Functional expression in xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43

doi:10.1074/jbc.M311438200

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Papers In Press, published online ahead of print December 14, 2003
J. Biol. Chem, 10.1074/jbc.M311438200
Submitted on October 17, 2003
Revised on December 9, 2003
Accepted on December 14, 2003

Functional expression in xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43

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

Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0437

Corresponding Author: galtenbe{at}utmb.edu

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 be potentially 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, 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 PKC 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 Cys-less Cx43 forms regulated functional hemichannels, and therefore it is a useful tool for future structural studies.

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