High-level expression and functional reconstitution of Shaker K+ channels.
Voltage-gated K+ channels were expressed in COS cells transiently transfected with a plasmid carrying a cDNA for an inactivation-removed Shaker K+ channel driven by an adenovirus promoter. Channel expression was followed by immunological detection, binding of radioactive charybdotoxin (CTX), and functional reconstitution into planar lipid bilayers. About 10(7) channels per transfected cell are expressed on the plasma membrane. The expressed channels are glycosylated and competent to bind CTX with the expected characteristics. Channels observed after insertion into planar lipid bilayers displayed the voltage-dependent gating, conduction, and ion selectivity behavior expected for this channel. Channels were solubilized in several detergents without loss of CTX binding activity. The results make plausible a systematic attack on the purification of milligram-level amounts of functional K+ channels from a heterologous expression system.
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Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Naini
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AA
Miller
C
C
D000256:Adenoviridae / Q000235:genetics; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018999:Charybdotoxin; D018076:DNA, Complementary / Q000235:genetics; D003902:Detergents; D005033:Ethylmaleimide / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D008051:Lipid Bilayers / Q000378:metabolism; D009994:Osmolar Concentration; D010957:Plasmids; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012604:Scorpion Venoms / Q000378:metabolism; D012995:Solubility; D014162:Transfection
D018076:DNA, Complementary; D003902:Detergents; D008051:Lipid Bilayers; D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin; D005033:Ethylmaleimide