AbstractHuman ether-à-go-go-related gene (hERG) potassium channels underlie the rapid delayed rectifier K+ current (IKr) and play an important role in repolarisation of cardiac action potentials (APs). Pathological events such as cardiac ischaemia can lead to a decrease in extracellular pH (acidosis). Extracellular acidosis is known to modulate the hERG current (IhERG) but the underlying mechanism(s) are not completely known. The aims of this study were: (1) to establish the effects of acidosis on macroscopic and single-channel IhERG, investigating both hERG1a and hERG1b isoforms; (2) to use an amino acid modifying reagent and site-directed mutagenesis to probe the molecular basis of proton modulation of IhERG, focusing on the hERG1a isoform.
Whole-cell and cell-attached patch-clamp recordings were made at ambient temperature of wild-type IhERG from mammalian cell lines (HEK-293 or CHO). When external pH (pHe) was reduced from 7.4 to 6.3, macroscopic IhERG amplitude and conductance decreased, activation was positively shifted, and deactivation kinetics were accelerated. Results obtained in the cell-attached configuration showed a reduction at pHe 6.3 in single-channel IhERG amplitude and conductance, decreased open- and burst-durations and increased closed-time durations. These effects at the single-channel level account for the modulation of macroscopic IhERG by acidic pHe. The first known single-channel recordings from hERG1b showed that this isoform retained sensitivity to acidic pHe, indicating that N-terminal differences between the two isoforms are not critical for proton sensitivity.
Experiments completed with a range of extracellular pH values (4.5 – 8.0) revealed that different features of IhERG have distinct pKa values, suggesting multiple sites of proton modulation. Titratable residues located in the pore region of the hERG1a channel were mutated to determine if they were responsible for pH sensitivity. The double mutation E575Q/H578N appeared to remove the proton reduction of channel conductance, thus identifying a novel proton sensor on the hERG channel.
|Date of Award||20 Mar 2018|
|Supervisor||Jules C Hancox (Supervisor) & Neil V Marrion (Supervisor)|