A conducting state with properties of a slow inactivated state in a ShakerK+ channel mutant

In Shaker K+ channel, the amino terminus deletion Delta6-46 removes fast in activation (N-type) unmasking a slow inactivation process. In Shaker Delta6 -46 (Sh-IR) background, two additional mutations (T449V-I470C) remove slow inactivation, producing a noninactivating channel. However, despite the fac t that Sh-IR-T449V-I470C mutant channels remain conductive, prolonged depol arizations (1 min, 0 mV) produce a shift of the QV curve by about -30 mV su ggesting that the channels still undergo the conformational changes typical of slow inactivation. For depolarizations longer than 50 ms, the tail curr ents measured during repolarization to -90 mV display a slow component that increases in amplitude as the duration of the depolarizing pulse increases . We found that the slow development of the QV shift had a counterpart in t he amplitude of the slow component of the ionic tail current that is not pr esent in Sh-IR. During long depolarizations, the time course of both the in crease in the slow component of the tail current and the change in voltage dependence of the charge movement could be well fitted by exponential funct ions with identical time constant of 459 Ins. Single channel recordings rev ealed that after prolonged depolarizations, the channels remain conductive for long periods after membrane repolarization. Nonstationary autocovarianc e analysis performed on macroscopic current in the T449V-I470C mutant confi rmed that a novel open state appears with increasing prepulse depolarizatio n time. These observations suggest that in the mutant studied, a new open s tate becomes progressively populated during long depolarizations (>50 ms). An appealing interpretation of these results is that the new open state of the mutant channel corresponds to a slow inactivated state of Sh-IR that be came conductive.