Functional reconstitution of cell-free synthesized purified Kv channels

Abstract

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active K1.1 and K1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using K1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of K1.1 and K1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

Probing membrane protein properties using droplet interface bilayers

“…Renauld et al attempted to electrically characterize the potassium channel Kv1.1 produced via cell-free protein synthesis using a DIB composed of DPhPC. 36 They observed notably higher conductance values for the channel compared to previously published studies, along with the loss of voltage gating behavior. The authors speculate that this behavior was due to a loss of associated lipid derived function that could not be replicated in the synthetic DPhPC bilayer.…”

“…The potassium channels Kv1.1, KcsA, and Kcv, the mechanosensitive channel MscL, and the disaccharide transporter LacY have all been incorporated into DIBs using this method. 9,15,36,40,44 In this method, liposomes are swollen by mixing with a detergent such as octyl glucoside (OG) before mixing with detergent solubilized protein. Excess detergent is then removed, often using adsorbent beads such as BioBeads, leaving proteoliposomes containing the protein of interest.…”

Strategies for Successful Over-Expression of Human Membrane Transport Systems Using Bacterial Hosts: Future Perspectives

“…Each of the parameters previously listed, also including growth temperature, E. coli strain, lipid composition, and detergent, have to be taken into consideration due to their influence on protein expression. Indeed, the addition of lipids or detergent was successfully adopted for the cell-free synthesis of a G protein–coupled receptor [ 142 ], two potassium channels [ 143 , 144 ], the human aquaglyceroporin 3 protein (AQP3) [ 145 ], three isoforms of the mitochondrial uncoupling protein (UCP1-3) [ 146 ], two isoforms of the mitochondrial ADP/ATP carrier (hAAC1 and hAAC3) [ 147 ], the human mitochondrial voltage-gated ion channel (VDAC) [ 148 ], and the human proton-couple folate transporter (PCFT) [ 149 ]. Interestingly, an E. coli cell-free expression system was successfully adopted for the production, purification, and crystallization of the human VDAC1 [ 150 ].…”

Development of Artificial Cell Models Using Microfluidic Technology and Synthetic Biology

“…The Kv1.1 and Kv1.3 channels were inserted into 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC)-based liposome, and the functions of these channels were observed by electrophysiological measurements [131]. A double-spanning mechanosensitive channel of large conductance (MscL) channel, a mechanosensitive channel in the inner membrane of bacteria, can be synthesized using the cell-free synthesis system of E coli.…”