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

Protein Expr Purif. 2018 Jun:146:61-68. doi: 10.1016/j.pep.2018.01.015. Epub 2018 Feb 7.

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-Kir6.2Δ26. 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.

Keywords: Black lipid membrane; Electrophysiology; Kir6.2; Recombinant ion channel; Reconstitution.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Chromatography, Affinity
  • Chromatography, Gel
  • Gene Expression
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / isolation & purification
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Lipid Bilayers / metabolism
  • Patch-Clamp Techniques
  • Potassium Channels, Inwardly Rectifying / analysis
  • Potassium Channels, Inwardly Rectifying / genetics*
  • Potassium Channels, Inwardly Rectifying / isolation & purification
  • Potassium Channels, Inwardly Rectifying / metabolism*
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Solubility
  • Transfection / methods

Substances

  • Kir6.2 channel
  • Lipid Bilayers
  • Potassium Channels, Inwardly Rectifying
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Adenosine Triphosphate