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Voltage dependence of theChara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide

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Summary

It is generally agreed that solute transport across theChara plasma membrane is energized by a proton electrochemical gradient maintained by an H+-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H+-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the trueI-V profile for the pump when treatments alter the thermodynamic pressure on transport.

We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cellI-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl3, so that steady-state currents could be recorded under voltage clamp between −400 and +100 mV. Exposures to 1mm NaCN (CN) and 0.4mm salicylhydroxamic acid (SHAM) depolarized (positive-going)Chara membrane potentials by 44–112 mV with a mean half time of 5.4±0.8 sec (n=13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3±0.9 sec ([ATP] t=0, 0.74±0.3mm; [ATP] t=x , 0.23±0.02mm). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses ofdI-V curves (±CN+SHAM) as well as of families ofI-V curves taken at times during CN+SHAM exposures indicated a stoichiometry for the pump of one charge (H+) transported per ATP hydrolyzed and an equilibrium potential near −420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60–75% of the total membrane conductance nearV m. Complementary results were obtained also in fitting previously publishedI-V data gathered over the external pH range 4.5–7.5 Kinetic features deduced for the pump were dominated by a slow step preceding H+ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium. These characteristics predict, in marked contrast to the situation forNeurospora, that cytoplasmic acid loads inChara should shift the pumpI-V curve negative-going along the voltage axis with little change in maximum current output at positive voltages.

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References

  • Amoury, A., Goffeau, A., McIntosh, D.B., Boyer, P.D. 1982. Exchange of oxygen between phosphate and water catalyzed by the plasma membrane ATPase from the yeastSchizosaccharomyces pombe.J. Biol. Chem. 257:12509–12516

    Google Scholar 

  • Bahinski, A., Nakao, M., Gadsby, D.C. 1988. Potassium translocation by the Na+/K+ pump is voltage insensitive.Proc. Natl. Acad. Sci. (USA) 85:3412–3416

    Google Scholar 

  • Beilby, M.J. 1984. Current-voltage characteristics of the protein pump atChara plasmalemma: I. pH dependence.J. Membrane Biol. 81:113–126

    Google Scholar 

  • Beilby, M.J. 1986. Factors controlling the K+ conductance inChara.J. Membrane Biol. 93:187–193

    Google Scholar 

  • Beilby, M.J. 1990. Current-voltage curves for plant membrane studies: A critical analysis of the method.J. Exp. Bot. (in press)

  • Beilby, M.J., Beilby, B.N. 1983. Potential dependence of the admittance ofChara plasmalemma.J. Membrane Biol. 74:229–245

    Google Scholar 

  • Beilby, M.J., Blatt, M. 1986. Simultaneous measurements of cytoplasmic K+ concentration and the plasma membrane electrical parameters in single membrane samples ofChara.Plant Physiol. 82:417–422

    Google Scholar 

  • Bisson, M.A. 1986. Inhibitors of proton pumping: Effect on passive proton transport.Plant Physiol. 81:55–59

    Google Scholar 

  • Blatt, M.R. 1986. Interpretation of steady-state current-voltage curves: Consequences and implications of current subtraction in transport studies.J. Membrane Biol. 92:91–110

    Google Scholar 

  • Blatt, M.R. 1987. Electrical characteristics of stomatal guard cells: The contribution of ATP-dependent, “electrogenic” transport revealed by current-voltage and difference-current-voltage analysis.J. Membrane Biol. 98:257–274

    Google Scholar 

  • Blatt, M.R. 1988a. Mechanisms of fusicoccin action: A dominant role for secondary transport in a higher-plant cell.Planta 174:187–200

    Google Scholar 

  • Blatt, M.R. 1988b. Potassium-dependent, bipolar gating of K+ channels in guard cells.J. Membrane Biol. 102:235–246

    Google Scholar 

  • Blatt, M.R., Rodriguez-Navarro, A., Slayman, C.L. 1987. Potassium-proton symport inNeurospora: Kinetic control by pH and membrane potential.J. Membrane Biol. 98:169–187

