Andersen-Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1

Reem Handklo-Jamal; Eshcar Meisel; Daniel Yakubovich; Leonid Vysochek; Roy Beinart; Michael Glikson; Julie R McMullen; Nathan Dascal; Eyal Nof; Shimrit Oz

doi:10.3389/fphar.2020.00672

Andersen-Tawil Syndrome Is Associated With Impaired PIP₂ Regulation of the Potassium Channel Kir2.1

Front Pharmacol. 2020 May 15:11:672. doi: 10.3389/fphar.2020.00672. eCollection 2020.

Authors

Reem Handklo-Jamal¹, Eshcar Meisel^{1

2}, Daniel Yakubovich^{1

3}, Leonid Vysochek², Roy Beinart^{1

2}, Michael Glikson^{1

2}, Julie R McMullen⁴, Nathan Dascal¹, Eyal Nof^{1

2}, Shimrit Oz^{1

2}

Affiliations

¹ Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
² Heart Center, Sheba Medical Center, Ramat-Gan, Israel.
³ Neonatology Department, Schneider Children's Medical Center, Petah-Tikva, Israel.
⁴ Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.

Abstract

Andersen-Tawil syndrome (ATS) type-1 is associated with loss-of-function mutations in KCNJ2 gene. KCNJ2 encodes the tetrameric inward-rectifier potassium channel Kir2.1, important to the resting phase of the cardiac action potential. Kir-channels' activity requires interaction with the agonist phosphatidylinositol-4,5-bisphosphate (PIP₂). Two mutations were identified in ATS patients, V77E in the cytosolic N-terminal "slide helix" and M307V in the C-terminal cytoplasmic gate structure "G-loop." Current recordings in Kir2.1-expressing HEK cells showed that each of the two mutations caused Kir2.1 loss-of-function. Biotinylation and immunostaining showed that protein expression and trafficking of Kir2.1 to the plasma membrane were not affected by the mutations. To test the functional effect of the mutants in a heterozygote set, Kir2.1 dimers were prepared. Each dimer was composed of two Kir2.1 subunits joined with a flexible linker (i.e. WT-WT, WT dimer; WT-V77E and WT-M307V, mutant dimer). A tetrameric assembly of Kir2.1 is expected to include two dimers. The protein expression and the current density of WT dimer were equally reduced to ~25% of the WT monomer. Measurements from HEK cells and Xenopus oocytes showed that the expression of either WT-V77E or WT-M307V yielded currents of only about 20% compared to the WT dimer, supporting a dominant-negative effect of the mutants. Kir2.1 sensitivity to PIP₂ was examined by activating the PIP₂ specific voltage-sensitive phosphatase (VSP) that induced PIP₂ depletion during current recordings, in HEK cells and Xenopus oocytes. PIP₂ depletion induced a stronger and faster decay in Kir2.1 mutant dimers current compared to the WT dimer. BGP-15, a drug that has been demonstrated to have an anti-arrhythmic effect in mice, stabilized the Kir2.1 current amplitude following VSP-induced PIP₂ depletion in cells expressing WT or mutant dimers. This study underlines the implication of mutations in cytoplasmic regions of Kir2.1. A newly developed calibrated VSP activation protocol enabled a quantitative assessment of changes in PIP₂ regulation caused by the mutations. The results suggest an impaired function and a dominant-negative effect of the Kir2.1 variants that involve an impaired regulation by PIP₂. This study also demonstrates that BGP-15 may be beneficial in restoring impaired Kir2.1 function and possibly in treating ATS symptoms.

Keywords: Andersen–Tawil syndrome; BGP-15; G-loop; PIP2; cardiac arrhythmia; inward-rectifier potassium channel; slide helix; voltage-sensitive phosphatase.