Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer Roxana Chiriac Æ Tudor Luchian Received: 6 August 2008 / Accepted: 18 September 2008 / Published online: 11 October 2008 Ó Springer Science+Business Media, LLC 2008 Abstract In this experimental work we employed single- molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC–chol–bSM) compared to cases when bilayers were made of POPC–chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data dem- onstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capac- itance of ternary lipid mixtures (POPC–chol–bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC–chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane. Keywords Lipid raft  Alamethicin  Electrophysiology  Planar lipid membrane Introduction As a result of the complex biochemical composition of eukaryotic plasma membranes, an interesting concept emerged even at the dawn of modern membrane biology which considered that most cell membranes are highly heterogeneous structures, whereby lipids and proteins are organized in functional domains (Singer and Nicolson 1972). During the past decade such domains, called ‘‘lipid rafts,’’ have constituted a major focus of studies of eukaryotic membrane structure. Concisely speaking, the lipid raft paradigm is based on the fact that selected, nat- urally occurring lipids aggregate in the plane of the membrane due to intermolecular interactions, such as van der Waals interactions between the nearly fully saturated chains of sphingomyelin and glycosphingolipids as well as hydrogen bonds between adjacent glycosyl moieties of glycosphingolipids (Simons and Ikonen 1997); and this entails the existence of relatively highly organized ‘‘islands’’ of lipid patches that ‘‘float’’ in a liquid-crystal- line two-dimensional lipid phase. The very existence of such domains was originally promoted by the fact that assemblies of sphingolipids and cholesterol apparently survived Triton-X 100 extraction at 4°C (Brown and Rose 1992), whereas methyl-b-cyclo- dextrin-induced cholesterol depletion led to loss of detergent resistance (Ilangumaran and Hoessli 1998; Scheiffele et al. 1997). Within a raft-containing membrane, cholesterol was shown to induce the existence of a so-called liquid ordered phase (lo) (Sankaram and Thompson 1990), in which lipid R. Chiriac  T. Luchian (&) Laboratory of Biophysics and Medical Physics, Faculty of Physics, Alexandru I. Cuza University, Blvd. King Carol I, No. 11, Iasi 700506, Romania e-mail: luchian@uaic.ro 123 J Membrane Biol (2008) 224:45–54 DOI 10.1007/s00232-008-9131-7