    Google Scholar 

  • Blatt, M.R., Slayman, C.L. 1987. Role of “active” potassium transport in the regulation of cytoplasmic pH by non-animal cells.Proc. Natl. Acad. Sci. USA 84:2737–2741

    Google Scholar 

  • Chapman, J.B., Johnson, E.A., Kootsey, J.M. 1983. Electrical and biochemical properties of an enzyme model of the sodium pump.J. Membrane Biol. 74:139–153

    Google Scholar 

  • DeWeer, P., Gadsby, D.C., Rakowski, R.F. 1988. Voltage dependence of the Na−K pump.Annu. Rev. Physiol. 50:225–241

    Google Scholar 

  • Felle, H. 1981. A study of the current-voltage relationships of electrogenic active and passive membrane elements inRiccia fluitans.Biochim. Biophys. Acta 646:151–160

    Google Scholar 

  • Gadsby, D.C., Nakao, M. 1989. Steady-state current-voltage relationship of the Na/K pump in guinea-pig ventricular myocytes.J. Gen. Physiol. (in press)

  • Goldfarb, V., Sanders, D., Gradmann, D. 1984. Reversal of electrogenic Cl− pump inAcetabularia increases level and32P labelling of ATP.J. Exp. Bot. 35:645–658

    Google Scholar 

  • Gradmann, D., Hansen, U.-P., Long, W.S., Slayman, C.L., Warncke, J. 1978. Current-voltage relationships for the plasma membrane and its principal electrogenic pump inNeurospora crassa: I Steady-state conditions.J. Membrane Biol. 39:333–367

    Google Scholar 

  • Gradmann, D., Hansen, U.-P., Slayman, C.L. 1982. Reaction kinetic analysis of current-voltage relationships for electrogenic pumps inNeurospora andAcetabularia.Curr. Top. Membr. Transp. 16:257–281

    Google Scholar 

  • Gradmann, D., Klieber, H.-G., Hansen, U.-P. 1987. Reaction kinetic parameters for ion transport from steady-state current-voltage curves.Biophys. J. 51:569–585

    Google Scholar 

  • Hager, K.M., Mandala, S.M., Davenport, J.W., Speicher, D.W., Benz, E.J., Slayman, C.W. 1986. Amino acid sequence of the plasma membrane ATPase ofNeurospora crassa: Deduction from genomic and cDNA sequences.Proc. Natl. Acad. Sci. USA 83:7693–7697

    Google Scholar 

  • Hansen, U.-P., Gradmann, D., Sanders, D., Slayman, C.L. 1981. Interpretation of current-voltage relationships for “active” ion transport systems: I. Steady-state reaction-kinetic analysis of class-I mechanisms.J. Membrane Biol. 63:165–190

    Google Scholar 

  • Hill, T.L., Eisenberg, E. 1981. Can energy transduction be localized at some crucial part of the enzymatic cycle?Q. Rev. Biophys. 14:463–511

    Google Scholar 

  • Hodgkin, A.L., Katz, B. 1949. The effect of sodium ions on the electrical activity of the giant axon of the squid.J. Physiol. (London) 108:37–77

    Google Scholar 

  • Jennings, I., Rea, P., Leigh, R., Sanders, D. 1988. Quantitative and rapid estimation of H+ fluxes in membrane vesicles.Plant. Physiol. 86:125–133

    Google Scholar 

  • Karlish, S.J.D., Yates, D.W., Glynn, I.M. 1978. conformational transitions Na+=bound and K+-bound forms of the (Na++K+)-ATPase studied with formycin nucleotides.Biochim. Biophys. Acta 525:252–264

    Google Scholar 

  • Kawamura, G., Shimmen, T., Tazawa, M. 1980. Dependence of the membrane potential ofChara cells on external pH in the presence or absence of internal adenosinetriphosphate.Planta 149:213–218

    Google Scholar 

  • Keifer, D.W., Spanswick, R.M. 1978. Activity of the electrogenic pump inChara corallina as inferred from measurements of the membrane potential, conductance and potassium permeability.Plant Physiol. 62:653–661

