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.