Rapid Activity Screening of Ion Channels Expressed in Cell-Free Systems Using a Lipid Bilayer Array Pcb-Device

Abstract

Cell-free expression is a rapidly developing tool for the efficient production of membrane proteins with enormous potential. Here we report on the design of a setup for parallel high-resolution electrophysiological recordings allowing for the screening of functional activity of ion channels synthesized in-vitro in various lipid environments.The developed recording chamber is based on a SU-8 coated printed circuit board containing 4 cavities (50µm) with individual microelectrodes (Micro-Electrode-Cavity-Array (MECA) as well as a common ground electrode. 4 suspended lipid bilayers can be self-assembled on the nonpolar chip surface from phospholipids in organic solvent. The MECA-PCB is connected to a multichannel amplifier capable of simultaneous recording of electrical activity from the ion channels functionally reconstituted into the bilayers with high-bandwidth and low noise (<1 pA rms ≅ 10 kHz).To test the capability of the MECA-PCB-chip, the tetrameric potassium channel KcsA was expressed in-vitro with co-translational insertion into supplied liposomes containing di-myristoyl-glycerophosphocholine or asolectin. The collected proteoliposomes were extruded through 100 nm polycarbonate filter and fused with preformed bilayers. Activity from the single channels inserted in different bilayers was recorded in parallel under identical conditions. We show thatKcsA expressed in-vitro displays the typical single-channel kinetic behavior and sensitivity to blockers (Ba2+) described for the protein isolated from cellular membrane preparations. Channel open times and open probability are shown to be dependent on lipid environment with negatively-charged phospholipids stabilizing the open channel conformation.The combination of the cell-free protein expression system and MECA-PCB-chip electrophysiology allows for functional characterization of the synthesized ion channel within several hours starting from the DNA template. The current PCB-chip design could be easily adapted for higher throughput and automated bilayer formation as recently reported1.1Del Rio Martinez et.al. Small 2014.