Block of the inactivating potassium channel by clofilium and hydroxylamine depends on the sequence of the pore region.

Cardiac antiarrhythmic compounds are a diverse group divided into classes that differ in their mechanisms of action. Recent attention has focused on class III compounds, which prolong the action potential by blocking K+ channels. The purpose of this study was to characterize the mechanisms of actions of a class III compound, clofilium, and a simple analog, hydroxylamine, on an inactivating K+ channel. The defined system used a cloned inactivating K+ channel (Shaker-B) expressed in Xenopus oocytes. This channel is similar in physiological properties and core sequence to the inactivating K+ channel cloned from mammalian heart. Results presented here demonstrate that clofilium (100 microM) and hydroxylamine (10 mM) can cause use-dependent block, depending on the sequence of the pore region. A mutation of the pore known to influence selectivity and tetraethylammonium binding (threonine-441 to serine) confers use-dependent sensitivity to hydroxylamine and clofilium. Hybrid channels were formed from the coinjection of wild-type and mutant channel mRNAs; the analysis of block with the hybrid channels suggests that binding of hydroxylamine involves all subunits of the tetrameric channel, whereas clofilium affects channels containing as few as one mutant subunit. The simplest interpretation is that all four subunits contribute to an internal binding site for blockers such as clofilium and hydroxylamine and threonine-441 influences this binding site. The effectiveness of clofilium, unlike hydroxylamine, on the hybrid channels may reflect its structural complexity, which could allow interaction with a broader receptor site. Future studies will test this idea using other class III-related compounds.