Abstract
Ion channels are known to be promising drug targets, therefore sensitive and reliable screening assays for ion channel activity are needed for development of new drugs. In this work two different ion channels, a cystic fibrosis transmembrane conductance regulator (CFTR) and a transient receptor potential cation channel, subfamily V (TRPV1)were shown to be expressed in vitro with sufficient yield to record their functional activity after reconstitution into planar bilayer arrays on the Micro Electrode Cavity Array (MECA) chip. Eukaryotic cell free expression system enabled correct folding and co-translational integration of synthesized membrane proteins into endogenous endoplasmic reticulum derived vesicles named microsomes as well as a posttranslational phosphorylation of CFTR. For the functional characterization of the in vitro expressed ion channels the collected microsomal membranes were either directly fused to planar lipid bilayers or reconstituted after the solubilization in detergent and subsequent relipidation in proteoliposomes of defined composition. The activity from the single ion channels inserted in different bilayers on the MECA chip was recorded in parallel. The in vitro expressed TRPV1 was shown to display typical single-channel kinetic behavior with steep channel activation above 40 °C as described for the protein isolated from cellular membrane preparations. In vitro synthesized CFTR showed typical conductance values, as well as ATP-dependent activation pattern previously described by the patch-clamp technique. Production of functional mammalian ion channels in eukaryotic cell-free systems together with the possibility of reliable functional activity screening using artificial membrane arrays have a potential to improve drug development pipelines for the search of new inhibitors of ion channel activity.
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