Native membranes on subwavelentgh holes for membrane receptors analysis
How membrane receptors transduce external signals across the membrane and activate intracellular processes is a key question for fundamental research and for therapeutic purposes. Reliable in situ investigations requiest to preserve the full biological activity, the organization and the lateral mobility of proteins in the lipid membrane. Here we demonstrate the immobilization of native cell membranes on microstructured devices consisting of an array of 200 nm holes in a free standing planar SiN support. This represents an attractive procedure to study membrane proteins in a physiologically relevant environment (original lipid composition, proteins orientations and populations) while providing the full accessibility to both intra- and extracellular sides of the receptors. We used fluorescence-base detection approach to characterize the neurokinin-1 receptor, a representative G-protein coupled receptor, and the 5HT3 receptor, a neurotransmitter-gated ion channel. The stability and the low fluorescence background of the patches allow for further development of chip-based screening assays. Preliminary experiments on aluminium surfaces show that the method established here can be extended to a zero-mode waveguides configuration where subwavelength holes span in a metal film . Therefore, single-molecule experiments on membrane receptors can be performed at micromolar concentrations of fluorescent ligands.
Record created on 2007-03-14, modified on 2016-08-08