Rapid functional characterization of ion channels with droplet interface bilayer...
Rapid functional characterization of ion channels with droplet interface bilayers
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. W...
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Descripción del proyecto
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.