Production and purification of ATP-sensitive potassium channel particles for cryo-electron microscopy

Abstract

ATP-sensitive potassium (K_ATP) channels are multimeric protein complexes made of four inward rectifying potassium channel (Kir6.x) subunits and four ABC protein sulfonylurea receptor (SURx) subunits. Kir6.x subunits form the potassium ion conducting pore of the channel, and SURx functions to regulate Kir6.x. Kir6.x and SURx are uniquely dependent on each other for expression and function. In pancreatic β-cells, channels comprising SUR1 and Kir6.2 mediate glucose-stimulated insulin secretion and are the targets of antidiabetic sulfonylureas. Mutations in genes encoding SUR1 or Kir6.2 are linked to insulin secretion disorders, with loss- or gain-of-function mutations causing congenital hyperinsulinism or neonatal diabetes mellitus, respectively. Defects in the K_ATP channel in other tissues underlie human diseases of the cardiovascular and nervous systems. Key to understanding how channels are regulated by physiological and pharmacological ligands and how mutations disrupt channel assembly or gating to cause disease is the ability to observe structural changes associated with subunit interactions and ligand binding. While recent advances in the structural method of single-particle cryo-electron microscopy (cryoEM) offers direct visualization of channel structures, success of obtaining high-resolution structures is dependent on highly concentrated, homogeneous K_ATP channel particles. In this chapter, we describe a method for expressing K_ATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying K_ATP channels using an affinity tag to the SURx subunit for cryoEM structural studies.

Keywords: K(ATP) channel; Kir6.2; Structure; Sulfonylurea receptor; cryoEM.

Fig. 1

Overall Topology and CryoEM structure of the K_ATP channel. K_ATP channels are hetero-octamers of four Kir6.x subunits that form the K⁺ conducting pore and four regulatory SURx subunits. (A) Topology of SUR1 and Kir6.2 with domains labeled and colored to match the 3D structures shown below. (B) Structural model of the pancreatic K_ATP channel generated using coordinates from PDB ID 6BAA (Martin, Kandasamy, DiMaio, Yoshioka, & Shyng, 2017), with the core Kir6.2 tetramer forming the potassium ion pore (Forest green). The outer regulatory proteins SUR1 has three transmembrane domains, TMD0 (pale cyan), TMD1 (sky blue) and TMD2 (pale green), two cytoplasmic nucleotide binding domains, NBD1 (sky blue) and NBD2 (pale green), and a cytoplasmic linker L0 (teal) that connects TMD0 to the ABC core structure of the protein. Each Kir6.2 subunit has two transmembrane helices, the inner helix and outer helix, an intermembrane pore helix, and cytoplasmic N- and C-terminus. ATP is bound to Kir6.2 (yellow spheres), and the K⁺ ion path is closed. Sulfonylureas also bind and inhibit the channel, and glibenclamide (GBC) is captured bound to the channel (purple spheres). Sulfonylureas and ATP binding help stabilize the full-channel complex in the closed conformation represented in this figure.

Fig. 2

Glibenclamide and carbamazepine increase SUR1 maturation as evidenced by SUR1 glycosylation. Western blot of whole cell lysates of INS-1 clone 832/13 cells transduced with recombinant Kir6.2 plus FLAG-tagged SUR1 (f-SUR1) adenovirus and the tTA virus. SUR1, a glycoprotein, shows two bands in immunoblots: a lower core-glycosylated (immature) form that has not exited the ER and an upper complex-glycosylated (mature) band that has trafficked through the Golgi and is the form expressed in the plasma membrane. Incubation of cells with glibenclamide (GBC) or carbamazepine (CBZ) increases the levels of the mature band compared to those treated with DMSO control. This demonstrates the ability of sulfonylureas and carbamazepine to enhance expression and maturation of the K_ATP channel.

Fig. 3

Analysis of K_ATP channel post-purification by single particle imaging. (A) SDS PAGE gel of purified sample showing SUR1 (lower band: core-glycosylated and upper band: complex-glycosylated forms) and Kir6.2 as the dominant bands. (B) A blue native gel confirms the ~1 mDa (arrow) size of the (Kir6.2/SUR1)4 complex. Both of these gel images were published previously (Martin et al., 2017). (C) Two-dimensional class averages of negative-stained K_ATP channels with two side views (left) and one top/down view (right). (D) A representative negative stain micrograph at 68,000 × showing monodispersed K_ATP channel particles. (E) A typical cryoEM image of purified K_ATP channel sample on an UltrAuFoil grid used in our published studies to resolve the first Kir6.2/SUR1 structure (Martin, Yoshioka, et al., 2017). A top/down view and a side view particles are highlighted with red circles. Scale bar: 20 nm. Note the protein concentration used for negative stain was about 1/5–1/10 of that used for the cryoEM grids. Reproduced from Martin, G. M., Yoshioka, C., Rex, E. A., Fay, J. F., Xie, Q., Whorton, M. R. et al. (2017). Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating. eLife, 6. e24149. doi:10.7554/eLife.24149.