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

Camden M. Driggers, Show Ling Shyng

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

ATP-sensitive potassium (KATP) 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 KATP 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 KATP channel particles. In this chapter, we describe a method for expressing KATP channels in mammalian cell culture, solubilizing the channel in detergent micelles and purifying KATP channels using an affinity tag to the SURx subunit for cryoEM structural studies.

Original languageEnglish (US)
Title of host publicationIon Channels
Subtitle of host publicationChannel Production and Optical Methods
EditorsDaniel L. Minor, Daniel L. Minor, Daniel L. Minor, Daniel L. Minor, Daniel L. Minor, Henry M. Colecraft
PublisherAcademic Press Inc.
Pages121-150
Number of pages30
ISBN (Print)9780323853767
DOIs
StatePublished - Jan 2021
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume653
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • K channel
  • Kir6.2
  • Structure
  • Sulfonylurea receptor
  • cryoEM

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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