Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system

• Published in: Biosensors & bioelectronics 2014-09, Vol.59, p.174-183
• Main Authors: Dondapati, Srujan Kumar, Kreir, Mohamed, Quast, Robert B., Wüstenhagen, Doreen A, Brüggemann, Andrea, Fertig, Niels, Kubick, Stefan
• Format: Article
• Language: English
• Subjects: Animals; Assessments; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnology; Cell Line; Cell-free; Channels; Cloning; Cloning, Molecular; Fundamental and applied biological sciences. Psychology; Ion channel; Ion channels; KcsA; Membrane protein expression; Membranes; Microsome; Microsomes - metabolism; Potassium; Potassium Channels, Voltage-Gated - chemistry; Potassium Channels, Voltage-Gated - genetics; Potassium Channels, Voltage-Gated - metabolism; Protein Biosynthesis; Protein Multimerization; Proteins; Quantitative analysis; Single-channel current; Streptomyces lividans - chemistry; Streptomyces lividans - genetics; Streptomyces lividans - metabolism
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2014.03.004

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba2+). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient. •The potassium channel KcsA was expressed in a eukaryotic cell-free system.•KcsA expression was monitored using 14C-labeled leucine in the reaction mixture.•KcsA presented a native tetramer configuration in endogenous microsomes.•Electrophysiology measurements demonstrated the functional activity of KcsA.•Functional membrane protein could be expressed directly from linear DNA within 90min.