Mechanistic Insight into Human ether-a-go-go-related Gene (hERG) K+ Channel Activation and Deactivation gating

hERG encodes the pore-forming α-subunit of the voltage-gated potassium channel that underlies the rapid delayed rectifier current, IKr, in the heart, which is essential for normal cardiac electrical activity and rhythm. Inherited mutations in, or pharmacological blockade of, hERG channels deplete the cardiac repolarization reserve, increasing the risk of life-threatening arrhythmias. The molecular bases of hERG gating events and drug binding are poorly understood. hERG channels display unique gating characteristics critical for their physiological function. They activate and deactivate slowly, yet inactivate and recover from inactivation rapidly. In addition, the promiscuous nature of drug interactions with hERG channels presents a therapeutic challenge for drug design and development. My thesis provides novel mechanistic and structural characterization of the unusual activation and deactivation gating processes of hERG. In my first study, I used a proline scan approach to define the activation gate region in hERG channels. Proximal substitutions (I655P-Q664P) impeded gate closure, trapping channels in the open state, while distal substitutions (R665P-Y667P) preserved normal gating, suggesting that Q664 marks the position of the activation gate in hERG. This is more than one helical turn lower than in related channels, which may allow for drug docking. Using two different approaches to measure voltage sensor gating in trapped open channels, I then demonstrated that slow activation is an intrinsic property of the voltage-sensing unit of hERG. In my second study, I showed that voltage-sensor stabilization slows hERG channel deactivation gating. I characterized the temporal sequence of events leading to voltage-sensor stabilization upon membrane depolarization. I showed that this occurs via two separable mechanisms, one derived from pore-gate-opening and the other from the voltage-sensing unit itself. In addition, I show that voltage sensor return in hERG channels is less energetically favourable than pore closure during repolarization and thus is what limits deactivation. Finally, I characterize the use of voltage clamp fluorimetry as a technique to track conformational rearrangements of the hERG voltage sensor associated with gating. These findings provide novel and in depth understanding regarding how hERG channels function and foundational knowledge relevant to finding targets for the treatment and management of cardiac arrhythmias.