title: Functional expression in Xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43.
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.
authors.affiliation: Membrane Protein Laboratory, Sealy Center for Structural Biology, and Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, Texas 77555-0437, USA.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Bao
authors.forename: Yongyue
authors.initials: Y
authors.lastname: Chen
authors.forename: Luis
authors.initials: L
authors.lastname: Reuss
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Altenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018031:Connexin 43 / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D003545:Cysteine / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D014982:Xenopus laevis
chemical_list: D018031:Connexin 43; D005452:Fluoresceins; D018075:RNA, Complementary; C024098:6-carboxyfluorescein; D011493:Protein Kinase C; D003545:Cysteine