Single-channel electrophysiology of cell-free expressed ion channels by direct incorporation in lipid bilayers

Abstract

Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERG S5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.Electrophysiology is the gold standard for the functional characterization of ion channel proteins, including screening of new drugs that target this pharmaceutically important class of membrane proteins. 1,2 The patch clamp technique involves intimate contact of an electrode-containing glass pipette with the membrane of a cell in which the channel of interest is overexpressed, and is used most widely for ensemble current measurements of channel populations. The alternative 'bilayer lipid membrane' (BLM) approach, in which puried ion channels are introduced by proteoliposome fusion into a pure-lipid membrane that separates two electrode-containing aqueous compartments, is typically employed for current measurements of single channels. 3,4 Automated instruments for mediumthroughput patch clamp electrophysiology have entered the market in the last decade, 5,6 whereas systems for automated and/or parallel BLM electrophysiology are under development by several research groups and companies. [7][8][9] Although there are recent developments toward patch clamping of giant proteoliposomes, 10 patch clamping typically relies on eukaryotic cells that are able to overexpress the ion channel of interest with concomitant cell membrane incorporation. 2 However, this is not possible when the expressed channel aggregates or when membrane incorporation is toxic to the cell. Proteoliposome formation, for patch clamping or for the BLM method, also requires ion channel overexpression, but, at the cell culture stage, not necessarily as solubilized or membrane-incorporated protein, and is not restricted to eukaryotic cells. 3 This offers more options to obtain the desired ion channel but at the cost of having to purify it from cell culture, with subsequent reconstitution from detergent solution into liposomes, which is a laborious process that requires a relatively high protein yield.In recent years, considerable progress has been made with the cell-free expression of membrane proteins, 11 which bypasses any cytotoxicity problems and facilitates protein purication. Bacterial lysates that contain the ribosomal machinery and are supplemented with amino acids, a metabolic energy supply and a protein-encoding plasmid, have been shown to express a large variety of membrane proteins, either as precipitates or solubilized in detergent micelles. 12-14 Interestingly, when liposomes are added to the cell-free reaction mixture, spontaneous reconstitution has been demonstrated for a variety of cell-free expressed membrane proteins, including stearyl-CoA desaturase, glucan synthase, ATP synthase, DesK thermosensor, endothelin rece...

Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K + channel: The power of native nanodiscs

“…5), implying spontaneous fusion events of single nanodiscs with the bilayer. The recorded traces show both high and low open probability states that are characteristic for KcsA monitored under similar conditions by single-channel recordings of KcsA fused from liposomes (33) or incorporated via a cell-free expression protocol (34). The results are also comparable with those obtained by patch clamp studies of giant unilamellar vesicles with KcsA reconstituted from detergent (35).…”

Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore

“…We observed a linear relation between the duplex concentration and the event frequency (Figure 4c), in agreement with previous miRNA studies with solid-state and αHL pores, resistive pulse sensing with droplet-in-oil systems, which can also be implemented as arrays for high-throughput assays. [64][65][66][67] Alternatively, a sample could be probed with multiple electrically independent nanopores, an approach that is similar to the development of high-throughput nanopore DNA sequencing. 68,69…”

Micro‐ and Nano‐Technologies for Lipid Bilayer‐Based Ion‐Channel Functional Assays

“…De Planque et al. applied this approach to the functional incorporation of hERG S5−S6 , the pore domain of hERG channels . Another direct method was also introduced by Bayley et al., in which ion channels are directly transferred from a bacterial colony to BLMs with an agarose‐coated tip or a glass probe .…”

“…A detergent‐free purification method using amphipathic styrene–maleic acid copolymers has been reported as an alternative to solubilizing ion channels directly from biological membranes to lipid nanodiscs shielded by polymers . Another detergent‐free approach is the direct insertion of ion channel proteins from cell‐free expression systems . Although these new techniques currently can only be used for prokaryotic or partially expressed eukaryotic channels, dramatic progress in these fields will enable detergent‐free purification or detergent‐free direct insertion into BLMs.…”