title: Functional hemichannels formed by human connexin 26 expressed in bacteria.
abstract: Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: Mariana C
authors.initials: MC
authors.lastname: Fiori
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: D Marien
authors.initials: DM
authors.lastname: Cortes
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: Mauricio A
authors.initials: MA
authors.lastname: Retamal
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: Luis
authors.initials: L
authors.lastname: Reuss
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Altenberg
authors.affiliation: *Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430-6551, U.S.A.
authors.forename: Luis G
authors.initials: LG
authors.lastname: Cuello
mesh_terms: D000072259:Connexin 26; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D011994:Recombinant Proteins; D000072259:Connexin 26