Characterization and regulation of wild‐type and mutant TASK‐1 two pore domain potassium channels indicated in pulmonary arterial hypertension

Abstract

Key points The TASK‐1 channel gene (KCNK3) has been identified as a possible disease‐causing gene in heritable pulmonary arterial hypertension (PAH). In the present study, we show that novel mutated TASK‐1 channels, seen in PAH patients, have a substantially reduced current compared to wild‐type TASK‐1 channels. These mutated TASK‐1 channels are located at the plasma membrane to the same degree as wild‐type TASK‐1 channels. ONO‐RS‐082 and alkaline pH 8.4 both activate TASK‐1 channels but do not recover current through mutant TASK‐1 channels. We show that the guanylate cyclase activator, riociguat, a novel treatment for PAH, enhances current through TASK‐1 channels but does not recover current through mutant TASK‐1 channels. Abstract Pulmonary arterial hypertension (PAH) affects ∼15–50 people per million. KCNK3, the gene that encodes the two pore domain potassium channel TASK‐1 (K2P3.1), has been identified as a possible disease‐causing gene in heritable PAH. Recently, two new mutations have been identified in KCNK3 in PAH patients: G106R and L214R. The present study aimed to characterize the functional properties and regulation of wild‐type (WT) and mutated TASK‐1 channels and determine how these might contribute to PAH and its treatment. Currents through WT and mutated human TASK‐1 channels transiently expressed in tsA201 cells were measured using whole‐cell patch clamp electrophysiology. Localization of fluorescence‐tagged channels was visualized using confocal microscopy and quantified with in‐cell and on‐cell westerns. G106R or L214R mutated channels were located at the plasma membrane to the same degree as WT channels; however, their current was markedly reduced compared to WT TASK‐1 channels. Functional current through these mutated channels could not be restored using activators of WT TASK‐1 channels (pH 8.4, ONO‐RS‐082). The guanylate cyclase activator, riociguat, enhanced current through WT TASK‐1 channels; however, similar to the other activators investigated, riociguat did not have any effect on current through mutated TASK‐1 channels. Thus, novel mutations in TASK‐1 seen in PAH substantially alter the functional properties of these channels. Current through these channels could not be restored by activators of TASK‐1 channels. Riociguat enhancement of current through TASK‐1 channels could contribute to its therapeutic benefit in the treatment of PAH.

Effects of the ventilatory stimulant, doxapram on human TASK‐3 (KCNK9, K2P9.1) channels and TASK‐1 (KCNK3, K2P3.1) channels

“…54 Vectors cloned with the gene of interest and a similar vector encoding the cDNA for GFP were added to each well at a concentration of 0.5 µg per well, with the exception being for murTASK-3, where 0.125 µg per well was used. 54 Vectors cloned with the gene of interest and a similar vector encoding the cDNA for GFP were added to each well at a concentration of 0.5 µg per well, with the exception being for murTASK-3, where 0.125 µg per well was used.…”

“…54 Briefly, all experiments were conducted at room temperature (20-24°C) using an external solution composed of 145 mM of NaCl, 2.5 mM of KCL, 3 mM of MgCl 2 , 1 mM of CaCl 2 and 10 mM of HEPES (pH 7.4, using NaOH) and an intracellular pipette solution composed of 150 mM of KCL, 3 mM of MgCl 2 , 5 mM of EGTA and 10 mM of HEPES (pH adjusted to 7.4 with KOH). 54 Briefly, all experiments were conducted at room temperature (20-24°C) using an external solution composed of 145 mM of NaCl, 2.5 mM of KCL, 3 mM of MgCl 2 , 1 mM of CaCl 2 and 10 mM of HEPES (pH 7.4, using NaOH) and an intracellular pipette solution composed of 150 mM of KCL, 3 mM of MgCl 2 , 5 mM of EGTA and 10 mM of HEPES (pH adjusted to 7.4 with KOH).…”

“…Point mutations (L122D, G236D, L239D, V242D or a stop codon) were introduced by site-directed mutagenesis into TASK-3 cDNA using the Quikchange kit (Stratagene) as previously described. 54…”

“…All experiments were performed using a modified human embryonic kidney 293 cell line, tsA201 (ECACC; Sigma-Aldrich), prepared and maintained as previously described. 54…”

“…For the electrophysiological experiments, cells were transiently transfected using a modified calcium phosphate protocol, following a process as previously described. 54 Vectors cloned with the gene of interest and a similar vector encoding the cDNA for GFP were added to each well at a concentration of 0.5 µg per well, with the exception being for murTASK-3, where 0.125 µg per well was used.…”