Ion Channel Reconstitution on a Pore-Suspending Membrane on Microstructured Glass Chip: Towards Artificial Gap Junction Formation Between Bilayers and Cells

Abstract

Chip based automated patch clamping is an attractive biophysical tool for studying ion channel proteins. Solvent-free planar lipid bilayers can be formed in an automated fashion by positioning and subsequent bursting of giant unilamellar lipid vesicles containing membrane proteins on micron-sized apertures in a borosilicate glass substrate. The use of proteoliposomes for bilayer formation on such chips allows for the direct recording of single channel activity without need for commonly difficult reconstitution of membrane protein after bilayer formation. This approach is specifically attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles or proteins from bacteria.Here, the biophysical and pharmacological characterization of different membrane proteins was performed. A wide variety of ion channels have been studied with this technique, for example potassium channels (KcsA, Kv1.2), sodium channels (NachBac) as well as other ligand-dependent (IP3 receptor) or mechanosensitive channels (MscL, TRP channels). Also, screening for influx of antibiotics through porins (OmpF, OmpC) was done to elucidate the uptake kinetics of antibiotics through porins.One main set of experimental data to be presented is on connexins proteins. Connexins are widely distributed in mammalian tissues and serve to join cells together into larger, functional units. We investigated the properties of hemichannels from Cx26 and Cx43 which were isolated biochemically and reconstituted into synthetic lipid membranes. In this study, preliminary data suggest the formation of gap junctions between cells and synthetic bilayer membranes. This opens possibilities to access the cytoplasm of living cells for biochemical or electrical studies, and especially to develop novel automated techniques for electrophysiological studies.