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In the present work the authors studied the conductance changes associated with …


Biology Articles » Biophysics » Molecular Biophysics » Voltage-Induced Nonconductive Pre-Pores and Metastable Single Pores in Unmodified Planar Lipid Bilayer » Introduction

Introduction
- Voltage-Induced Nonconductive Pre-Pores and Metastable Single Pores in Unmodified Planar Lipid Bilayer

Barrier function, one of the most important functions of cell membranes, is based on the continuity of membrane lipid bilayers. However, diverse physiological processes including cell lysis and membrane rearrangements in endocytosis and exocytosis require transient breaking of bilayer structure and apparently involve formation of non-bilayer intermediates (Bechinger, 1999; Chernomordik, 1996; Nanavati et al., 1992; Schmidt et al., 1999). A local through-going pore in a membrane lipid bilayer is among the simplest examples of these hypothetical intermediates. In contrast to proteinaceous ionic channels, the edge of the lipidic pores is formed mainly by lipid molecules. Proteins involved in membrane fusion (Chernomordik et al., 1994, 1998; Melikyan et al., 1997), and apoptosis (Basanez et al., 1999), and cytolytic peptides (Bechinger, 1999; Matsuzaki et al., 1998; Miteva et al., 1999) were hypothesized to facilitate formation and expansion of these pores.

To better understand the mechanisms of the protein-mediated disruption of the continuity of lipid bilayers one may study the properties of the lipidic pores in a well-defined experimental system of protein-free bilayer lipid membranes (BLMs). Local disruption of BLMs can be readily achieved by applying high electric fields. Opening and expansion of lipidic pores is thought to underlie the well-known phenomenon of permeabilization of protein-free bilayers (BLMs and liposomes) under high electric field (also referred to as electroporation and electrical breakdown) (Abidor et al., 1979; Weaver and Chizmadzhev, 1996). Two types of membrane behavior under electrical stress, irreversible and reversible electrical breakdown, are distinguished in literature. In the case of irreversible membrane breakdown observed for BLMs of any lipid composition, a measurable increase in membrane conductance rapidly leads to mechanical rupture of the membrane (Abidor et al., 1979; Genco et al., 1993; Wilhelm et al., 1993). On the other hand, BLMs of some specific compositions (for instance, bilayers formed from oxidized cholesterol) exposed to a short pulse of high electric field demonstrate so-called reversible breakdown. In this case even after five to six orders of magnitude increase, BLM conductance quickly drops to the initial level upon voltage decrease (Benz et al., 1979; Glaser et al., 1988). It has been hypothesized that in the case of irreversible breakdown few pores are formed before the first one of them reaches a critical radius and starts irreversible expansion leading to membrane rupture. In contrast, in the case of reversible breakdown a large population of pores accumulates under high voltage before the onset of BLM rupture. These studies have indicated that voltage application to BLMs of any lipid composition provides a convenient way to promote pore formation and simultaneously offers a very fast and sensitive assay for pore detection by the conductance measurements.

A number of models were proposed to describe formation and development of pores under electric field (Krassowska and Neu, 1994; Moroz and Nelson, 1997; Needham and Hochmuth, 1989; Partenskii et al., 1998; Winterhalter and Helfrich, 1987) and the properties of large irreversibly expanding pores (Needham and Hochmuth, 1989; Sukharev et al., 1983; Wilhelm et al., 1993; Zhelev and Needham, 1993). However the lack of experimental data on the properties of single pores of small, sub-critical radii limited the theoretical analysis of the early stages of pore formation.

In the present work we studied the conductance changes associated with the formation of the metastable transient single pores in unmodified BLMs under high electric field. Sizes of these pores and kinetic characteristics of the pore evolution were evaluated. The character of observed electrical activity suggests that opening and closing of the metastable lipidic pore proceed through a nonconductive pre-pore.



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