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.
Bullock
J O
JO
Department of Physiology, University of Missouri-Columbia 65212.
Armstrong
S K
SK
Shear
J L
JL
Lies
D P
DP
McIntosh
M A
MA
D003001:Cloning, Molecular; D003087:Colicins / Q000276:immunology / Q000378:metabolism*; D004553:Electric Conductivity; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D010477:Perfusion; D015183:Restriction Mapping
D003087:Colicins; D007106:Immune Sera; D007473:Ion Channels; D008051:Lipid Bilayers