Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43

Xiaoyong Bao, Yongyue Chen, Luis Reuss, Guillermo A. Altenberg

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Gap-junctional channels are formed by two connexons or gap-junctional hemichannels in series, with each connexon conformed by six connexin molecules. As with other membrane proteins, structural information on connexons can potentially be obtained with techniques that take advantage of the highly specific thiol chemistry by positioning Cys residues at locations of interest, ideally in an otherwise Cys-less protein. It has been shown that conserved Cys residues located in the extracellular loops of connexins are essential for the docking of connexons from adjacent cells, preventing the formation of functional gap-]junctional channels. Here we engineered a Cys-less version of connexin 43 (Cx43) and assessed its function using a Xenopus oocyte expression system. The Cys-less protein was expressed at the plasma membrane and did not form gap-junctional channels but formed hemichannels that behave similarly to those formed by Cx43 in terms of permeation to carboxyfluorescein. The carboxyfluorescein permeability of Cys-less hemichannels was increased by protein kinase C inhibition, like the wild-type Cx43 hemichannels. We generated a protein with a single Cys in a position (residue 34) thought to face the channel pore and show that thiol modification of the Cys abolishes the carboxyfluorescein permeability. We conclude that Cysless Cx43 forms regulated functional hemichannels, and therefore Cys-less Cx43 is a useful tool for future structural studies.

Original languageEnglish (US)
Pages (from-to)9689-9692
Number of pages4
JournalJournal of Biological Chemistry
Volume279
Issue number11
DOIs
StatePublished - Mar 12 2004

Fingerprint

Connexin 43
Xenopus
Oocytes
Cysteine
Connexins
Sulfhydryl Compounds
Permeability
Proteins
Cell membranes
Permeation
Protein Kinase C
Membrane Proteins
Cell Membrane
Molecules
6-carboxyfluorescein

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43. / Bao, Xiaoyong; Chen, Yongyue; Reuss, Luis; Altenberg, Guillermo A.

In: Journal of Biological Chemistry, Vol. 279, No. 11, 12.03.2004, p. 9689-9692.

Research output: Contribution to journalArticle

Bao, Xiaoyong ; Chen, Yongyue ; Reuss, Luis ; Altenberg, Guillermo A. / Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 11. pp. 9689-9692.
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