Abstract
E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.
Highlights
Mammalian voltage-gated sodium channels are composed of a single transcript encoding 4 domains (DI-DIV), each with 6 transmembrane segments (S1-S6)
Using a tetrameric model of the sodium channel, fluorescence recordings of labelled S4 voltage-sensors, and experiments on fast-inactivation deficient channels, we show that the depolarizing shift in the conductance curve is likely due to the mutant-dependent hyperpolarization and acceleration of fast inactivation
The conductance curve is shifted to more depolarized membrane potentials and the voltage-dependence of S4 movement is shifted to more hyperpolarized membrane potentials in E1784K
Summary
Mammalian voltage-gated sodium channels are composed of a single transcript encoding 4 domains (DI-DIV), each with 6 transmembrane segments (S1-S6). The first 4 transmembrane segments of each domain form a voltage-sensing domain (VSD), whereas S5, S6, and the extracellular linker between these segments form the channel pore [1]. In response to membrane depolarization, the positively charged S4 segments rotate and move towards the outside of the membrane, preceding channel pore opening. Outward S4 movements produce a small but measurable gating current that can be used to map the conformational changes of the voltage sensors en masse [2].
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