Site-directed glycosylation tagging of functional Kir2.1 reveals that the putative pore-forming segment is extracellular.
Inwardly rectifying K+ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K+ channels, Kvs, and this stretch is called the signature sequence of K+ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K+ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation sites, NX(S/T), at positions Ser(128) in E1, and Gln(140), Ileu(143), and Phe(147) in the H5 sequence of a strong inwardly rectifying K+ channel, Kir2.1. Furthermore, we show that biotinylated channel proteins with N-linked oligosaccharides attached at positions 140 and 143 in the signature sequence are located at the cell surface. Mutant channels were functional as detected by whole-cell and single-channel recordings. Unlike Kir1.1, position Lys(117) was not occupied. We conclude that, for yet another K+ channel, the invariant G(Y/F)G sequence is extracellular rather than intramembranous.
Schwalbe
Ruth A
RA
Division of Nephrology, Hypertension, and Transplantation, University of Florida, and the Nephrology Section, Department of Veterans Affairs Medical Center, Gainesville, Florida 32610, USA. schwara@medicine.ufl.edu
Rudin
Alicia
A
Xia
Shen-Ling
SL
Wingo
Charles S
CS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D005973:Glutamine; D006031:Glycosylation; D007532:Isoleucine; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D016133:Polymerase Chain Reaction; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D018411:Spodoptera; D014162:Transfection
D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D007532:Isoleucine; D005973:Glutamine; D012694:Serine; D010649:Phenylalanine