Distinct mechanisms of block of Kv1.5 channels by tertiary and quaternary amine clofilium compounds

The quaternary ammonium compound clofilium and its tertiary amine derivativ e LY97241 were used to analyze mechanisms of block in a voltage-gated potas sium channel. Wild-type and mutant Kv1.5 channels expressed in Xenopus oocy tes were recorded by two-electrode voltage clamp. Open-channel block to 20% of the control current amplitude was induced reversibly by 50 muM clofiliu m or 200 AM LY97241, and was seen as an acceleration of the macroscopic cur rent decay. Although blockers remained present after application, channels recovered from block during each interpulse interval. The optimum voltage f or recovery (-45 mV at pH 7.3) at the threshold for channel activation indi cated that clofilium block and recovery occurred principally through the op en channel state. In contrast, LY97241 appeared to exit from the closed sta te and the open state. In an acid-tolerant Kv1.5 mutant channel (H452Q), ex ternal pH was used to titrate LY97241. At low pH, which protonates the LY97 241 amine group, recovery from block at hyperpolarized potentials was impai red in a manner similar to that seen with clofilium. Recovery from clofiliu m block was reduced at negative potentials independent of pH, an effect att ributed to trapping of the permanently charged compound within the closed c hannels.