TY - JOUR
T1 - Transmembrane Topology of the Sulfonylurea Receptor SUR1
AU - Conti, Lisa R.
AU - Radeke, Carolyn M.
AU - Shyng, Show Ling
AU - Vandenberg, Carol A.
PY - 2001/11/2
Y1 - 2001/11/2
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M106555200
DO - 10.1074/jbc.M106555200
M3 - Article
C2 - 11546780
AN - SCOPUS:0035798623
SN - 0021-9258
VL - 276
SP - 41270
EP - 41278
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 44
ER -