Purification of Ca2+‐activated K+ channel protein on calmodulin affinity columns after detergent solubilization of luminal membranes from outer renal medulla

FEBS Letters

Petersen, Janne¹  Klærke, Dan A.¹  Jørgensen, Peter L.¹ 

[1] Institute of Physiology, Aarhus University, 8000 Aarhus C, Denmark

关键词: Ca2+ activation;    K+ channel;    Calmodulin inhibitor;    Calmodulin;    Affinity chromatography;    Phosphorylation;    cyclic AMP dependence;    Protein kinase;    ATP;    adenosine 5′-triphosphate;    CHAPS;    3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate;    DTT;    DL-dithiothreitol;    metrizamide;    2-(3-acetamido-5-N-methylacetamido-2;    4;    6-triiodobenzamido)-2-deoxy-D-glucose;    Mops;    3-(N-morpholino)-propanesulfonic acid;    PMSF;    phenylmethylsulfonyl fluoride;    TAL;    thick ascending limb of Henle's loop;    TFP;    trifluoperazine;   

DOI  :  10.1016/0014-5793(87)80691-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 (K 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.

【 授权许可】

Unknown