The a-Dendrotoxin Footprint on a Mammalian Potassium Channel*

a-Dendrotoxin, a 59-amino acid basic peptide from the venom of Dendroaspis angusticeps (green mamba snake), potently blocks some but not all voltage-dependent potassium channels. Here we have investigated the relative contribution of the individual a-subunits constituting functional Kv1.1 potassium channels to a-dendrotoxin binding. Three residues critical for a-dendrotoxin binding and located in the loop between domains S5 and S6 were mutated (A352P, E353S, and Y379H), and multimeric cDNAs were constructed encoding homoand heterotetrameric channels composed of all possible ratios of wild-type andmutant a-subunits. Completemutant channels were about 200-fold less sensitive for the a-dendrotoxin block than complete wild-type channels, which is attributable to a smaller association rate. Analysis of the bimolecular reaction between a-dendrotoxin and the different homoand heteromeric channel constructs revealed that the association rate depends on the number of wild-type a-subunits in the functional channel. Furthermore, we observed a linear relationship between the number of wild-type a-subunits in functional channels and the free energy for a-dendrotoxin binding, providing evidence that all four a-subunits must interact with a-dendrotoxin to produce a high affinity binding site.