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...

No abstract

Photoformation of metalloporphyrin cations in a lipid bilayer increases the ionic currents of negative and decreases those of positive hydrophobic ions. At low concentrations of the mobile hydrophobic ion, a 30% change in conductivity is observed that decreases with increasing concentration of positive tetraphenylphosphonium ion and increases drastically with increasing concentration of negative tetraphenylboride ion. In the region of saturated conductance of boride ion, the increase in conductivity is 3.6-fold. A 15-fold increase is observed with the protonophore carbonyl cyanide 3-chlorophenylhydrazone. In this case the net charge gated is 300 times greater than the photogenerated charge in the bilayer membrane. Thus there is a net gain in this organic field effect phototransistor. The gating can also be accomplished by continuous light or chemical oxidants. Photogating is explained as space charge effects inside the bilayer.There is great interest in the movement of ions across cell membranes since these currents are associated with the activity ofall metabolically functional cells. In addition, there is much work aimed at understanding the chemical properties ofthese membranes (1). The planar lipid bilayer membrane as developed by Mueller et al. (2) has proven to be remarkably useful as a model ofthe cell membrane. It was discovered that small ions cross these membranes by two mechanisms: ion channels that can be gated by voltage or receptors (3, 4) and ion carriers that are not gated (5). We now show that the latter ionic currents, exemplified by currents of large hydrophobic ions, can be gated by photoinduced charge generation inside the membrane. The photogating effect is explained by local electrostatic effects in the bilayer membrane. The pigment/ bilayer/hydrophobic ion system is an example of an organic field effect phototransistor (6).Much work has been carried out on the mechanism by which ion carriers or hydrophobic ions cross the lipid bilayer (7-11). The large radius of these ionic species decreases the Born electrostatic charging energy of the ion, thus enabling them to traverse the hydrocarbon core of the membrane (10). It is striking that for hydrophobic ions of similar size, the conductivity of negative ions is 102-103 times that of positive ions (7,8,12,13). This observation has been explained by a dipolar potential, originating in ordered ester carbonyl groups, which is positive toward the hydrocarbon core (12). Most studies of the kinetics of ion crossing monitor either current relaxations after an applied voltage step or voltage relaxations after a charge pulse (7,9). These techniques, although very useful, are limited by capacitive transients and by ambiguity of interpretation (14). The present technique avoids the former problem and allows a direct measure of the transients in the change of conductivity. EXPERIMENTALThe 4-ml plastic membrane cell was separated by a Teflon divider with a 1-mm2 hole in the center and had glass slides for windows. A bilayer was formed from a solut...

The ionic currents of positive and negative hydrophobic ions across a -7 n,m thick lipid bilayer can be gated by the photoformation of metalloporphyrin cations in the ester region of 1336--1EEE ENGINEERING IN MEDICINE L BIOLOGY SOCIETY llTH ANNUAL INTERNATIONAL CONFERENCE

No abstract