Mechanisms Underlying the Loss-Of-Functional Kir6.1 KATP Channel Mutations in Sudden Infant Death Syndrome

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. KCNJ8-encoded Kir6.1 KATP channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. Previously, we identified two KCNJ8 mutations (E332del and V346I) in a large SIDS cohort that exhibited a marked loss-of-function phenotype and reduction of cell surface expression. Here we further investigate the mechanisms underlying the loss-of-functional Kir6.1 KATP channel mutations in SIDS. Methods and Results: A hemagglutinin (HA) epitope was inserted in an extracellular loop of Kir6.1 wild type (WT), Kir6.1-E332del and Kir6.1-V346I channels. HEK293 cells were co-transfected with cDNA encoding HA-tagged Kir6.1-WT, HA-tagged Kir6.1-E332del or -V346I and SUR2A in a ratio of 1:1:2. Cell surface expression was assessed by Flow-cytometry with FITC-conjugated anti-HA antibody. Apoptosis assays were performed on HEK293 cells transfected with IRES-GFP constructs containing Kir6.1-WT or mutant (E332del or V346I) with SUR2A. After staining 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. The cell-counting studies showed that the cell surface expression of Kir6.1-WT was suppressed 40% to 70% when co-expressed with Kir6.1-E332del or Kir6.1-V346I. The apoptosis assay data indicated that the apoptotic ratio was increased significantly for E332del (38.9%) compared to WT (4.02%) and for V346I (11.2%) compared to WT (3.04%). The Caspase-3/7 activity was also increased 2.1 fold for E332del and 1.6 fold for V346I over WT. Conclusions: The loss-of-functional Kir6.1 KATP channel mutations found in SIDS display a dominant-negative effect on Kir6.1-WT channels and induce apoptosis in heterologous expression system.