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, and two membrane-spanning segments, M1 and M2, connected by an intramembranous pore-forming segment, H5. The sequence of H5 is similar in voltage-dependent K+ channels and features a strictly conserved GY/FG in its mid-region, which has been proposed as the selectivity filter of the 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 Tyr144 and Phe146, 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 contrary to the model, we observed that position 257 was glycosylated and surrounding positions at 199, 222, and 298 were unglycosylated. N-Glycosylation 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 extracellular. The COOH terminus appears to form two additional membrane-spanning segments and to contribute to the ion conduction pathway.
Schwalbe
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The Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio 44109-1998, USA. rschwalbe@research.mhmc.org
Bianchi
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Brown
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D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D017124:Conserved Sequence; D006031:Glycosylation; D007668:Kidney / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D008961:Models, Structural; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014443:Tyrosine
D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D014415:Tunicamycin; D001464:Barium; D014443:Tyrosine; D010649:Phenylalanine