Transmembrane topology of the sulfonylurea receptor SUR1.
Sulfonylurea receptors (SURx) are multi-spanning transmembrane proteins of the ATP-binding cassette (ABC) family, which associate with Kir6.x to form ATP-sensitive potassium channels. Two models, with 13-17 transmembrane segments, have been proposed for SURx topologies. Recently, we demonstrated that the amino-terminal region of SUR1 contains 5 transmembrane segments, supporting the 17-transmembrane model. To investigate the topology of the complete full-length SUR1, two strategies were employed. Topology was probed by accessibility of introduced cysteines to a membrane-impermeable biotinylating reagent, biotin maleimide. Amino acid positions 6/26, 99, 159, 337, 567, 1051, and 1274 were accessible, therefore extracellular, whereas many endogenous and some introduced cysteines were inaccessible, thus likely cytoplasmic or intramembrane. These sites correspond to extracellular loops 1-3, 5-6, and 8 and the NH2 terminus, and intracellular loops 3-8 and COOH terminus in the 17-transmembrane model. Immunofluorescence was used to determine accessibility of epitope-tagged SUR1 in intact and permeabilized cells. Epitopes at positions 337 and 1050 (putative external loops 3 and 6) were labeled in intact cells, therefore external, whereas positions 485 and 1119 (putative internal loops 5 and 7) only were accessible after permeabilization and therefore internal. These results are compatible with the 17-transmembrane model with two pairs of transmembrane segments as possible reentrant loops.
Conti
L R
LR
Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
Radeke
C M
CM
Shyng
S L
SL
Vandenberg
C A
CA
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001710:Biotin / Q000378:metabolism; D019556:COS Cells; D003545:Cysteine / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D001710:Biotin; D003545:Cysteine