MAPPING THE KIDNEY POTASSIUM CHANNEL ROMK1 - GLYCOSYLATION OF THE PORE SIGNATURE SEQUENCE AND THE COOH TERMINUS

ROMK1, also known as Kir 1.1, is an inwardly rectifying K+ channel and is the prototypical member of the large Kir gene family. The accepted model of Kir topology predicts intracellular NH2 and COOH termini, an d two membrane-spanning segments, M1 and M2, connected by an intramemb ranous pore-forming segment, H5. The sequence of H5 is similar in volt age-dependent K+ channels and features a strictly conserved GY/FG in i ts mid-region, which has been proposed as the selectivity filter of th e pore. We have been using N-glycosylation substitution mutants to map the extracellular topology of ROMK1 biochemically and have described several loci in H5 that were glycosylated. We now report glycosylation at loci Tyr(144) and Phe(146), which indicates that the signature GYG sequence (143-145) rather than being intramembranous is extracellular , The COOH terminus was predicted to begin at position 178, but contra ry to the model, we observed that position 257 was glycosylated and su rrounding positions at 199, 222, and 298 were unglycosylated. N-Glycos ylation sequon substitution at the latter three positions abolished K/Na+ selectivity. Our results suggest a major revision of the topology of ROMK1 with H5 and the pore signature sequence now completely extra cellular. The COOH terminus appears to form two additional membrane-sp anning segments and to contribute to the ion conduction pathway.