AbstractThe hERG1a potassium channel is a voltage-gated potassium channel which underlies the fast inwardly rectifying current in the cardiac action potential. The channel’s voltage sensing domain (the S4 helix) contains positively charged residues which enable it’s movement in response to membrane depolarisation to open the channel. The neutralisation of R528, one of the positive S4 residues, should alter S4 movement and therefore the gating currents used to record said movement.
Whole-cell voltage clamp was used to characterise the ionic and gating current of WT-hERG1a and R528C-hERG1a, and the gating currents of the N588K-hERG1a inactivation-deficient mutant when transiently expressed in mammalian tsA201 cells. The voltage-dependence of R528C-hERG1a activation and inactivation was positively shifted relative to WT-hERG1a current, reflecting the loss of a positively charged S4 residue. R528C-hERG1a current had accelerated activation and inactivation kinetics but slower deactivation kinetics, indicating that the mutation had destabilised the deactivated state of the S4 helix and accelerated its movement into the activated state. The reversal potential of R528C-hERG1a was also positively shifted, reflecting the ~1.5-fold increase in the PNa/PK ratio of the mutant, providing evidence of an interaction between S4 movement and the channel’s selectivity filter.
The majority of WT-hERG1a gating currents were classed as asymmetric with the on gating current being of smaller amplitude than the off, while the majority of R528C-hERG1a and N588K-hERG1a gating currents were classed as symmetric. This was attributed to WT-hERG1a channels opening slowly over the pulse resulting in a smaller on gating current, in contrast to the fast activation of R528C-hERG1a and N588K-hERG1a which resulted in a larger initial on gating current.
Atomic Force Microscopy was used to image a tetrameric structure on the fixed cell membrane of the hERG-HEK293 stable cell line, which was believed to be the extracellular structure of the hERG channel in its native conformation.
|Date of Award||23 Jan 2020|
|Supervisor||Mervyn Miles (Supervisor) & Neil V Marrion (Supervisor)|