title: hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping.
abstract: The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.
authors.affiliation: AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zhang
authors.forename: Thai
authors.initials: T
authors.lastname: Phung
authors.forename: James
authors.initials: J
authors.lastname: Dunlop
authors.forename: Julie
authors.initials: J
authors.lastname: Dalziel
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D061987:Sf9 Cells; D018411:Spodoptera; D014701:Veratridine / Q000494:pharmacology
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D008051:Lipid Bilayers; D008081:Liposomes; D026902:Potassium Channel Blockers; D014701:Veratridine; D011188:Potassium