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Title: Deactivation gating and pharmacology of hERG potassium channel
Author: Thomson, Steven James
ISNI:       0000 0004 2721 3006
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2012
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hERG (Kv11.1) encodes the α-subunit of the potassium (K+) channel that carries IKr, an important current for repolarisation of the cardiac action potential. Alterations of hERG current, either through inherited mutations that alter gating or through drugs that block the pore, are associated with Long QT syndrome, cardiac arrhythmias and sudden death. The N-terminus has an important role in regulating deactivation, a gating process that is important for timing of the hERG current during cardiac action potentials. Removing the entire N-terminus accelerates deactivation. A crystal structure of part of the N-terminus (residues 26-135) was solved in 1998 and showed it contained a PAS domain, but it did not resolve the structure of the functionally important first 26 residues (NT 1-26). Here we present an NMR structure of residues 1-135. The structure reveals that residues 1-10 are unordered and residues 11-24 form an amphipathic helix one face of which is positively charged. Neutralising the positive charge accelerates deactivation to similar rates as if the whole of the N-terminus has been removed. Neutralising negative charge in the C-terminus also accelerates deactivation. We propose a model where the N and C-termini interact to stabilise the open state of the channel and slow deactivation. Exactly how changes in membrane voltage are transduced into movement of the activation gate is not fully understood. In hERG, the mutation V659A dramatically slows deactivation. Val659 is located in a region where hERG’s activation gate is believed to lie. From the structure of Kv2.1 it can be seen the S4-S5 linker forms a cuff around S6 where the activation gate is thought to be. Using cysteine cross-linking experiments we show that V659C interacts with E544C and Y545C in the S4-S5 linker to lock the channel in the open state. Trapping of drugs in the inner cavity of hERG has been an important model used to help explain why hERG is blocked by so many drugs and with high potency. A series of derivatives of E-4031, a well characterised high-affinity hERG blocker, were made that progressively increased the length of the molecule. Results in this thesis showed these compounds had binding kinetics completely different from E-4031 and none were trapped in the inner cavity. An alternative model of strongly state-dependent drug binding rather than drug-trapping is proposed. Together, the results in this thesis present new insights on the structural basis for deactivation gating and drug binding in hERG channels.
Supervisor: Mitcheson, John Sponsor: BBSRC ; Novartis
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available