Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells.

ABSTRACT: 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-Kir6.2?26. eGFP-Kir6.2?26 was expressed in HEK293?cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2?26 retains native single channel conductance (64?±?3.3?pS), mean open times (?1?=?0.72?ms, ?2?=?15.3?ms) and ATP affinity (IC50?=?115?±?25??M) 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-Kir6.2?26 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-?-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5?mol ratio. Reconstituted eGFP-Kir6.2?26 displayed similar single channel conductance (61.8?±?0.54?pS) compared to eGFP-Kir6.2?26 expressed in HEK293 membranes; however, channel mean open times increased (?1?=?7.9?ms, ?2?=?61.9?ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2?26 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.

SUBMITTER: Agasid MT 

PROVIDER: S-EPMC5864551 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature