Cecropins, positively charged antibacterial peptides found in the cecropia moth, and synthetic peptide analogs form large time-variant and voltage-dependent ion channels in planar lipid membranes in the physiological range of concentration. Single-channel conductances of up to 2.5 nS (in 0.1 M NaCl) were observed, which suggests a channel diameter of 4 nm. Channels formed by the peptides cecropin AD and MP3 had a permeability ratio of Cl-/Na+ = 2:1 in 0.1 M NaCI. A comparative study of the three cecropins, cecropins A, B, and D, and of six synthetic analogs allowed determination of structural requirements for pore formation. Shorter amphipathic peptides did not form channels, although they adsorbed to the bilayer. A flexible segment between the N-terminal amphipathic region and the C-terminal more hydrophobic region of the peptide was required for the observation of a time-variant, voltage-dependent conductance. Cecropin AD was the most effective voltage-dependent poreforming peptide and was also the most potent antibacterial peptide against several test organisms. A positive surface charge or cholesterol in the bilayer reduced the conductances caused by cecropin AD or MP3 by at least 5-fold. This behavior is consistent with the known insensitivity of eukaryotic cells to cecropins. Our observations suggest that the broad antibacterial activity of cecropins is due to formation of large pores in bacterial cell membranes.The immune system of Hyalophora cecropia and other silkworms responds very effectively to bacterial infections by the induced synthesis of 15-20 hemolymph proteins including lysozyme and two classes ofantibacterial compounds, named cecropins and attacins. The antibacterial spectrum of cecropins is broad and includes both Gram-positive and Gramnegative bacteria, whereas all eukaryotic cell types tested so far are resistant (for review, see ref. 1). The primary structures of the three principal cecropins (namely, cecropins A, B, and D containing 37, 35, and 36 residues, respectively) have been determined (2), and their secondary structures have been predicted. They show an unusually polarized amphipathic helix in the N-terminal segment (residues 1-11) (3,4), which is indicative of a strong membrane association potential (5). The central portion of cecropin A has some potential for a 8-turn at residues 14-17 and contains the strong helix breakers glycine and proline. The C-terminal portion is hydrophobic and has some potential for an amphipathic helix.Studies with synthetic cecropin A analogs (6, 7) and cecropin D analogs (J.F., H. G. Boman, and R.B.M., unpublished results) showed the importance of the amphipathic helix as a structural element for cell lysis occurring on the cell membrane level. The molecular mechanism of lysis is still poorly understood. Model studies with liposomes showed cecropin-induced leakage of carboxyfluorescein, but evidence for membrane disruption was ambiguous (8).For the present study, several peptides have been designed and synthesized in order to extend the struc...
