%A Handklo-Jamal,Reem %A Meisel,Eshcar %A Yakubovich,Daniel %A Vysochek,Leonid %A Beinart,Roy %A Glikson,Michael %A McMullen,Julie R. %A Dascal,Nathan %A Nof,Eyal %A Oz,Shimrit %D 2020 %J Frontiers in Pharmacology %C %F %G English %K Andersen-Tawil syndrome,cardiac arrhythmia,Inward-rectifier potassium channel,slide-helix,G-loop,voltage-sensitive phosphatase,PIP2 – phosphatidylinositol-4,5-bisphosphate %Q %R 10.3389/fphar.2020.00672 %W %L %M %P %7 %8 2020-May-15 %9 Original Research %+ Shimrit Oz,Sackler School of Medicine, Tel Aviv University,Israel,shimrit.oz.fr@gmail.com %+ Shimrit Oz,Heart Center, Sheba Medical Center,Israel,shimrit.oz.fr@gmail.com %# %! Regulation of Kir2.1 in Andersen-Tawil syndrome %* %< %T Andersen–Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1 %U https://www.frontiersin.org/articles/10.3389/fphar.2020.00672 %V 11 %0 JOURNAL ARTICLE %@ 1663-9812 %X 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.