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.