Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1.
In this study, we compared the channel intrinsic stability of the bacterial K(+)-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at approximately 45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.
Raja
Mobeen
M
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstr 69, 4040 Linz, Austria. mraja@pmcol.ualberta.ca
Vales
Elisabeth
E
D001426:Bacterial Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying