Voltage-dependent, monomeric channel activity of colicin E1 in artificial membrane vesicles

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Abstract
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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