Locked-Open Activation Gate Prevents the Recovery of Shaker K+ Channels from Slow Inactivation

Abstract

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation. It has been shown earlier that Cd2+ traps the V476C Shaker channels in the open state, even at very negative voltages, by forming a metal bridge between a cysteine in one subunit and a native histidine (H486) in a neighboring subunit (Webster et al., Nature, 2004). However, none of the previous studies determined the relationship between inactivation/recovery from inactivation and the locked-open activation gate. The current experiments tested the hypothesis that locking the activation gate in the open configuration prevents recovery from inactivation. To address this hypothesis we compared the extent of recovery from inactivation for control conditions and in the presence of 20 µM Cd2+. V476C/IR channels contained an alanine in position 449 to facilitate the entry of the channels into the slow-inactivated state. All ionic current experiments were performed with excised inside-out patches. A fast-step perfusion system was used for rapid solution exchange. 2.0-s-long depolarizing pulses from a holding potential of −120 mV to +50 mV were applied and when applicable, 20 µM Cd2+ was added to the fully inactivated channels. The duration of the Cd2+ pulse was 1.0 s long which was started 800 ms after the start of the depolarization. Under control conditions 100% of the channels recovered from inactivation within 60 s at −120 mV, whereas upon Cd2+ application less than 10 % of the current recovered under identical conditions (n=3). The lack of recovery from inactivation of the locked-open channels suggests that closure of the activation gate is essential for the recovery from slow inactivation.