UBC Theses and Dissertations
Mutagenesis studies of charged residues in the hERG K+ channel Wu, Yue
The loss-of-function mutations in the human ether-à-go-go–related gene (hERG) K+ channel serve as one of the primary genetic substrates for congenital long QT syndrome (LQTS), and unlike most Kv channels with fast-activating kinetics, hERG channels have unusually slow activation kinetics. Thus, it is a necessary task to understand mechanisms of hERG gating, especially since the charge-rich S4 segments play such an important role in channel opening in response to membrane potential changes. Despite the structural homology to other Kv channels, how individual S4 charges in hERG are positioned and moved under the influence of membrane voltage changes remains controversial. Therefore, this thesis focuses on the molecular basis of individual charge residues underlying S4 movement. To monitor S4 movement associated with the voltage- and time-dependence accessibility by the sulfhydryl-specific agent MTSET, we mutated a series of S4 charges (K525-K538) to uncharged glutamine (Q) in a mutant in which cysteine replaced the isoleucine at position 521 near the top of S4. We found that K525, R528, and R534 mainly secure the S4 position in the resting state. Our results on the rate of S4 movement suggest that R528, R531, and R537 potentially facilitate the transition of S4 from the closed to the active state. Conversely, neutralization of the bottom lysine K538 significantly accelerates the rate of S4 movement, implying an important constraint on S4 by K538. To further investigate this unique role of K538, we measure the gating charge contributions of K538 and D411 (a potential ion-pairing in S1) with the cut-open vaseline gap technique. Our data show that mutations of both charges to neutral residues (K538Q and D411N) accelerate the time dependence of charge movement while both mutant channels are gated with faster kinetics than control over a physiologically relevant range of depolarizations. This highlights the substantial contribution of both residues to slow hERG gating.
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