Novel sites of N-glycosylation in ROMK1 reveal the putative pore-forming segment H5 as extracellular.
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.
Schwalbe
R A
RA
Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998, USA.
Wang
Z
Z
Bianchi
L
L
Brown
A M
AM
D000818:Animals; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space; D006031:Glycosylation / Q000187:drug effects; D015640:Ion Channel Gating; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000648:ultrastructure; D017924:Nucleopolyhedroviruses; D015221:Potassium Channels / Q000737:chemistry* / Q000648:ultrastructure; D024661:Potassium Channels, Inwardly Rectifying; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014415:Tunicamycin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D014415:Tunicamycin