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

Ruth A. Schwalbe; Zhiguo Wang; Barbara A. Wible; Arthur M. Brown

doi:10.1074/jbc.270.25.15336

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

From the (1)Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998

↵^§ To whom correspondence should be addressed: Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, 2500 MetroHealth Drive, Cleveland, OH 44109-1998. Tel.: 216-459-5955; Fax: 216-778-8282.

Abstract

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, p^o, 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.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants HL36930 and NS23877 (to A. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.