Hochohmige porenüberspannende Lipidmembranen: Elektrochemische Untersuchungen zur Aktivität von Gramicidin und Bacteriorhodpsin

Abstract

To study transmembrane proteins they need to be integrated in a lipid environment to preserve their functionality. For this purpose, model membranes have been developed which allow the investigation of ion channels and transporters in a defined environment. In recent years, a new artificial membrane system based on porous substrates has been developed, which exhibits on the one hand robustness and long-term stability and on the other hand allows for monitoring ion channel activity down to the single channel level. The drawback of this system is that transmembrane proteins can only be inserted from aqueous solution by dilution of a detergent solution. Furthermore, high protein densities, that are required for the investigation of membrane transporters with low turn over rates, cannot be obtained. Thus, the aim of this work was to develop a method to prepare lipid membranes on porous substrates, which allows the insertion of transmembrane proteins with high density. For this purpose, liposomes were used to generate lipid bilayers on gold coated porous alumina substrates, which were functionalised with the spacer lipid (cholesterylpolyethylenoxy)thiol CPEO3. Highly insulating and long-term stable pore-spanning membranes were obtained. By means of electrical impedance spectroscopy the membrane capacitance and resistance were elucidated. The obtained specific capacitance of (0,5 ± 0,1) µF cm -2 is in agreement with the expected value for lipid bilayer membranes. The membrane resistance was in the range of 10 6 -10 8 Ω. Partially ruptured membranes can be re-established by the fusion of vesicles. The pore-suspending membranes are very susceptible to the insertion of the detergent octyl-polyoxyetylene (o-POE), the membrane soluble proton carrier carbonyl-cyanide-m-chloro-phenylhydrazone (CCCP) and the bacterial outer membrane protein OmpF. The suitability of the established method for the transfer of integral membrane proteins from proteoliposomes into pore-spanning membranes was demonstrated by employing the well-investigated model ion channel gramicidin D and the light-driven proton transporter bacteriorhodopsin. The distinct ion channel activity of gramicidin D was investigated by electrical impedance spectroscopy. Gramicidin D-doped pore spanning membranes allowed for the read out of distinct ion channel properties such as the well-known selectivity for monovalent cations and the ion channel block by Ca 2+ in an integral manner. Furthermore, the mass transport -a result of the concentration gradient towards the Gramicidin D-doped membrane -was clearly discriminated. As an example for a transporter molecule the light driven proton pump Bacteriorhodopsin was integrated into pore-spanning membranes. Its specific activity was investigated by means of light induced photocurrent measurements.