A Novel Channelopathy in Pulmonary Arterial Hypertension

Abstract

BACKGROUND Pulmonary arterial hypertension is a devastating disease with high mortality. Familial cases of pulmonary arterial hypertension are usually characterized by autosomal dominant transmission with reduced penetrance, and some familial cases have unknown genetic causes. METHODS We studied a family in which multiple members had pulmonary arterial hypertension without identifiable mutations in any of the genes known to be associated with the disease, including BMPR2, ALK1, ENG, SMAD9, and CAV1. Three family members were studied with whole-exome sequencing. Additional patients with familial or idiopathic pulmonary arterial hypertension were screened for the mutations in the gene that was identified on whole-exome sequencing. All variants were expressed in COS-7 cells, and channel function was studied by means of patch-clamp analysis. RESULTS We identified a novel heterozygous missense variant c.608 G→A (G203D) in KCNK3 (the gene encoding potassium channel subfamily K, member 3) as a disease-causing candidate gene in the family. Five additional heterozygous missense variants in KCNK3 were independently identified in 92 unrelated patients with familial pulmonary arterial hypertension and 230 patients with idiopathic pulmonary arterial hypertension. We used in silico bioinformatic tools to predict that all six novel variants would be damaging. Electrophysiological studies of the channel indicated that all these missense mutations resulted in loss of function, and the reduction in the potassium-channel current was remedied by the application of the phospholipase inhibitor ONO-RS-082. CONCLUSIONS Our study identified the association of a novel gene, KCNK3, with familial and idiopathic pulmonary arterial hypertension. Mutations in this gene produced reduced potassium-channel current, which was successfully remedied by pharmacologic manipulation. (Funded by the National Institutes of Health.)

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery

“…Mutations were engineered into human KCNK3 cDNA in a pcDNA3.1+ expression vector by site‐directed mutagenesis using QuickChange (Stratagene) 3. Human KCNK9 cDNA in a pIRES‐GFP and pcDNA3.1+ vector was used.…”

“…GFP was co‐expressed or tagged to the C‐terminus of the channel as a marker of transfection. A previously established transfection protocol using Lipofectamine reagents was used in COS7 cells,3 with modifications to optimize efficiency in human PASMCs (hPASMCs).…”

“…For experiments in COS7 cells, solutions were prepared as previously reported3: pipette (internal) solution (in mmol/L) contained: 150 KCl, 3 MgCl2, 5 EGTA, 10 HEPES, adjusted to pH 7.2 with KOH. Bath (extracellular) solution (in mmol/L) contained: 150 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES adjusted to pH 6.4, 7.4, or 8.4 with NaOH.…”

“…We recently identified KCNK3 as the first ion channelopathy associated with PAH 3. Using exome sequencing, 6 distinct mutations in the KCNK3 potassium channel were identified in patients with hereditary PAH.…”

“…Notably, KCNK3 downregulation was recently identified as a pathogenic hallmark of PAH in humans and in a monocrotaline‐induced pulmonary hypertension rat model, and administration of ONO alleviated signs of pulmonary hypertension in the animal model, further implicating KCNK3 as a therapeutic target in PAH 3, 8…”