title: Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.
abstract: Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.
authors.affiliation: From the Biology Department, State University of New York Buffalo State, Buffalo, New York 14222.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Brennan
authors.affiliation: From the Biology Department, State University of New York Buffalo State, Buffalo, New York 14222.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Karcz
authors.affiliation: From the Biology Department, State University of New York Buffalo State, Buffalo, New York 14222.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Vaughn
authors.affiliation: the Clinical and Translational Research Center, State University of New York at Buffalo, Buffalo, New York 14214.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Woolwine-Cunningham
authors.affiliation: the Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263.
authors.forename: Adam D
authors.initials: AD
authors.lastname: DePriest
authors.affiliation: the Computational Biology Lab, Fundación Ciencia and Vida, 7780344 Santiago, Chile, and the Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, 2360102 Valparaíso, Chile.
authors.forename: Yerko
authors.initials: Y
authors.lastname: Escalona
authors.affiliation: the Computational Biology Lab, Fundación Ciencia and Vida, 7780344 Santiago, Chile, and the Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, 2360102 Valparaíso, Chile.
authors.forename: Tomas
authors.initials: T
authors.lastname: Perez-Acle
authors.affiliation: From the Biology Department, State University of New York Buffalo State, Buffalo, New York 14222, skerreim@buffalostate.edu.
authors.forename: I Martha
authors.initials: IM
authors.lastname: Skerrett
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D018031:Connexin 43 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D017629:Gap Junctions / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014982:Xenopus laevis; D000097002:Gap Junction beta-1 Protein
chemical_list: D018031:Connexin 43; D017630:Connexins; D008051:Lipid Bilayers; D021122:Protein Subunits; D011994:Recombinant Proteins; D014364:Tryptophan; D000097002:Gap Junction beta-1 Protein; C580592:Gja1 protein, rat
keywords: Cx32; TM domain interactions; comparative model; connexin; gap junction; gating; hemichannel; mutagenesis; transmembrane domain; tryptophan scanning