    Google Scholar 

  • Keifer, D.W., Spanswick, R.M. 1979. Correlation of adenosine triphosphate levels inChara corallina with the activity of the electrogenic pump.Plant Physiol. 64:165–168

    Google Scholar 

  • Kishimoto, U., Kami-ike, N., Takeuchi, Y., Ohkawa, T. 1984. A kinetic analysis of the electrogenic pump ofChara corallina: I. Inhibition of the pump by DCCD.J. Membrane Biol. 80:175–183

    Google Scholar 

  • Komor, E., Tanner, W. 1980. Proton-cotransport of sugars in plants.In: Plant Membrane Transport: Current Conceptual Issues. R. Spanswick, W. Lucas, and J. Dainty, editors. pp. 247–257. Elsevier, Amsterdam

    Google Scholar 

  • Läuger, P., Stark, G. 1970. Kinetics of carrier-mediated ion transport across lipid bilayer membranes.Biochim. Biophys. Acta 211:458–466

    Google Scholar 

  • Lew, R., Spanswick, R.M. 1984. Characterization of the electrogenicity of soybean roots.Plant Physiol. 75:1–6

    Google Scholar 

  • Lucas, W.J. 1982. Mechanism of acquisition of exogenous bicarbonate by internodal cells ofChara corallina.Planta 156:181–192

    Google Scholar 

  • Marquardt, D. 1963. An algorithm for least-squares estimation of nonlinear parameters.J. Soc. Ind. Appl. Math. 11:431–441

    Google Scholar 

  • Mitchell, P. 1969. Chemiosmotic coupling and energy transduction.Theor. Exp. Biophys. 2:159–216

    Google Scholar 

  • Nelder, J.A., Mead, R. 1965. A simplex method for function minimization.Comput. J. 7:308–313

    Google Scholar 

  • Press, W., Flannery, B., Teukolsky, S., Vetterling, W. 1986. Numerical Recipies: The Art of Scientific Computing. Cambridge University Press, Cambridge

    Google Scholar 

  • Raven, J.A. 1976. Transport in algal cells.In: Encyclopedia of Plant Physiology, N.S. U. Lüttge and M. Pitman, editors. Vol. 2A, pp. 129–188. Springer Verlag, Berlin-Heidelberg-New York

    Google Scholar 

  • Reid, R.J., Waler, N.A. 1983. Adenylate concentrations inChara: Variability, effects of inhibitors and relationship to protoplasmic streaming.Aus. J. Plant Physiol. 10:373–383

    Google Scholar 

  • Richards, J.L., Hope, A.B. 1974. The role of protons in determining membrane electrical characteristics inChara corallina.J. Membrane Biol. 16:121–144

    Google Scholar 

  • Roberts, J.K.M., Lane, A., Clark, R., Nieman, R. 1985. Relationships between rate of synthesis of ATP and the concentrations of reactants and products of ATP hydrolysis in maize root tips, determined by31P-NMR.Arch. Biochem. Biophys. 240:712–722

    Google Scholar 

  • Sanders, D., Hansen, U.-P., Gradmann, D., Slayman, C.L. 1984. Generalized kinetic analysis of ion-driven cotransport systems: A unified interpretation of selective ionic effects on Michaelis parameters.J. Membrane Biol. 77:123–152

    Google Scholar 

  • Sanders, D., Hansen, U.-P., Slayman, C.L. 1981. Role of the plasma membrane proton pump in pH regulation in non-animal cells.Proc. Natl. Acad. Sci. USA 78:5903–5907

    Google Scholar 

  • Sanders, D., Slayman, C.L. 1982. Control of intracellular pH: Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganismNeurospora.J. Gen. Physiol. 80:377–402

    Google Scholar 

  • Serrano, R., Kielland-Brandt, M.C., Fink, G.R. 1986. Yeast plasma membrane ATPase is essential for growth and has homology with (Na++K+), K+- and Ca2+-ATPases.Nature (London) 319:689–693

