title: Topology of transmembrane channel-like gene 1 protein.
abstract: Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N- and C-termini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with that of the shaker-TRP superfamily of ion channels.
authors.affiliation: Molecular Biology and Genetics Section, Otolaryngology Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, USA.
authors.forename: Valentina
authors.initials: V
authors.lastname: Labay
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Weichert
authors.forename: Tomoko
authors.initials: T
authors.lastname: Makishima
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Griffith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D014162:Transfection
chemical_list: D000906:Antibodies; D008565:Membrane Proteins; C514628:TMC1 protein, mouse