Interrogating permeation and gating of Orai channels using chemical modification of cysteine residues

Megumi Yamashita, Murali Prakriya*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Chemical modification of ion channels using the substituted cysteine accessibility method has a rich and successful history in elucidating the structural basis of ion channel function. In this approach, cysteine residues are introduced in regions of interest into the protein and their accessibility to water soluble thiol-reactive reagents is determined by monitoring ion channel activity. Because a wide range of these reagents are available with differing size, charge, and membrane solubility, the physio-chemical environment of the introduced cysteine residue and therefore the protein domain of interest can be probed with great precision. The approach has been widely employed for determining the secondary structure of specific ion channel domains, the location and nature of the channel gate, and the conformational rearrangements in the channel pore that underlie the opening/closing of the pore. In this chapter, we describe the use of these and related approaches to probe the functional architecture and gating of store-operated Orai1 channels.

Original languageEnglish (US)
Title of host publicationIon Channels
Subtitle of host publicationChannel Biochemistry, Reconstitution, and Function
EditorsDaniel L. Minor, Daniel L. Minor, Daniel L. Minor, Daniel L. Minor, Daniel L. Minor, Henry M. Colecraft
PublisherAcademic Press Inc
Pages213-239
Number of pages27
ISBN (Print)9780323853743
DOIs
StatePublished - Jan 2021

Publication series

NameMethods in Enzymology
Volume652
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Funding

The work described here was supported by NIH grant NS057499.

Keywords

  • CRAC channels
  • Cysteine accessibility analysis
  • Gating
  • Ion channel
  • Ion permeation
  • Orai1
  • Pore residues
  • STIM1

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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