Purification and reconstitution of functional Shaker K+ channels assayed with a light-driven voltage-control system.
Voltage-dependent potassium channels are integral membrane proteins that control the excitability of nerve and muscle. The cloning of genes for K+ channels has led to structure/function analysis using a combination of site-directed mutagenesis and electrophysiology. As a result, much has been learned about how these proteins work. A deeper understanding of their function will require detailed structural characterization, however. We now report the purification of Shaker K+ channels from an insect expression system using immunoaffinity methods. The purified channels have been reconstituted, assayed using a novel, light-driven, vesicular voltage-control system, and shown to be functional. This approach will enable us to compare and optimize methods for protein production and purification. Purification of active protein is a prerequisite for detailed structural analysis, since activity is the key indication that the structural integrity of the channel has been preserved during biochemical procedures. Thus, this work represents a first step toward the determination of the structure of Shaker K+ channels.
Santacruz-Toloza
L
L
Department of Physiology (School of Medicine), University of California at Los Angeles 90024-1751.
Perozo
E
E
Papazian
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001436:Bacteriorhodopsins / Q000502:physiology; D016367:Baculoviridae / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D004331:Drosophila melanogaster / Q000235:genetics; D004553:Electric Conductivity; D004563:Electrochemistry; D008027:Light; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D015221:Potassium Channels / Q000302:isolation & purification* / Q000502:physiology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012414:Rubidium Radioisotopes / Q000378:metabolism; D014162:Transfection
D008081:Liposomes; D015221:Potassium Channels; D011994:Recombinant Proteins; D012414:Rubidium Radioisotopes; D001436:Bacteriorhodopsins