Novel sites of N-glycosylation in ROMK1 reveal the putative pore- forming segment H5 as extracellular

Ruth A. Schwalbe, Zhiguo Wang, Laura Bianchi, Arthur M. Brown

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Inwardly rectifying K + channels (IRKs) maintain resting membrane potential, excitability, and K + exchange. The proposed topological model of IRKs consists of intracellular amino and carboxyl termini and two transmembrane segments (M1 and M2) linked by a pore-forming segment (H5). Structure-function studies have identified critical pore determinants in M2 and the carboxyl terminus but not as expected by analogy with voltage- dependent K + channels, in H5. We investigated the topology of the IRK ROMK1 by substituting novel N-glycosylation sites which act as markers for extracellular segments. N-Glycosylation, before and after an N-glycosylation inhibitor, tunicamycin, was measured directly by gel shift assays and changes in membrane currents. Tunicamycin produced gel shifts and changes in membrane currents that correlated exactly. N-Glycosylation sites substituted into the amino and carboxyl termini and the M1 segment gave results consistent with the proposed model. N-Glycosylation sites were distributed throughout H5 and its flanking regions indicating that H5 is mainly extracellular. Thus, the linker between M1 and M2 has little or no intramembranous component.

Original languageEnglish
Pages (from-to)24201-24206
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number39
DOIs
StatePublished - Oct 10 1996
Externally publishedYes

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Glycosylation
Tunicamycin
Membranes
Gels
Inwardly Rectifying Potassium Channel
Membrane Potentials
Assays
Topology
Electric potential

ASJC Scopus subject areas

  • Biochemistry

Cite this

Novel sites of N-glycosylation in ROMK1 reveal the putative pore- forming segment H5 as extracellular. / Schwalbe, Ruth A.; Wang, Zhiguo; Bianchi, Laura; Brown, Arthur M.

In: Journal of Biological Chemistry, Vol. 271, No. 39, 10.10.1996, p. 24201-24206.

Research output: Contribution to journalArticle

Schwalbe, Ruth A. ; Wang, Zhiguo ; Bianchi, Laura ; Brown, Arthur M. / Novel sites of N-glycosylation in ROMK1 reveal the putative pore- forming segment H5 as extracellular. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 39. pp. 24201-24206.
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