AN ENGINEERED CYSTEINE IN THE EXTERNAL MOUTH OF A K+ CHANNEL ALLOWS INACTIVATION TO BE MODULATED BY METAL-BINDING

Substitution of a cysteine in the extracellular mouth of the pore of the Shaker-Delta K+ channel permits allosteric inhibition of the channel by Zn2+ or Cd2+ ions at micromolar concentrations. Cd2+ binds weakly to the open state but drives the channel into the slow (C-type) inactivated state, which has a K-d for Cd2+ of similar to 0.2 mu M. There is a 45,000-fold increase in affinity when the channel changes from open to inactivated. These results indicate that C-type inactivation involves a structural change in the external mouth of the pore. This structural change is reflected in the T449C mutant as state-dependent metal affinity, which may result either from a change in proximity of the introduced cysteine residues of the four subunits or from a change of the exposure of this residue on the surface of the protein.