SEGMENTAL EXCHANGES DEFINE 4-AMINOPYRIDINE BINDING AND THE INNER MOUTH OF K+ PORES

4-Aminopyridine (4AP) blocks the intracellular mouth of voltage-gated K+ channels. We identified critical regions for 4AP binding with chime ric channels in which segments of a low affinity clone (Kv2.1, IC50 = 18 mM) were replaced with those of a high affinity clone (Kv3.1, IC50 = 0.1 mM). 4AP sensitivity was not tranferred with the S5-S6 linker (p ore or P region). Instead, a chimera of the cytoplasmic half of S6 inc reased block 20-fold, without affecting gating. A double chimera of th e cytoplasmic halves of S5 and S6 fully transferred 4AP sensitivity. B ecause 4AP block was inhibited by tetrapentylammonium, we conclude tha t determinants of 4AP binding lie in the S6 segment that forms the cyt oplasmic vestibule of the pore and that this site may overlap a quater nary ammonium site.