RESUMO
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.
RESUMO
Mutations in the SCN5A gene, encoding the cardiac voltage-gated sodium channel NaV1.5, are associated with inherited cardiac arrhythmia and conduction disease. Ca2+-dependent mechanisms and the involvement of ß-subunit (NaVß) in NaV1.5 regulation are not fully understood. A patient with severe sinus-bradycardia and cardiac conduction-disease was genetically evaluated and compound heterozygosity in the SCN5A gene was found. Mutations were identified in the cytoplasmic DIII-IV linker (K1493del) and the C-terminus (A1924T) of NaV1.5, both are putative CaM-binding domains. These mutants were functionally studied in human embryonic kidney (HEK) cells and HL-1 cells using whole-cell patch clamp technique. Calmodulin (CaM) interaction and cell-surface expression of heterologously expressed NaV1.5 mutants were studied by pull-down and biotinylation assays. The mutation K1493del rendered NaV1.5 non-conductive. NaV1.5K1493del altered the gating properties of co-expressed functional NaV1.5, in a Ca2+ and NaVß1-dependent manner. NaV1.5A1924T impaired NaVß1-dependent gating regulation. Ca2+-dependent CaM-interaction with NaV1.5 was blunted in NaV1.5K1493del. Electrical charge substitution at position 1493 did not affect CaM-interaction and channel functionality. Arrhythmia and conduction-disease -associated mutations revealed Ca2+-dependent gating regulation of NaV1.5 channels. Our results highlight the role of NaV1.5 DIII-IV linker in the CaM-binding complex and channel function, and suggest that the Ca2+-sensing machinery of NaV1.5 involves NaVß1.