Membrane topology of the amino-terminal region of the sulfonylurea receptor.
The sulfonylurea receptor (SUR) is a member of the ATP-binding cassette family that is associated with Kir 6.x to form ATP-sensitive potassium channels. SUR is involved in nucleotide regulation of the channel and is the site of pharmacological interaction with sulfonylurea drugs and potassium channel openers. SUR contains three hydrophobic domains, TM(0), TM(1), and TM(2), with nucleotide binding folds following TM(1) and TM(2). Two topological models of SUR have been proposed containing either 13 transmembrane segments (in a 4+5+4 arrangement) or 17 transmembrane segments (in a 5+6+6 arrangement) (Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P. t., Boyd, A. E., III, González, G., Herrera-Sosa, H., Nguy, K., Bryan, J., and Nelson, D. A. (1995) Science 268, 423-426; Tusnády, G. E., Bakos, E., Váradi, A., and Sarkadi, B. (1997) FEBS Lett. 402, 1-3; Aguilar-Bryan, L., Clement, J. P., IV, González, G., Kunjilwar, K., Babenko, A., and Bryan, J. (1998) Physiol. Rev. 78, 227-245). We analyzed the topology of the amino-terminal TM(0) region of SUR1 using glycosylation and protease protection studies. Deglycosylation using peptide-N-glycosidase F and site-directed mutagenesis established that Asn(10), near the amino terminus, and Asn(1050) are the only sites of N-linked glycosylation, thus placing these sites on the extracellular side of the membrane. To study in detail the topology of SUR1, we constructed and expressed in vitro fusion proteins containing 1-5 hydrophobic segments of the TM(0) region fused to the reporter prolactin. The fusion proteins were subjected to a protease protection assay that reported the accessibility of the prolactin epitope. Our results indicate that the TM(0) region is comprised of 5 transmembrane segments. These data support the 5+6+6 model of SUR1 topology.
Raab-Graham
K F
KF
Department of Molecular, Cellular, Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
Cirilo
L J
LJ
Boettcher
A A
AA
Radeke
C M
CM
Vandenberg
C A
CA
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D006224:Cricetinae; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000737:chemistry*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D011388:Prolactin / Q000235:genetics; D011487:Protein Conformation; D011955:Receptors, Drug / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D008565:Membrane Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D064233:Sulfonylurea Receptors; D011388:Prolactin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase