Phlorizin- and 6-Ketocholestanol-Mediated Antagonistic Modulation of Alamethicin Activity in Phospholipid Planar Membranes Tudor Luchian* and Loredana Mereuta Department of Biophysics and Medical Physics, Faculty of Physics, Alexandru I. Cuza UniVersity, BouleVard Carol I, no 11, Iasi, Romania, R-6600 ReceiVed May 16, 2006. In Final Form: August 1, 2006 As a result of the interfacial chemical heterogenity, membrane-penetrating peptides will experience a dramatic variation in environmental polarity manifested via electrical interactions with the surface and dipole potential of membranes prone to modulate the membrane insertion and folding of different peptides and proteins. Herein we present evidence demonstrating that roughly a 30 mV, phlorizin-induced lowering of the magnitude of the dipole potential of a phosphatidyilcholine membrane leads to a 4-fold increase in the electrical activity of embedded alamethicin. The effect is voltage-independent, implying that the dipole potential affects the barrier of alamethicin adsorption to the membrane rather than the translocation of it across the hydrophobic core. Our interpretation points to an enhanced interfacial accumulation of alamethicin monomers on the cis side of the membrane caused by a lower value of the cis dipole potential, which will promote an elevated activity of alamethicin oligomers across the membrane. As expected for a modestly selective ion channel, the enhancing effect of such dipole potential changes on the electrical conductivity is limited (80 ( 3 pS before and 100 ( 2 pS after phlorizin addition to the membrane, for the first conductive state of the channel). Our study emphasizes the possibility that, by manipulating at will the sign of change and the magnitude of the interfacial dipole field, it is possible to modulate the extent of the membrane penetration of ion-channel-forming peptides and thereby provide deeper insights into mechanisms of protein-lipid and protein- protein interactions within membranes. Introduction Regardless of its biochemical composition, the overall electrical profile of a biomembrane combines contributions from the transmembrane potential, dipole potentials, and surface potentials at both sides of a membrane. 1,2 Of these, the membrane dipole potential has received particular attention mainly due to the extremely high electric field associated with it over the interface between the aqueous phase and the hydrocarbon region of a biomembrane (10 8 -10 9 V‚m -1 ). A large amount of both structural and functional data have unraveled the two main factors that underlie the origin of the dipole potential: the orientation of dipolar groups located on the lipid molecule (i.e., the dipole of the carbonyl group of the ester bond and the P - -N + dipole of the headgroup) and the dipoles of oriented water molecules at the membrane-water interface. 3-5 Dipole potential has often been cited as being among other factors that have powerful influences on membrane-protein interactions, including protein insertion and functioning, 6 kinetics of the gramicidin channel, 7 modulation of the activity of phospholipase A2, 8 and electrical conductance of certain aqueous protein pores. 9 Recent data have demonstrated that the emission intensity of a widely used fluorescent moiety, 7-nitro-2,1,3-benzoxadiazol- 4-yl (NBD) labeling covalently either the headgroup of DPPC lipids (DPPN) or the acyl chains of PC lipids (NBD-PC) aggregated as liposomes, is sensitive to the membrane dipole potential and that the rate of the reduction of NDB in these probes by dithionite can be controlled by the dipole potential. 10 Moreover, dipole potential was shown to affect the membrane insertion and folding of a model amphiphilic peptide 11 as well as the extent of the membrane fusion. 12 In a similar line of reasoning and knowing that an essential feature of membrane penetration by various membrane toxins 13,14 and fusion peptides 15 is changing from a water-soluble state to a membrane-associated state, lipid contribution and variation of the dipole potential may critically affect the state of peptide aggregation within lipid membranes and have profound implications for their biological activity. Bearing in mind the relevance of the membrane dipole potential on cellular function, it has been established that by manipulating its value, the influence of the dipole potential on protein-protein interactions may be explored. 16 It was even possible to modulate the extent of the penetration of peptides within membranes and the manner in which they adopt different secondary structures. 17 Membrane insertion of such peptides and proteins requires a refolding process that is poorly understood at the molecular level, but the membrane interfacial region (IF) is expected to play a critical role in the refolding event, as described previously. 18,19 * Corresponding author. Tel: +040232 201191. Fax: +040232 201151. E-mail: luchian@uaic.ro. (1) Franklin, J. C.; Cafiso, D. S. Biophys. J. 1993, 65, 289. (2) Cevc, G. Biochim. Biophys. Acta 1990, 1031, 311. (3) Zheng, C.; Vanderkooi, G. Biophys. J. 1992, 63, 935. (4) Gawrisch, K.; Ruston, D.; Zimmerberg, J.; Parsegian, A.; Rand, R. 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