Summary
The dependence of colicin channel activity on membrane potential and peptide concentration was studied in large unilamellar vesicles using colicin E1, its COOH-terminal thermolytic peptide and other channel-forming colicins. Channel activity was assayed by release of vesicle-entrapped chloride, and could be detected at a peptide: lipid molar ratio as low as 10−7. The channel activity was dependent on the magnitude of atrans-negative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than −60mV (K +in /K +out ≥10), addition of valinomycin resulted in a 10-fold increase in the rate of Cl− efflux. A delay in Cl− efflux observed when the peptide was added to vesicles in the presence of a membrane potential implied a potential-independent binding-insertion mechanism. The initial rate of Cl− efflux was about 1% of the single-channel conductance, implying that only a small fraction of channels were initially open, due to the delay or latency of channel formation known to occur in planar bilayers.
The amount of Cl− released as a function of added peptide increased monotonically to a concentration of 0.7 ng peptide/ml, corresponding to release of 75% of the entrapped chloride. It was estimated from this high activity and consideration of vesicle number that 50–100% of the peptide molecules were active. The dependence of the initial rate of Cl− efflux on peptide concentration was linear to approximately the same concentration, implying that the active channel consists of a monomeric unit.
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Ames, B.N., Dubin, D.T. 1960. The role of polyamines in the neutralization of bacteriophage DNA.J. Biol. Chem. 235:769–775
Bishop, L.J., Cohen, F.S., Davidson, V.L., Cramer, W.A. 1986. Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1.J. Membrane Biol. 92:237–245
Bruggemann, E.P., Kayalar, C. 1986. Determination of the molecularity of the colicin E1 channel by stopped-flow ion kinetics.Proc. Natl. Acad. Sci. USA 83:4273–4276
Brunden, K.R., Cramer W.A., Cohen, F.S. 1984. Purification of a small receptor-binding peptide from the central region of the colicin E1 molecule.J. Biol. Chem. 259:190–196
Bullock, J.O., Cohen, F.S. 1986. Octyl glucoside promotes incorporation of channels into neutral planar phospholipid bilayers. Studies with colicin la.Biochim. Biophys. Acta 856:101–108
Bullock, J.O., Cohen, F.S., Dankert, J.R., Cramer, W.A. 1983. Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide.J. Biol. Chem. 258:9908–9912
Cleveland, B.M., Slatin, S., Finkelstein, A., Levinthal, C., 1983. Structure-function relationships for a voltage-dependent ion channel: Properties of C-terminal fragments of colicin E1.Proc. Natl. Acad. Sci. USA 80:3706–3710
Dankert, J.R. 1982. On the mechanism of penetration of the colicin E1 molecule through the cell envelope. Ph.D. Thesis, Purdue University, 117 pp.
Dankert, J.R., Uratani, Y., Grabau, C., Cramer, W.A., Hermodson, M. 1982. On a domain structure of colicin E1.J. Biol. Chem. 257:3857–3863
Davidson, V.L., Brunden, K.R., Cramer, W.A. 1985. Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: Relevance to the mechanism of action of colicins and certain toxins.Proc. Natl. Acad. Sci. USA 82:1386–1390
Davidson, V.L., Cramer, W.A., Bishop, L.J., Brunden, K.R. 1984. Dependence of the activity of colicin E1 in artificial membrane vesicles on pH, membrane potential, and vesicle size.J. Biol. Chem. 259:594–600
Farid-Sabet, S. 1982. Interaction of125I-labeled colicin E1 withEscherichia coli.J. Bacteriol. 150:1383–1390
Guy, H.R. 1983. A model of colicin E1 membrane channel protein structure.Biophys. J. 41:363a
Jacob, F., Simonovitch, L., Wollman, E. 1952. Sur la biosynthèse d'une colicine et sur son mode d'action.Ann. Inst. Pasteur 83, 295–315
Kagawa, Y., Yacker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation: XXV. Reconstitution of vesicles catalyzing32P t -adenosine triphosphate exchange.J. Biol. Chem. 246:5477–5487
Kayalar, C., Erdheim, G.R., Shanafelt, A., Goldman, K. 1984. Colicin channels and cellular immunity.Curr. Topics Cell. Reg. 24:301–312
Kayalar, C., Luria, S.E. 1979. Channel formation by colicin K on liposomes.In: Membrane Bioenergetics. C.P. Lee, G. Schatz, and L. Ernster, editors, pp. 297–306. Addison-Wesley, New York
Liu, Q.R., Crozel, V., Levinthal, F., Slatin, S., Finkelstein, A., Levinthal, C. 1986. A very short peptide makes a voltage-dependent ion channel: The critical length of the channel domain of colicin E1.Proteins 1:218–229
Martinez, M.C., Lazdunski, C., Pattus, F. 1983. Isolation, molecular and functional properties of the C-terminal domain of colicin A.EMBO J 2:1501–1507
Ohno-Iwashita, Y., Imahori, K. 1982. Assignment of the functional loci in the colicin E1 molecule by characterization of its proteolytic fragments.J. Biol. Chem. 257:6446–6451
Olson, F., Hunt, C.A., Szoka, F.C., Vail, W.J., Papahadjopoulos, D. 1979. Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.Biochim. Biophys. Acta 557:9–23
Pattus, F., Heitz, F., Martinez, C., Provencher, S.W., Lazdunski, C. 1985. Secondary structure of the pore-forming colicin A and its C-terminal fragment.Eur. J. Biochem. 152:681–689
Pattus, F., Martinez, M.C., Dargent, B., Cavard, D., Verger, R., Lazdunski, C. 1983. Interaction of colicin A with phospholipid monolayers and liposomes.Biochemistry 22:5698–5703
Peterson, A.A., Cramer, W.A. 1987. Membrane-potential and concentration-dependence of colicin E1 channel formation in artificial membrane vesicles.Biophys. J. 51:249a
Pressler, V., Braun, V., Wittmann-Liebold, B., Benz, R. 1986. Structural and functional properties of colicin B.J. Biol. Chem. 261:2654–2659
Schein, S., Kagan, B., Finkelstein, A. 1978. Colicin K acts by forming voltage-dependent channels in phospholipid bilayer membranes.Nature (London) 276:159–163
Schwartz, S.A., Helinski, D.R. 1971. Purification and characterization of colicin E1.J. Biol. Chem. 246:6318–6327
Shiver, J.W., Peterson, A.A. Widger, W.R., Cramer, W.A. 1987. Prediction of bilayer spanning domains of hydrophobic and amphipathic membrane proteins: Applications to the cytochromeb and colicin families.Meth. Enzymol. (in press)
Slatin, S., Raymond, L., Finkelstein, A. 1986. Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: The role of protein translocation.J. Membrane Biol. 92:247–254
Tokuda, H., Konisky, J. 1978. Effect of colicin Ia and E1 on ion permeability of liposomes.Proc. Natl. Acad. Sci. USA 75:6167–6171
Veatch, W.R., Mathies, R., Eisenberg, M., Stryer, L. 1975. Simultaneous fluorescence and conductance of planar bilayer membranes containing a highly active and fluorescent analog of gramicidin A.J. Mol. Biol. 99:75–92
Youkharibache, P., Fine, R., Levinthal, C. 1987. Possible conformations of the colicin E1 voltage-switchable channel.Biophys. J. 51:83a
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Peterson, A.A., Cramer, W.A. Voltage-dependent, monomeric channel activity of colicin E1 in artificial membrane vesicles. J. Membrain Biol. 99, 197–204 (1987). https://doi.org/10.1007/BF01995700
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DOI: https://doi.org/10.1007/BF01995700