Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC269

Poster Communications

Coupling of the Voltage Sensing Domain to the Pore Domain in the hERG K+ Potassium Channel

S. Thomson1, S. Dalibalta2, J. Mitcheson1

1. University of Leicester, Leicester, United Kingdom. 2. University of Oxford, Oxford, United Kingdom.


The gating of voltage gated potassium (K) channels is a fundamental process for regulating cellular excitability. Voltage gated K+ channels are tetramers with each subunit containing six transmembrane spanning helices (S1 - S6). S1-S4 forms the voltage sensing domain (VSD) and is joined via the S4-S5 linker (S4S5L) to the pore of the channel formed by S5-S6. How the VSD couples to the pore domain is not well understood, but is thought to involve interactions between the S4-S5 linker and S6. hERG channel gating is characterised by rapid inactivation gating and slow deactivation gating and these processes are important for regulating the physiological time course of this current during the cardiac action potential. When the Val at position 659, in the C-terminal end of the S6, was mutated to Ala during an Ala scan of S6 it profoundly slowed deactivation. Our aim was to investigate the molecular basis for the slow deactivation of V659A. Site directed mutagenesis was used to mutate Val659 to a range of amino acids and effects on gating were studied by two-electrode voltage clamp in Xenopus oocytes. Mutation to bulky hydrophobic residues like Trp, Ile & Phe had little effect on deactivation. However, mutation to smaller residues like Cys & Ala slowed deactivation (τ’s at -140mV, WT 222±13ms, 659Ile 202±17ms, 659Ala 3181±156ms, n=6). V659G would not deactivate even at -200mV indicating that the pore was no longer coupled to the VSD. The crystal structure of Kv1.2 shows the S4S5L passes across S6 close to the activation gate. To test if Val659 was in close proximity to the S4S5L we investigated if the oxidising agent tert-Butyl hydroperoxide (2mM) could facilitate disulphide bonds between 659Cys and Cys residues substituted into different positions on the S4S5L. We ran a current-voltage (I-V) protocol, holding at -120mV and stepping to test potentials from -90mV to +30mV then back to the holding potential in the presence of the oxidising agent. The I-V relationship for the double mutant V659C:E544C became linear and there was a large increase in instantaneous current (the current 5ms after stepping to the test potential) suggesting that the channel was been locked open with the addition of the oxidising agent. This effect was not reversible on switching back to the control solution & was inhibited by pre-treating the cells with a reducing agent (10 mM Dithiothreitol). We investigated if this effect was happening when the channel was in the open or closed state by holding the channel closed at -120mV or open at +40mV and washing on the oxidising agent for 5 minutes and measuring the instantaneous current at +20mV. In the closed state current increased 18±6% but in the open state the current increased 178±5% (n=4). Our results suggest that Val659 couples to the S4-S5L in the open state forming an interaction that is required for closing the activation gate of hERG.

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