TY - JOUR
T1 - Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells
AU - Agasid, Mark T.
AU - Wang, Xuemin
AU - Huang, Yiding
AU - Janczak, Colleen M.
AU - Bränström, Robert
AU - Saavedra, S. Scott
AU - Aspinwall, Craig A.
N1 - Funding Information:
The authors would like to thank Joseph Bryan (Pacific Northwest Research Institute, WA) for the generous gift of Kir6.2 cDNA, Dr. Paula Campbell and Dr. John Finch at the University of Arizona UACC/ARL Cytometry Core Facility for sorting transfected cells, and Dr. Christopher Atcherley for assistance with with LabView data collection programming. Research reported in this publication was partially supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Numbers R01EB007047 and R21EB022297 , the Office of the Director, National Institutes of Health under award number S10OD013237 , and the Cancer Center Support Grant (CCSG - CA 023074 ). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2018 Elsevier Inc.
PY - 2018/6
Y1 - 2018/6
N2 - The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ± 3.3 pS), mean open times (τ1 = 0.72 ms, τ2 = 15.3 ms) and ATP affinity (IC50 = 115 ± 25 μM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ± 0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ1 = 7.9 ms, τ2 = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC50 = 3.14 ± 0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.
AB - The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ± 3.3 pS), mean open times (τ1 = 0.72 ms, τ2 = 15.3 ms) and ATP affinity (IC50 = 115 ± 25 μM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), L-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ± 0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ1 = 7.9 ms, τ2 = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC50 = 3.14 ± 0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.
KW - Black lipid membrane
KW - Electrophysiology
KW - Kir6.2
KW - Recombinant ion channel
KW - Reconstitution
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U2 - 10.1016/j.pep.2018.01.015
DO - 10.1016/j.pep.2018.01.015
M3 - Article
C2 - 29409958
AN - SCOPUS:85041660514
VL - 146
SP - 61
EP - 68
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -