Characterisation of the Two Pore Domain Potassium Ion Channels, TASK1 and TASK3, Associated with Pulmonary Arterial Hypertension and KCNK9 Imprinting Syndrome

This study investigated the regulation and function of two-pore domain potassium (K2P) channels in disease. TASK family of K2P ion channels are predominantly expressed at the cell surface where they maintain the resting membrane potential to regulate neuronal excitability. TASK channel variants associated with diseases such as pulmonary arterial hypertension (PAH) (TASK1_G106R/L214R) and KCNK9 Imprinting Syndrome (TASK3_G236R/Y205C/Y205Δ) demonstrate reduced activity. Due to the constitutively active nature of TASK channels expressed at the plasma membrane, changes in the level of surface expression can alter current density and subsequently function. Using a combination of imaging, biochemical and electrophysiological techniques, this study explored the effects of pathogenic variants on channel trafficking to the plasma membrane and other organelles. The findings presented here show that the reduced plasma membrane expression of the KCNK9 Imprinting channel mutation, TASK3_G236R cannot explain all the pathogenic consequences of this mutation. The G236R channels expressed were functionally altered when compared to WT, as interaction with activated Gαq/11 remains, yet G protein-coupled receptor (GPCR) inhibition is lost. The plasma membrane expression of Y205C and Y205Δ pathogenic variants was unaffected, but the channels were electrophysiologically silent. Low arousal is one symptom of KCNK9 Imprinting Syndrome which is linked to mitochondrial dysfunction. As membrane expression of TASK3 variants were indistinguishable from the WT, this symptom is potentially a consequence of an inability to respond to GPCR activation by wakeful neurotransmitters such as histamine. TASK3 is regulated by hormones and transmitters acting through GPCR via the Gαq/11 subclass. Without a crystal structure, TASK3 functional mechanisms remain subjective. Removal of an extreme proximal [VLRFLT] motif from the cytosolic C-terminus abolishes regulation, reminiscent of that seen in KCNK9 Imprinting syndrome. Since it is unclear whether this motif behaves as a binding site for regulatory molecules or is essential for intracellular signal transduction, this study details the production of the C-terminal fragment. Synchrotron radiation circular dichroism confirmed that the produced fragment was conformationally correct proven by the direct binding of Gαq/11 to the C-terminus, although the role of the VLRFLT as a binding site or transducer of signal remains to be elucidated. Reduced activity of TASK1 is a key event in PAH pathogenesis. In this study I show that the nonfunctional G106R and L214R TASK1 channel variants are translated and expressed at the plasma membrane as efficiently as the WT. This study also explored the hypoxic characteristics inherent to PAH. TASK1 expression in mitochondria was investigated and confirmed for the first time. The expression of the G106R and L214R mutations appeared to be no different to the WT, implying that the presence of these mutations in the mitochondria may not contribute to the progression of PAH.