NOVEL SITES OF N-GLYCOSYLATION IN ROMK1 REVEAL THE PUTATIVE PORE-FORMING SEGMENT H5 AS EXTRACELLULAR

Inwardly rectifying K+ channels (IRKs) maintain resting membrane poten tial, excitability, and K+ exchange. The proposed topological model of IRKs consists of intracellular amino and carboxyl termini and two tra nsmembrane 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 v oltage-dependent K+ channels, in H5. We investigated the topology of t he 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 ge l 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 a nd the M1 segment gave results consistent with the proposed model. N-G lycosylation sites were distributed throughout H5 and its flanking reg ions indicating that H5 is mainly extracellular. Thus, the linker betw een M1 and M2 has little or no intramembranous component.