A point mutation turns an inactivated potassium channel into a hyperpolarization activated cation channel.

Abstract

In potassium channels, the slow inactivation (C-type) process decreases the ionic conduction by affecting the selectivity filter. The W434F mutation in Shaker potassium channel stabilizes it in the inactivated conformation. The recent determination of the structure of the Shaker W434F channel shows a dilated selectivity filter, compared to Shaker WT structure, reminiscent to the hyperpolarization activated cyclic nucleotide gated channel (HCN). This suggests a common link between inactivation and hyperpolarization gating, which is explored in this work. In Shaker the P475D (Sh-P475D) is a mutation in the bundle crossing region that stabilizes the activation gate in the open conformation over a wide voltage range (−140 mV to +100). Sh-P475D was expressed in Xenopus oocytes and inactivation was favored by removing external potassium. Under these conditions, the channel displays robust hyperpolarization activated currents reminiscent of HCN. (Similar currents are observed when the W434F mutation is added in Sh-P475D background.) The currents elicited by hyperpolarizing voltages in the Sh-P475D mutant are non-selective (PK/PNa∼1) and even N-methyl-D-glucamine and calcium are permeable in this mutant. We analyzed the role of residues known to affect pore inactivation, the contribution of the selectivity filter, and the influence of the voltage sensor movement on the kinetics and steady state characteristics of the P475D-induced hyperpolarization activated currents. Our results suggest a mechanism of hyperpolarization gating and inactivation at the selectivity filter that seems to be common among voltage gated cation channels. Supported by NIH GM03076.