Characterisation of human TASK-1 mutations in atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting over two million Americans and this is set to triple by 2050, incurring an estimated U.S. treatment cost of $6.65 billion yearly. AF is a chronic condition associated with electrical changes within the heart that are related to atrial remodeling caused by variation in gene expression. The TWIK related acid sensitive potassium channel 1 (TASK-1, K2P3.1) is a protein with the ability to induce AF. The manifestation of TASK-1 induced AF has been categorized by three factors: incorrect protein trafficking, defective K2P3.1 channel assembly and TASK-1 electrophysiological dysfunction. However, the genomic variations that can induce these changes is not well documented, and so this project is aimed at characterising TASK-1 in human AF. This was conducted by the creation of mutant TASK-1 constructs pathogenic in AF. These included mutation of a transmembrane domain (V123L), and two Kozak mutations (-3A>U and -4G>A). These mutants were proposed to effect channel conformation and protein translation. For those reasons these mutations were picked for investigation, along with a C-terminally truncated protein, and a N-terminally mutated protein as to investigate the effects of phosphorylation sites and 14.3.3 binding in the trafficking of the protein. These constructs were individually or co-expressed on the membrane of tsA-201 cells; where they underwent electrophysiological analysis and confocal microscopy imaging. Overall TASK-1 mutants were shown, to present themselves in varied states. The C-terminus truncation and -3A mutations suggested an increased expression, by providing a significant increase in current compared to the wildtype. Whilst V123L proved to be potentially pathogenic, by demonstrating a significantly decreased current plus a significantly depolarized reversal potential. However, the mutational effects of V123L were rectified by heterodimerization with wildtype TASK-1, as would occur in native atrial tissue. Additionally, TASK-1 proved dominant in heterodimer formation with TASK-3, reducing the current amplitude close to that of TASK-1 homodimers, yet, the reversal potential was between the two reversal potential figures for homodimers of TASK-1 and TASK-3. Confocal imaging experiments showed that wildtype TASK-1 channels were expressed at the cell membrane. However, both the N-terminus and Kozak mutations imaged, proved even localization at the membrane, with no intracellular retention visible, suggesting that by use of another protocol, a quantifiable increase in expression maybe provided compared to wildtype.