BLM experimentation towards a better understanding of (electro)pore formation

Motivation

Cell membranes are very complex structures that are composed of a bilayer of a great variety of phospholipids, cholesterols and proteins (see figure 1, left). In order to study and understand fundamental properties of these membranes, simplification is required. A model that is often employed for this purpose is a planar lipid bilayer (bilayer lipid membrane or BLM) (see figure 1, right). BLMs have the advantage that their composition (in terms of phospholipids, cholesterol and possibly proteins) is easily controlled and adapted and that it is possible to access both sides of the membrane chemically as well as electrically. This enables straightforward electrical measurements across the membrane.

Figure 1. From cell membranes to model membranes.

Left: A schematic representation of a cell membrane consisting of a bilayer of many different types of phospholipids, cholesterol and proteins. Right: A planar model membrane (bilayer lipid membrane, BLM) is often employed to study cell membrane properties.

Goals

In this project, these advantages of BLMs are employed to get a better understanding of pore formation in the cell membrane during the process of electroporation. Understanding the fundamental principles involved in the pore formation process, which factors of the membrane affect this and how, results in an increased control over the process in real cells and consequently a higher yield.

Approach

Here, we study specifically the influence of the membrane composition on pore formation during electroporation. Membranes with an increasing complexity are employed. In a first step, bimolecular systems of two phospholipids, one phospholipid and cholesterol or one phospholipid and a protein (α-hemolysin) are investigated. Following this, a third component found in natural membranes (a sphingolipid) is added to the binary mixture of a glycerolipid and cholesterol to approximate such membranes even better. In a last step, the composition of the BLMs is again made more complex to mimic the composition of real cell membranes.

Results

We have found that there is a large effect of the membrane constituents on its resistance to pore formation. The constituents that strengthen the membranes the most are cholesterol and glycolipids. This effect is explained by the precise molecular interactions between the different molecules in the membrane [1-2].

Furthermore, we have found that the resistance of membrane to perturbations can be correlated to the phases the phospholipids are in [2]. This enables the derivation of a ternary phase diagram from relatively simple measurements (see figure 2). We foresee that this novel methodology to derive a ternary phase diagram of complex lipid mixtures will be a great tool to quickly scan the behavior of such systems, after which the interesting areas can be studied in more detail with the more conventional methods.

Figure 2. Electroporation results for ternary mixtures of a glycerolipid, a sphingolipid and cholesterol

Left: The dependence of the threshold voltage for electroporation of a 1:2 L-α-PC/SM mixture with increasing cholesterol content. The different zones correspond to the different phases the lipids are in. Right: Proposed ternary phase diagram for L-α-PC, SM and cholesterol mixtures, obtained using the membrane resistance to an applied electric field. The black striped line indicates where the plot on the left (the1:2 L-α-PC/SM mixtures) is found in the diagram.

Literature

[1] van Uitert, I. et al. (2010). The influence of different membrane components on the electrical stability of bilayer lipid membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1798 (1), pp. 21-31.

[2] van Uitert, I. et al. (2010). Determination of the electroporation onset of bilayer lipid membranes as a novel approach to establish ternary phase diagrams: example of the l-α-PC/SM/cholesterol system. Soft Matter. DOI: 10.1039/C0SM00181C.

Contact persons

Dr. ir. Iris van Uitert

Dr. Séverine Le Gac