Abstract
The efficacy and mechanism of α-dendrotoxin (DTX) block of K+ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K+ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K+ (K+) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K+ current (IK,in), but was ineffective in blocking current through the outward-rectifying K+ channels (IK,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for IK,in, neither altering its activation at voltages negative of −120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of IK,in followed a simple titration function with an apparent K1/2 for block of 2.2 nM in 3 mm K +o . However, DTX block was dependent on the external K+ concentration. Raising K+ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K i = 10 mm in 10 nm DTX). The effect of K+ in protecting I K,in was competitive with DTX and specific for permeant cations. A joint analysis of IK,in block with DTX and K+ concentration was consistent with a single class of binding sites with a K d for DTX of 240 pm. A K d of 410 μm for extracellular K+ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K+ (K1/2 ∼ 1 mm) for IK,in activation; they parallel features of K+ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K+ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K+ inward rectifier.
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Anderson, J.A., Huprikar, S.S., Kochian, L.V., Lucas, W.J., Gaber, R.F. 1992. Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89:3736–3740
Anderson, C.S., MacKinnon, R., Smith, C., Miller, C. 1988. Charybdotoxin block of single Ca2+-activated K+ channels. J. Gen. Physiol. 91:317–333
Balser, J.R., Roden, D.M., Bennett, P.B. 1991. Single inward rectifier potassium channels in guinea pig ventricular myocytes. Biophys. J. 59:150–161
Benoit, E., Dubois, J.-M. 1986. Toxin I from the snake Dendroaspis polylepis polylepis: a highly specific blocker of one type of potassium channel in myelinated nerve fiber. Brain Res. 377:374–377
Blatt, M.R. 1987a. Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes. Planta 170: 272–287
Blatt, M.R. 1987b. 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
Blatt, M.R. 1988. Potassium-dependent bipolar gating of potassium channels in guard cells. J. Membrane Biol. 102:235–246
Blatt, M.R. 1990. Potassium channel currents in intact stomatal guard cells: rapid enhancement by abscisic acid. Planta 180:445–455
Blatt, M.R. 1991a. Ion channel gating in plants: Physiological implications and integration for stomatal function. J. Membrane Biol. 124:95–112
Blatt, M.R. 1991b. A primer in plant electrophysiological methods. In: Methods in Plant Biochemistry. K. Hostettmann, editor, pp. 281–321. Academic, London
Blatt, M.R. 1992. K+ channels of stomatal guard cells: characteristics of the inward rectifier and its control by pH. J. Gen. Physiol. 99:615–644
Blatt, M.R., Armstrong, F. 1993. K+ channels of stomatal guard cells: abscisic acid-evoked control of the outward rectifier mediated by cytoplasmic pH. Planta 131:330–341
Blatt, M.R., Thiel, G. 1993. Hormonal control of ion channel gating. Annu. Rev. Plant Physiol. Mol. Biol. 44:543–567
Blatt, M.R., Thiel, G., Trentham, D.R. 1990. Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate. Nature 346:766–769
Blatz, A.L., Magleby, K.L. 1986. Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. Nature 323:718–720
Blatz, A.L., Magleby, K.L. 1987. Calcium-activated potassium channels. Trends Neurosci. 10:463–467
Bräu, M.E., Dreyer, F., Repp, H., Vogel, W. 1990. A K+ channel in Xenopus nerve fibres selectively blocked by bee and snake toxins: binding and voltage-clamp experiments. J. Physiol. 420:365–385
Candia, S., Garcia, M.L., Latorre, R. 1992. Mode of action of iberiotoxin, a potent blocker of the large conductance Ca2+-activated K+ channel. Biophys. J. 63:583–590
Catterall, W.A. 1988. Structure and function of voltage-sensitive ion channels. Science 242:50–61
Christensen, O., Zeuthen, T. 1987. Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential. Pfluegers Arch. 408:249–259
Garcia, M.L., Galvez, A., Garcia-Calvo, M., King, V.F., Vazquez, J., Kaczorowski, G.J. 1991. Use of toxins to study potassium channels. J. Bioenerg. Biomembr. 23:615–646
Hagiwara, S., Miyazaki, S., Rosenthal, N.P. 1976. Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. J. Gen. Physiol. 67:621–638
Hagiwara, S., Yoshii, M. 1979. Effects of internal potassium and sodium on the anomalous rectification of the starfish egg as examined by internal perfusion. J. Physiol. 292:251–265
Halliwell, J., Othman, I., Pelchen-Matthews, A., Dolly, J.O. 1986. Central action of dendrotoxin: selective reduction of a transient K conductance in hippocampus and binding to located acceptors. Proc. Natl. Acad. Sci. USA 83:493–497
Ho, K., Nichols, C.G., Lederer, W.J., Lytton, J., Vassilev, P.M., Kanazirska, M.V., Hebert, S.C. 1993. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature 362:31–38
Hodgkin, A.L., Huxley, A.F., Katz, B. 1952. Measurements of current-voltage relations in the membrane of the giant axon of Loligo. J. Physiol. 116:424–448
