Conserved extracellular cysteine residues in the inwardly rectifying potassium channel Kir2.3 are required for function but not expression in the membrane.
The mouse potassium channel Kir2.3 possesses conserved extracellular cysteine residues at positions 113 and 145. We have investigated the role of these cysteines in structure/function and membrane trafficking. Cysteine to serine mutations resulted in the absence of potassium currents in oocytes and co-expression of these mutants with wild-type channel showed a dominant negative inhibition of wild-type currents. FLAG-tagged channels expressed in oocytes were detected in the cell membrane by anti-FLAG antibody for wild-type and mutant channels. In vitro translation using the reticulocyte lysate system showed that mutation of these residues did not affect processing nor insertion into membranes. Cysteine residues at 113 and 145 are therefore required for function of the Kir2.3 channel but not for processing into the cell membrane; disulfide bonds between subunits are unlikely.
Bannister
J P
JP
School of Biomedical Sciences, University of Leeds, UK.
Young
B A
BA
Sivaprasadarao
A
A
Wray
D
D
D000818:Animals; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice; D008845:Microinjections; D009154:Mutation; D009842:Oligopeptides; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000235:genetics; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D014176:Protein Biosynthesis; D012333:RNA, Messenger; D012156:Reticulocytes; D014981:Xenopus
C490969:Kcnj4 protein, mouse; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; C085706:FLAG peptide; D003545:Cysteine