Expression of human BK ion channels in Sf9 cells, their purification using metal affinity chromatography, and functional reconstitution into planar lipid bilayers.
This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250-300 pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals.
Dalziel
Julie E
JE
Biomembrane Laboratory, AgResearch Limited, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North 4442, New Zealand. Julie.Dalziel@agresearch.co.nz
Wong
Shu Shyan
SS
Phung
Thai
T
Zhang
Yan Li
YL
Dunlop
James
J
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008670:Metals; D009842:Oligopeptides; D024681:Potassium Channels, Calcium-Activated / Q000302:isolation & purification* / Q000378:metabolism*
C471213:His-His-His-His-His-His; D008051:Lipid Bilayers; D008670:Metals; D009842:Oligopeptides; D024681:Potassium Channels, Calcium-Activated; D006639:Histidine