Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel

Methods Mol Biol. 2018:1843:147-166. doi: 10.1007/978-1-4939-8704-7_13.

Abstract

ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Here we describe a convenient yet powerful cross-linking approach to examine the pore architecture of CRAC channels using ORAI1 proteins engineered to contain one or two cysteine residues. The generalizable cross-linking in situ approach can also be readily extended to study other integral membrane proteins expressed in various types of cells.

Keywords: Calcium release-activated calcium channel; Calcium signaling; Cross-linking; Disulfide bond; Lymphocyte activation; Membrane protein; ORAI1; Store-operated calcium entry; Stromal interaction molecule; Structure.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calcium
  • Calcium Release Activated Calcium Channels / chemistry*
  • Calcium Release Activated Calcium Channels / physiology*
  • Calcium Signaling
  • Cloning, Molecular
  • Cysteine
  • Gene Expression
  • Genetic Engineering
  • HEK293 Cells
  • Humans
  • Ion Channel Gating
  • Mutagenesis, Site-Directed
  • Plasmids / genetics
  • Protein Multimerization
  • Structure-Activity Relationship

Substances

  • Calcium Release Activated Calcium Channels
  • Cysteine
  • Calcium