Membrane Topology of the Amino-terminal Region of the Sulfonylurea Receptor

• Published in: The Journal of biological chemistry Vol. 274; no. 41; pp. 29122 - 29129
• Main Authors: Raab-Graham, Kimberly F., Cirilo, Laura J., Boettcher, Anne A., Radeke, Carolyn M., Vandenberg, Carol A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.10.1999; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; ATP-Binding Cassette Transporters; Cricetinae; Glycosylation; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Membrane Proteins - chemistry; Mutagenesis, Site-Directed; Potassium Channels - chemistry; Potassium Channels, Inwardly Rectifying; Precipitin Tests; Prolactin - genetics; Protein Conformation; Receptors, Drug - chemistry; Recombinant Fusion Proteins - chemistry; Sulfonylurea Receptors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.41.29122

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, TM0, TM1, and TM2, with nucleotide binding folds following TM1 and TM2. 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 TM0 region of SUR1 using glycosylation and protease protection studies. Deglycosylation using peptide-N-glycosidase F and site-directed mutagenesis established that Asn10, near the amino terminus, and Asn1050 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 TM0 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 TM0 region is comprised of 5 transmembrane segments. These data support the 5+6+6 model of SUR1 topology.