Voltage-dependent, monomeric channel activity of colicin E1 in artificial membrane vesicles.
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 a transnegative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than -60 mV (K+in/K+out greater than or equal to 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.
D002118:Calcium / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism; D003087:Colicins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism*; D008564:Membrane Potentials; D008567:Membranes, Artificial; D009994:Osmolar Concentration; D014634:Valinomycin / Q000378:metabolism
D002712:Chlorides; D003087:Colicins; D007473:Ion Channels; D008567:Membranes, Artificial; D014634:Valinomycin; D002118:Calcium