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 Kf 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.