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Rapid functional characterization of ion channels .. (DIB screening)
Rapid functional characterization of ion channels with droplet interface bilayers
(DIB screening)
Date du début: 1 juin 2010,
Date de fin: 31 mai 2012
PROJET
TERMINÉ
Of the estimated 400 ion channels encoded in the human genome, ~70 are potassium (K+) channels. Their implication in a number of human diseases, e.g., cardiac arrhythmia, cystic fibrosis, makes K+ channels relevant drug targets. Whereas the number of high-resolution structures of membrane proteins has consistently increased over the last few years, their functional characterization using screening approaches has not kept pace with that of water-soluble proteins. In this context, reliable and informative rapid screening assays for membrane proteins are needed. By further developing the recently reported droplet interface bilayer (DIB) system, I intend to develop a rapid automatable platform for screening wild-type channels as well as libraries of mutant ion channels using Kcv (viral), KvAP (prokaryotic) and Kv1.2 (eukaryotic) as model systems. My blueprint for such a screening device is analogous to an assembly line consisting of a network of microchannels to; (i) construct lipid monolayer-encased aqueous droplets, (ii) synthesize ion channels inside these nanobioreactors by coupled in vitro transcription-translation (IVTT), (iii) form bilayers with other droplets containing channel blockers, (iv) subsequently measure single-channel conductance to determine activity. The assay will be capable of screening one channel against hundreds of blockers or screen a library of mutants against one or a few blockers. The development of this nanoscale-streamlined process offers the possibility of producing powerful lab-on-chip instruments for membrane protein assays, which have previously proven intractable.
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