Potassium Channel Structure and Function as Reported by a Single Glycosylation Sequon (∗)

• Published in: The Journal of biological chemistry 1995-06, Vol.270 (25), p.15336-15340
• Main Authors: Schwalbe, Ruth A., Wang, Zhiguo, Wible, Barbara A., Brown, Arthur M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Baculoviridae; Cell Line; Cell Membrane - physiology; Cell Membrane - ultrastructure; Consensus Sequence; DNA Primers; Glycosylation; Kidney - physiology; Membrane Potentials - drug effects; Membrane Potentials - physiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Potassium Channels - biosynthesis; Potassium Channels - chemistry; Potassium Channels - physiology; Potassium Channels, Inwardly Rectifying; Protein Conformation; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spodoptera; Transfection; Tunicamycin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.25.15336

Inwardly rectifying K+ channels (IRKs) are highly K+-selective, integral membrane proteins that help maintain resting the membrane potential and cell volume. Integral membrane proteins as a class are frequently N-glycosylated with the attached carbohydrate being extracellular and perhaps modulating function. However, dynamic effects of glycosylation have yet to be demonstrated at the molecular level. ROMK1, a member of the IRK family is particularly suited to the study of glycosylation because it has a single N-glycosylation consensus sequence (Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Herbert, S. C. (1993) Nature 362, 31-38). We show that ROMK1 is expressed in a functional state in the plasmalemma of an insect cell line (Spodoptera frugiperda, Sf9) and has two structures, glycosylated and unglycosylated. To test functionality, glycosylation was abolished by an N117Q mutation or by treatment with tunicamycin. Whole cell currents were greatly reduced in both of the unglycosylated forms compared to wild-type. Single channel currents revealed a dramatic decrease in opening probability, po, as the causative factor. Thus we have shown biochemically that the N-glycosylation sequon is extracellular, a result consistent with present topological models of IRKs, and we conclude that sequon occupancy by carbohydrate stabilizes the open state of ROMK1.