108 Biochimica et Biophysica Acta, 727 (1983) 108-114 Elsevier Biomedical Press BBA 71498 MELITFIN AND A CHEMICALLY MODIFIED TRICHOTOXIN FORM ALAMETHICIN-TYPE MULTI-STATE PORES WOLFGANG HANKE a, CHRISTOPH METHFESSEL a, HANS-ULRICH WILMSEN a, ELLEN KATZ b GONTHER JUNG b and GONTHER BOHEIM a., a Lehrbereich Zellphysiologie, Ruhr-Universiti~t Bochum, Postfach 102148, D 4630 Bochum and b lnstitut fi~r Organische Chemie, Universiti~t Tiibingen, A uf der Morgenstelle, D 7400 Ti~bingen (F. R. G.) (Received May 17th, 1982) (Revised manuscript received August 2nd, 1982) Key words: Pore formation, A lamethicin; Lipid bilayer," Ion channel," Melittin; Trichotoxin; Voltage-dependent conductance The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 ttm2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal a-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores. Introduction The amphiphilic polypeptides alamethicin and melittin are structurally related to a large extent (Fig. 1) [1-4]. The N-terminal parts consist of lipophilic amino acids except for position [7]. A helix-breaking proline is found in position 14 and the C-termini have a quite polar character. The N-terminal regions adopt a-helical conformation in organic solvents [2,3], in lipid bilayer systems [4], in aqueous solutions at high ionic strength [8] and in the crystallized state [9]. a-Helices bear an intrinsic dipole moment. Indeed, a dipole moment of 67 D [10] or 75 D [l 1], respectively, per * To whom correspondence should be addressed. 0005-2736/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press alamethicin molecule was found. Dipoles tend to arrange in an antiparallel fashion. At 6 A resolu- tion, crystallized melittin exhibits a two-fold axis of symmetry and rod-like segments which run antiparaUel to each other [9,12]. Similar results were obtained for a helix-forming undecapeptide analogue of alamethicin [13]. 1H-NMR spectra of the melittin tetrameric aggregate reveal proximity of isoleucine (position 2) and tryptophan (position 19)~ which is not observed with monomeric a-heli- cal melittin [14]. In detergent micelles, the amino- terminal and carboxy-terminal halves of the primary structure constitute separate, compact do- mains within the conformation of monomeric micelle-bound melittin [ 15,16]. Adsorption experiments with alamethicin [17] and desformyl melittin [18] at a lipid/water inter-