    Google Scholar 

  • Scott, I.R., Ellar, D.J. 1978. Metabolism and the triggering of germination ofBacillus megaterium.Biochem. J. 174:627–634

    Google Scholar 

  • Shimmen, T., Tazawa, M. 1977. Control of membrane potential and excitability ofChara cells with ATP and Mg2+.J. Membrane Biol. 37:167–192

    Google Scholar 

  • Shimmen, T., Tazawa, M. 1980. Dependence of H+ efflux on ATP in cells ofChara australis.Plant Cell Physiol. 21:1007–1013

    Google Scholar 

  • Slayman, C.L., Long, W.S., Lu, C.Y.-H. 1973. The relationship between ATP and an electrogenic pump in the plasma membrane ofNeurospora crassa.J. Membrane Biol. 14:305–338

    Google Scholar 

  • Slayman, C.L., Sanders, D. 1984. pH-dependence of proton pumping inNeurospora.In: Hydrogen Ion Transport in Epithelia. pp. 47–56. J.G. Forte, D. G. Warnock, and F. C. Rector, editors. Wiley-Interscience, New York

    Google Scholar 

  • Slayman, C.L., Sanders, D. 1985. Steady-state kinetic analysis of an electroenzyme.Symp. Soc. Biochem. 50:11–29

    Google Scholar 

  • Spanswick, R.M. 1972. Evidence for an electrogenic ion pump inNitella translucens. I. The effects of pH, K+, Na+, light and temperature on the membrane potential and resistance.Biochim. Biophys. Acta 288:73–89

    Google Scholar 

  • Spanswick, R.M. 1974. Evidence for an electrogenic ion pump inNitella translucens. II. Control of the light-stimulated component of the membrane potential.Biochim. Biophys. Acta 332:387–398

    Google Scholar 

  • Spanswick, R.M. 1981. Electrogenic ion pumps.Annu. Rev. Plant Physiol. 32:267–312

    Google Scholar 

  • Spear, D.J., Barr, J.K., Barr, C.E. 1969. Localization of hydrogen ion and chloride ion fluxes inNitella.J. Gen. Physiol. 54:397–414

    Google Scholar 

  • Stelzer, A., Kay, A.R., Wong, R.K.S. 1988. GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors.Science 241:339–341

    Google Scholar 

  • Sze, H. 1985. H+-translocating ATPasesAnnu. Rev. Plant Physiol. 36:175–208

    Google Scholar 

  • Takeshige, K., Shimmen, T., Tazawa, M. 1986. Quantitative analysis of ATP-dependent H+ efflux and pump current driven by an electrogenic pump inNitellopsis obtusa.Plant Cell Physiol. 27:337–348

    Google Scholar 

  • Takeuchi, Y., Kishimoto, U., Ohkawa, T., Kami-ike, N. 1985. A kinetic analysis of the electrogenic pump ofChara corallina: II. Dependence of the pump activity on external pH.J. Membrane Biol. 86:17–26

    Google Scholar 

  • Tester, M. 1988. Pharmacology of K+ channels in the plasma-lemma of the green algaChara corallina.J. Membrane Biol. 103:159–169

    Google Scholar 

  • Tracey, A., Gresser, M. 1986. Interaction of vanadate with phenol and tyrosine: Implications for the effects of vanadate on systems regulated by tyrosine phosphorylation.Proc. Natl. Acad. Sci. USA 83:609–613

    Google Scholar 

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  1. Michael R. Blatt

    Present address: Department of Biochemistry and Biological Sciences, Wye College, University of London, TN25 5AH, Wye (Ashford), Kent, England

  2. Mary J. Beilby

    Present address: School of Biological Sciences, University of Sydney, 2006, Sydney, N.S.W., Australia

Authors and Affiliations

  1. Botany School, University of Cambridge, CB2 3EA, Cambridge, England

    Michael R. Blatt, Mary J. Beilby & Mark Tester

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Blatt, M.R., Beilby, M.J. & Tester, M. Voltage dependence of theChara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide. J. Membrain Biol. 114, 205–223 (1990). https://doi.org/10.1007/BF01869215

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