Loss-Of-Functional Kir6.1 KATP Channel Mutations Induce Cell Apoptosis through ROS Production and Mitochondrial Dysfunction

Abstract

INTRODUCTION: Hypoxia-induced apoptosis and arrhythmia are the important cause of sudden infant death syndrome (SIDS). ATP-sensitive K+ (KATP) channels are known to provide a functional linkage between the electrical activity of cell membrane and metabolism. The KCNJ8-encoded Kir6.1 KATP channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. Previously, we reported two KCNJ8 mutations (E332del, V346I) in a large SIDS cohort that exhibited a marked loss-of-function phenotype, reduction of cell surface expression and increased cell death. Here we further investigate the effect of the mutants on apoptosis.METHODS AND RESULTS: HEK293 cells transfected with IRES-GFP constructs containing Kir6.1-WT or mutant (Kir6.1-E332del or Kir6.1-V346I) with SUR2A in a ratio of 1:1 for 48 hrs. For apoptosis assays, cells were stained with PE-Annexin V and 7-AAD, the apoptotic cells were measured by Flow-cytometry within gated GFP (+) cells. Caspase-3/7 activity was measured with Apo-ONE Homogenous caspase 3/7 assay kit. It showed the apoptotic ratio was significantly increased for mutants Kir6-1-E332del and Kir6.1-V346I compared to Kir6.1-WT. The Caspase-3/7 activity was also increased 1.5∼2.2 folds for Kir6.1-E332del and Kir6.1-V346I over Kir6.1-WT. Furthermore, the mitochondrial superoxide levels was measured by using MitoSOX Red and the basal level of superoxide were significantly higher in mutant Kir6.1-E332del and Kir6.1-V346I compared to Kir6.1-WT (p < 0.05). The mitochondrial membrane potential of Kir6.1-E332del, Kir6.1-V346I and Kir6.1-WT transfected cells were also measured by JC-1 staining with flow cytometry analysis. The results showed that the green fluorescent signal in the mutant cells were increased 1.5 ∼2.5 folds over Kir6.1-WT, indicating the mutants induced mitochondrial membrane damage.CONCLUSIONS: The loss-of-functional Kir6.1 KATP channel mutations found in SIDS cohort induce cell apoptosis through ROS production and mitochondrial dysfunction in heterologous expression system.