHP and otherwise is non-aggressive; and the classic Hawk and Dove strategies correspond to zero and infinite thresholds, respectively. For RHPs drawn at random from an arbitrary Gamma distribution there is a unique evolutionarily stable strategy or ESS, which depends on a parameter measuring the reliability of RHP as a predictor of the outcome of a fight, on the ratio of the valueV of winning to the costC of losing (both measured in units of reproductive fitness) and on the mean µ and variance ² of the RHP distribution. In a population at this ESS, ifV/C < 1 then the threshold is 1 and there is no fighting. AsV/C increases beyond 1 to a second critical value , however, the threshold decreases steadily from 1 to 0 and remains 0 forV/C > ; is an increasing function of , but a decreasing function of ². That a lower variance of RHP can imply a lower escalation frequencyp is a novel insight of the analysis. The prediction is at first counterintuitive, because if the aggression threshold were fixed then larger variance would imply lowerp (dispersion effect of variance). When natural selection acts on the threshold, however, increasing the variance not only reduces the probability that an animal with larger RHP will be attacked by an animal with lower RHP at the existing threshold, but also reduces the expected costs of adopting that particular threshold, so that a mutant with a somewhat lower threshold can invade the population (selection effect of variance). Forp, the selection effect dominates toward the upper end of the interval 1 V/C .     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide     

Chemiosmotic coupling of ion transport in the yeast vacuole: Its role in acidification inside organelles

Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H⁺-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl^– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl^– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl^– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK _m for Cl^– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail.     

Effects of amphipathic peptides,including presequences,on the functional integrity of rat liver mitochondrial membranes

A number of amphipathic peptides were tested for their effects on structural and functional properties of isolated rat liver mitochondria. The peptides included the matrix targeting sequence of subunit IV of (yeast) cytochromec oxidase. Titration experiments in which the mitochondria were incubated with increasing concentrations of the peptides revealed two major stages in the interaction. First, at low peptide/mitochondria ratios, peptide binding to the outer membrane occurred which was accompanied by gradual lysis of the outer membrane at higher ratios. The latter was deduced from the release of adenylate kinase, the classical marker enzyme of the intermembrane space. Secondly, at still higher peptide/mitochondria ratios, the permeability of the inner membrane progressively increased, as evidenced by measurements of respiratory control and of the membrane potential. Complete uncoupling of respiration seemed to precede dissipation of the membrane potential.