A method for assessing ionic and molecular permeation in connexin hemichannels

Pablo S. Gaete, Jorge E. Contreras

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Connexin hemichannels are permeable to both atomic ions and small molecules. Yet, they have different selectivity for ions and signaling molecules critical for biological functions. Activity of connexin hemichannels in living cells is commonly evaluated by methods that include electrophysiology and fluorescence-based approaches. Although less common, luminescence and radioactivity-based uptake/release assays have been also successfully used to determine selectivity and permeability to different molecules. The current methods, however, have important technical and quantitative limitations that make them unsuitable for simultaneously evaluating ionic and molecular permeability using different stimuli that control channel gating (e.g., voltage or extracellular Ca2 +). To address this, we have recently designed a novel methodology that combines two-electrode voltage clamp (TEVC) and dye uptake assays in translucent Xenopus oocytes. This method allows for the evaluation of molecular transport kinetics in connexin hemichannels, and its utility can also be extended to other large pore channels, such as those formed by pannexin and CALHM. In this article, we describe step by step the protocol to perform the TEVC/Dye uptake assay.

Original languageEnglish (US)
Title of host publicationIon Channels
Subtitle of host publicationChannel Chemical Biology, Engineering, and Physiological Function
EditorsDaniel L. Minor, Henry M. Colecraft
PublisherAcademic Press Inc.
Pages271-293
Number of pages23
ISBN (Print)9780128239247
DOIs
StatePublished - Jan 2021
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume654
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • CALHM
  • Connexin
  • Large-pore channel
  • Molecular transport
  • Pannexin
  • Permeability assay
  • TEVC/Dye uptake assay

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