Jan, L.Y., Jan, Y.N. 1992. Structural elements involved in specific K+ channel functions. Annu. Rev. Physiol. 54:537–555
Jonas, P., Brau, M.E., Hermsteiner, M., and Vogel, W. 1989. Singlechannel recording in myelinated nerve fibers reveals one type of Na channel but different K channels. Proc. Natl. Acad. Sci. USA 86:7238–7242
Ketchum, K.A., Poole, R.J. 1990. Pharmacology of the Ca2+-dependent K+ channel in corn protoplasts. FEBS Lett. 274:115–118
Kubo, Y., Baldwin, T.J., Jan, Y.N., Jan, L.Y. 1993. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362:127–133
Laurido, C., Candia, S., Wolff, D., Latorre, R. 1991. Proton modulation of Ca2+-activated K+ channel from rat skeletal muscle incorporated into planar bilayers. J. Gen. Physiol. 98:1025–1043
Lucchesi, K., Ravindran, A., Young, H., Moczydlowski, E. 1989. Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca2+-activated K+ channels. J. Membrane Biol. 109:269–281
MacKinnon, R., Miller, C. 1988. Mechanism of charybdotoxin block of single Ca2+-activated K+ channels. J. Gen. Physiol. 91:335–349
MacKinnon, R., Miller, C. 1989. Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor. Science 245:1382–1385
Marquardt, D. 1963. An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math. 11:431–441
Miller, C., Moczydlowski, E., Latorre, R., Phillips, M. 1985. Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature 313:316–318
Moczydlowski, E., Lucchesi, K., Ravindran, A. 1988. An emerging pharmacology of peptide toxins targeted against potassium channels. J. Membrane Biol. 105:95–111
Parcej, D.N., Dolly, J.O. 1989. Dendrotoxin acceptor from bovine synaptic plasma membranes. Biochem. J. 257:899–903
Penner, R., Petersen, M., Pierau, F.-K., Dreyer, F. 1986. Dendrotoxin: a selective blocker of a non-inactivating potassium current in guinea-pig dorsal root ganglion neurones. Pfluegers Arch. 407: 365–369
Rehm, H., Pelzer, S., Cochet, C., Chambaz, E., Tempel, B.L., Trautwein, W., Pelzer, D., Lazdunski, M. 1989. Dendrotoxin binding brain membrane protein displays a K+ channel activity that is stimulated by both cAMP-dependent and endogenous phosphorylations. Biochemistry 28:6455–6460
Ruppersberg, J.P., Schroter, K.H., Sakmann, B., Stocker, M., Sewing, S., Pongs, O. 1990. Heteromultimeric channels formed by rat brain potassium-channel proteins. Nature 345:535–537
Schachtman, D.P., Schroeder, J.I., Lucas, W.J., Anderson, J.A., Gaber, R.F. 1992. Expression of an inward-rectifying potassium channel by the Arabidopsis KAT1 cDNA. Science 258:1654–1657
Schroeder, J.I., Hagiwara, S. 1989. Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Naure 338:427–430
Schweitz, H., Stansfeld, C.E., Bidard, J.-N., Fagni, L., Maes, P., Lazdunski, M. 1989. Charybdotoxin blocks dendrotoxin-sensitive voltage-activated K+ channels. FEBS Lett. 250:519–522
Scott, V.E., Parcej, D.N., Keen, J.N., Findlay, J.B.C., Dolly, J.O. 1990. Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. J. Biol. Chem. 65:20094–20097
Sentenac, H., Bnneaud, N., Minet, M., LaCroute, F., Salmon, J.M., Gaymard, F., Grignon, C. 1992. Cloning and expression in yeast of a plant potassium-ion transport system. Science 256:663–665
Stansfeld, C.E., Feltz, A. 1988. Dendrotoxin-sensitive K+ channels in dorsal root ganglion cells. Neurosci. Lett. 93:49–55
Stühmer, W., Ruppersberg, J.P., Schröter, K.H., Sakmann, B., Stocker, B., Giese, K.P., Perschke, A., Baumann, A., Pongs, O. 1989. Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain. EMBO J. 8:3235–3244
Talvenheimo, J.A. 1985. The purification of ion channels from excitable cells. J. Membrane Biol. 87:77–91
Tester, M. 1988. Pharmacology of K+ channels in the plasmalemma of the green alga Citara corallina. J. Membrane Biol. 103:159–169
Tester, M. 1990. Plant ion channels: whole-cell and single-channel studies. New Phytol. 114:305–340
Thiel, G., MacRobbie, E.A.C., Blatt, M.R. 1992. Membrane transport in stomatal guard cells: the importance of voltage control. J. Membrane Biol. 126:1–18
Weiser, T., Bentrup, F.W. 1991. Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium-rubrum L. Biochim. Biophys. Acta 1066:109–110
Weller, U., Bernhardt, U., Siemen, D., Dreyer, F., Vogel, W., Habermann, E. 1985. Electrophysiological and neurobiochemical evidence for the blockade of a potassium channel by dendrotoxin. Naunyn-Schmied. Arch. Pharmacol. 330:77–83
White, P.J., Tester, M.A. 1992. Potassium channels from the plasma membrane of rye roots characterised following incorporation into planar lipid bilayers. Planta 186:188–202
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We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship.
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Obermeyer, G., Armstrong, F. & Blatt, M.R. Selective block by α-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane. J. Membarin Biol. 137, 249–259 (1994). https://doi.org/10.1007/BF00232593
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DOI: https://doi.org/10.1007/BF00232593