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.
Highlights
Andersen–Tawil syndrome (ATS, known as long-QT7) is a channelopathy, typically characterized by a triad of symptoms: cardiac arrhythmias, periodic paralysis, and dysmorphic features (Tawil et al, 1994)
Kir2.1 Variants V77E and M307V Are Associated With ATS
The voltagesensitive phosphatase (VSP) effect, following a 5-s depolarization step to 100 mV, was 21% ± 4% in WT dimer and 54% ± 6%, 52% ± 64%, and 43% ± 65% in mutant dimers (Figure 6G), corresponding a normalized VSP effect of 262% ± 631%, 254% ± 625%, and 226% ± 621% in WT-V77E, WTM307V, and WT-R67W, respectively (Figure 6H). These results demonstrate that the mutant dimers WT-V77E, WT-M307V, and WT-R67W have an increased sensitivity to PIP2 depletion, compared to WT dimers of Kir2.1
Summary
Andersen–Tawil syndrome (ATS, known as long-QT7) is a channelopathy, typically characterized by a triad of symptoms: cardiac arrhythmias, periodic paralysis, and dysmorphic features (Tawil et al, 1994). Variable symptom severity and incomplete penetrance are associated with the syndrome, in addition to a phenotypic mimicry of catecholaminergic polymorphic ventricular tachycardia (CPVT) characterized by bidirectional ventricular tachycardia (VT) (Barajas-Martinez et al, 2011). Pathogenic variants of KCNJ2 gene account for 60-70% of clinical ATS cases, termed type-1 ATS (Plaster et al, 2001). Most of the pathogenic variants cause Kir2.1 loss-of-function and exert a dominant-negative effect, attributed to trafficking or gating defects (Tristani-Firouzi et al, 2002; Hosaka et al, 2003; Ma et al, 2011). Type-2 ATS is caused by mutations in genes other than KCNJ2 (Kokunai et al, 2014)
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