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.

Cysteine mutagenesis for the purpose of chemical labelling was applied to the K+ channel neurotoxin charybdotoxin, a 37-residue peptide with six functionally essential cysteines. An additional 'spinster cysteine' was introduced at a position far away in space from the toxin's known interaction surface where it contacts its K+ channel receptor. Despite the presence of the extra unpaired cysteine residue, the toxin still folds efficiently and may be labelled by fluorescent and radioactive reagents to give a functionally competent toxin.