Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

Karina Eckey,Eva Wrobel,Nathalie Strutz-Seebohm,Lutz Pott,Nicole Schmitt,Guiscard Seebohm

doi:10.1074/jbc.m114.589796

Abstract

Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.

Highlights

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates a variety of ion channels including Kv7 channels
Because oocyte batches may vary in their endogenous PIP2 levels and binding sites may be occupied to different extents, we included only those batches in which increased WT currents were observed after PIP2 application
The amplitudes at 40 mV were normalized to the respective current without the PIP2 analog diC8-PIP2 of each individual batch (Fig. 2B). diC8-PIP2 significantly increased the WT (Ϫcys) current amplitude to ϳ160% on average, as well as the amplitudes of four mutants shown in gray (H126C, R195C, K196C, and K358C)

Summary

Introduction

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates a variety of ion channels including Kv7 channels. Conclusion: PIP2 interacts with several Kv7.1 amino acids, indicating the existence of at least two binding pockets. Significance: Impaired molecular interaction between mutant Kv7.1 and PIP2 may underlie heart disease. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. We identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding

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