Purification of Ca2+-activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla.
A method is developed for purification of the protein of the Ca2+-activated K+ channel from outer renal medulla of pig kidney. The response of this K+ channel to physiological concentrations of Ca2+ is important for regulation of transtubular NaCl transport. In reconstituted vesicles direct addition of calmodulin doubles Ca2+ activation with sufficient affinity (K1/2 0.1 nM) for chromatographic purification of the protein. For purification luminal plasma membrane vesicles are isolated on metrizamide density gradients and solubilized in CHAPS. The fraction of soluble protein retained on calmodulin-Sepharose 4B columns in the presence of Ca2+ and eluted by EGTA is 0.7%. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into phospholipid vesicles. It distributes on two bands of 51 and 36 kDa after gel electrophoresis in SDS. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation of the channel. Phosphorylation from cAMP-dependent protein kinase strongly stimulates Ca2+-activated K+ channel activity and labels the 51 kDa band suggesting that this protein is involved in regulation of K+ channel opening.
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002793:Cholic Acids; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007473:Ion Channels; D007679:Kidney Medulla / Q000032:analysis*; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D010868:Pimozide / Q000494:pharmacology; D012995:Solubility; D013552:Swine
D002148:Calmodulin-Binding Proteins; D002793:Cholic Acids; D007473:Ion Channels; D008565:Membrane Proteins; D010750:Phosphoproteins; D010868:Pimozide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium