Referenced in: none

Automatically associated channels: Kir1.1

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

Authors: R A Schwalbe, Z Wang, L Bianchi, A M Brown

Journal, date & volume: J. Biol. Chem., 1996 Sep 27 , 271, 24201-6

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/8798662

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.