Ion selectivity of colicin E1: II. Permeability to organic cations.

Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time.

Formation of ion channels by colicin B in planar lipid bilayers.

The gene for the antibacterial peptide colicin B was cloned and transformed into a host background where it was constitutively overexpressed. The purified gene product was biologically active and formed voltage-dependent, ion-conducting channels in planar phospholipid bilayers composed of asolectin. Colicin B channels exhibited two distinct unitary conductance levels, and a slight preference for Na+ over Cl-. Kinetic analysis of the voltage-driven opening and closing of colicin channels revealed the existence of at least two conducting states and two nonconducting states of the protein. Both the ion selectivity and the kinetics of colicin B channels were highly dependent on pH. Excess colicin protein was readily removed from the system by perfusing the bilayer, but open channels could be washed out only after they were allowed to close. A monospecific polyclonal antiserum generated against electrophoretically purified colicin B eliminated both the biological and in vitro activity of the protein. Membrane-associated channels, whether open or closed, remained functionally unaffected by the presence of the antiserum. Taken together, our results suggest that the voltage-independent binding of colicin B to the membrane is the rate-limiting step for the formation of ion channels, and that this process is accompanied by a major conformational rearrangement of the protein.