A P-helix Mutant In A Shaker-type Kv Channel Converts The Inactivated State Into A Conducting One

Abstract

Voltage-gated potassium (Kv) channels are tetramers of α-subunits, each composed out of six membrane-spanning helices (S1-S6) with a pore loop between S5 and S6 that forms the channel's selectivity filter. The activation gate that seals off the ion conducting pore in a closed channel is controlled by the transmembrane potential. After channel activation most Kv channels display C-type inactivation, a process that is believed to involve reorientations of the selectivity filter and results in a non-conducting channel although the channel gate is open. hKv1.5 (a Shaker-type Kv channel) displays such C-type inactivation. Here we report that an alanine substitution for residue T480 that is located at the end of the pore-helix prevents hKv1.5 channels from entering the inactivated state. The mutant T480A had an isochronal activation curve similar to Wild Type hKv1.5 when determined with 250ms depolarizing steps. Longer depolarizations (∼5 seconds) caused WT channels to inactivate (∼58%) displaying an inactivation curve with a midpoint of −23.2 ± 1.2 mV and a slope factor of about 4. However, T480A did not inactivate and such long depolarizations caused an additional (slow) activation at more negative potentials thus generating an isochronal activation curve with properties that were similar to the inactivation process in WT channels. To investigate the structural changes that underlie the unusual behaviour of this mutant, we performed a series of MD simulations of the pore domain of WT hKv1.5 and T480A. Analysis of the trajectories shows that T480A affects the stability and flexibility of the filter region and the surrounding pore loop. These results show that residue T480 (located outside the pore region that determines the integrity of the selectivity filter) affects the stability of the filter and influences C-type inactivation.