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. 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.

Aims: The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK‐1 and TASK‐1/TASK‐3 heterodimer channels, than TASK‐3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK‐1 and TASK‐3.Methods: Whole‐cell patch clamp electrophysiology on tsA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK‐1, TASK‐3 and TASK‐2 channels. Mutations of amino acids in the pore‐lining region of TASK‐3 channels were introduced using site‐directed mutagenesis.Results: Doxapram was an equipotent inhibitor of human TASK‐1 and TASK‐3 channels, compared with mouse channel variants, where it was more selective for TASK‐1 and heterodimers of TASK‐1 and TASK‐3. The effect of doxapram could be attenuated by either the removal of the C‐terminus of human TASK‐3 channels or mutations of particular hydrophobic residues in the pore‐lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK‐3, zinc. The positive enantiomer of doxapram, GAL‐054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL‐053, had little inhibitory effect.Conclusion: These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK‐1 and TASK‐3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to the rhythmic firing of pacemaker neurons and cardiomyocytes. Mutations in HCN channels are associated with cardiac arrhythmia and epilepsy. HCN channels belong to the superfamily of voltage-gated K⁺ channels, most of which are activated by depolarization. HCN channels, however, are activated by hyperpolarization. The mechanism behind this reversed gating polarity of HCN channels is not clear. We here show that sea urchin HCN (spHCN) channels with mutations in the C-terminal part of the voltage sensor use the same voltage-sensor movement to either close or open in response to hyperpolarizations depending on the absence or presence of cAMP. Our results support that non-covalent interactions at the C-terminal end of the voltage sensor are critical for HCN gating polarity. These interactions are also critical for the proper closing of the channels because these mutations exhibit large constitutive currents. Since a similar voltage-sensor movement can cause both depolarization- and hyperpolarization-activation in the same channel, this suggests that the coupling between the voltage sensor and the pore is changed to create channels opened by different polarities. We also show an identical voltage-sensor movement in activated and inactivated spHCN channels and suggest a model for spHCN activation and inactivation. Our results suggest the possibility that channels open by opposite voltage dependence, such as HCN and the related EAG channels, use the same voltage-sensor movement but different coupling mechanisms between the voltage sensor and the gate.

In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause long QT syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain – which shifts the voltage dependence to more negative voltages – and the other in the pore domain – which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single-channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.

Voltage-gated potassium (Kv) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels share similar structures but have opposite gating polarity. Kv channels have a strong coupling (>10⁹) between the voltage sensor (S4) and the activation gate: when S4s are activated, the gate is open to >80% but, when S4s are deactivated, the gate is open <10⁻⁹ of the time. Using noise analysis, we show that the coupling between S4 and the gate is <200 in HCN channels. In addition, using voltage clamp fluorometry, locking the gate open in a Kv channel drastically altered the energetics of S4 movement. In contrast, locking the gate open or decreasing the coupling between S4 and the gate in HCN channels had only minor effects on the energetics of S4 movement, consistent with a weak coupling between S4 and the gate. We propose that this loose coupling is a prerequisite for the reversed voltage gating in HCN channels.