Electrophysiologcal recording of transmembrane currents enables the direct functional assessment of ion-conducting pores formed by proteins in membranes. Pore-formation can be followed in real time on sub-ms timescales and with single molecule resolution, allowing kinetic analysis as well as determination of basic structural and functional features (pore size and stability, ionic selectivity, pH- and salt-dependence).
As a means of rapid and direct functional characterization of pore-forming proteins, however, electrophysiology is currently heavily underused due to the technical difficulties of the formation of lipid membranes and the laborious nature of sequential one-by-one experiments. Especially when the phenomenology of pores is not clear-cut but presents a wide variety, as has been shown for a number of clinically important pore-formers, sequential experiments on single pores are not only time-consuming but also difficult to interpret because of possible time-dependent changes in the protein preparation and experimental conditions.
We report on the development and use of microfabricated bilayer array chips for high resolution and high-throughput electrophysiological analysis of pore-forming proteins. The Microelectrode Cavity Array (MECA) platform allows lipid bilayers to be formed repetitively and reliably by hand or in automated fashion on between 4 and currently 16 recording positions on a single chip. We have used this device to characterize the biological variability of a number pore-forming proteins, ranging from antimicrobial peptides such as gramicidin and alamethicin and well-characterized soluble bacterial toxins such as alpha-hemolysin and aerolysin through bacterial outer membrane proteins such as MspA, to viroporins and bacterial and mammalian ion channels. Even for the well-known beta-barrel pores alpha-hemolysin and aerolysin, simultaneous recordings of many single pores reveal variability in pore formation under identical exeprimental conditions.
We suggest that parallel recording from lipid bilayer arrays will prove an enabling technique for future studies of mechanisms of pore-formation.