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Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations

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ABSTRACT

KCNQ1 is a voltage-gated potassium channel that is modulated by the beta-subunit KCNE1 to generate IKs, the slow delayed rectifier current, which plays a critical role in repolarizing the cardiac action potential. Two KCNQ1 gain-of-function mutations that cause a genetic form of atrial fibrillation, S140G and V141M, drastically slow IKs deactivation. However, the underlying gating alterations remain unknown. Voltage clamp fluorometry (VCF) allows simultaneous measurement of voltage sensor movement and current through the channel pore. Here, we use VCF and kinetic modeling to determine the effects of mutations on channel voltage-dependent gating. We show that in the absence of KCNE1, S140G, but not V141M, directly slows voltage sensor movement, which indirectly slows current deactivation. In the presence of KCNE1, both S140G and V141M slow pore closing and alter voltage sensor-pore coupling, thereby slowing current deactivation. Our results suggest that KCNE1 can mediate changes in pore movement and voltage sensor-pore coupling to slow IKs deactivation and provide a key step toward developing mechanism-based therapies.

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In the absence of KCNE1, S140G slows both current and voltage sensor deactivation, whereas V141M slows neither.(a–c) Current (black) and fluorescence (red) traces for KCNQ1 (a), KCNQ1S140G (b), and KCNQ1V141M (c) using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +60 mV, followed by a repolarizing step to −40 mV. Cells were held at −80 mV. (d) Normalized current during deactivation at −100 mV for KCNQ1, KCNQ1S140G and KCNQ1V141M. Inset shows first 2 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −140 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −80 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 2 s of deactivation. (f) Time to half deactivation (t1/2). Data are shown as mean ± SEM (error bars). *P < 0.05.
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f1: In the absence of KCNE1, S140G slows both current and voltage sensor deactivation, whereas V141M slows neither.(a–c) Current (black) and fluorescence (red) traces for KCNQ1 (a), KCNQ1S140G (b), and KCNQ1V141M (c) using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +60 mV, followed by a repolarizing step to −40 mV. Cells were held at −80 mV. (d) Normalized current during deactivation at −100 mV for KCNQ1, KCNQ1S140G and KCNQ1V141M. Inset shows first 2 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −140 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −80 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 2 s of deactivation. (f) Time to half deactivation (t1/2). Data are shown as mean ± SEM (error bars). *P < 0.05.

Mentions: To study the gating effects of atrial fibrillation mutations using VCF, we employed a KCNQ1 construct containing a cysteine site at extracellular residue G219 that is close to S4 and can be labelled covalently with a fluorophore. This construct has been used in multiple VCF studies18192930, and we refer to it here as KCNQ1. We first used a single pulse protocol to determine whether the atrial fibrillation mutations S140G and V141M affect channel current and voltage sensor movement in the absence of KCNE1. From a holding potential of −80 mV, a prepulse to −140 mV was applied to deactivate voltage sensors to the resting position. Then, a single pulse to +60 mV was applied for 2 s followed by repolarization to −40 mV. In comparison to KCNQ1, both KCNQ1S140G and KCNQ1V141M exhibit similar activation kinetics in current and fluorescence at +60 mV (Fig. 1a–c). However, KCNQ1S140G appears to slow the deactivation kinetics of both current and fluorescence, whereas KCNQ1V141M does not appear to slow either. To determine the voltage dependence of activation, we applied the protocol at multiple voltages ranging from −140 mV to +60 mV and plotted the isochronal activation of fluorescence (FV) and conductance (GV) (Supplementary Fig. S1). Compared with KCNQ1 (FV1/2 = −29.3 ± 2.5 mV, GV1/2 = −27.2 ± 3.3 mV), KCNQ1S140G (FV1/2 = −47.2 ± 2.1 mV, GV1/2 = −44.8 ± 2.7 mV) causes a significant hyperpolarizing shift in in both FV and GV, whereas KCNQ1V141M (FV1/2 = −23.8 ± 1.3 mV, GV1/2 = −36.7 ± 2.0 mV) does not significantly shift either.


Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations
In the absence of KCNE1, S140G slows both current and voltage sensor deactivation, whereas V141M slows neither.(a–c) Current (black) and fluorescence (red) traces for KCNQ1 (a), KCNQ1S140G (b), and KCNQ1V141M (c) using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +60 mV, followed by a repolarizing step to −40 mV. Cells were held at −80 mV. (d) Normalized current during deactivation at −100 mV for KCNQ1, KCNQ1S140G and KCNQ1V141M. Inset shows first 2 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −140 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −80 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 2 s of deactivation. (f) Time to half deactivation (t1/2). Data are shown as mean ± SEM (error bars). *P < 0.05.
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f1: In the absence of KCNE1, S140G slows both current and voltage sensor deactivation, whereas V141M slows neither.(a–c) Current (black) and fluorescence (red) traces for KCNQ1 (a), KCNQ1S140G (b), and KCNQ1V141M (c) using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +60 mV, followed by a repolarizing step to −40 mV. Cells were held at −80 mV. (d) Normalized current during deactivation at −100 mV for KCNQ1, KCNQ1S140G and KCNQ1V141M. Inset shows first 2 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −140 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −80 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 2 s of deactivation. (f) Time to half deactivation (t1/2). Data are shown as mean ± SEM (error bars). *P < 0.05.
Mentions: To study the gating effects of atrial fibrillation mutations using VCF, we employed a KCNQ1 construct containing a cysteine site at extracellular residue G219 that is close to S4 and can be labelled covalently with a fluorophore. This construct has been used in multiple VCF studies18192930, and we refer to it here as KCNQ1. We first used a single pulse protocol to determine whether the atrial fibrillation mutations S140G and V141M affect channel current and voltage sensor movement in the absence of KCNE1. From a holding potential of −80 mV, a prepulse to −140 mV was applied to deactivate voltage sensors to the resting position. Then, a single pulse to +60 mV was applied for 2 s followed by repolarization to −40 mV. In comparison to KCNQ1, both KCNQ1S140G and KCNQ1V141M exhibit similar activation kinetics in current and fluorescence at +60 mV (Fig. 1a–c). However, KCNQ1S140G appears to slow the deactivation kinetics of both current and fluorescence, whereas KCNQ1V141M does not appear to slow either. To determine the voltage dependence of activation, we applied the protocol at multiple voltages ranging from −140 mV to +60 mV and plotted the isochronal activation of fluorescence (FV) and conductance (GV) (Supplementary Fig. S1). Compared with KCNQ1 (FV1/2 = −29.3 ± 2.5 mV, GV1/2 = −27.2 ± 3.3 mV), KCNQ1S140G (FV1/2 = −47.2 ± 2.1 mV, GV1/2 = −44.8 ± 2.7 mV) causes a significant hyperpolarizing shift in in both FV and GV, whereas KCNQ1V141M (FV1/2 = −23.8 ± 1.3 mV, GV1/2 = −36.7 ± 2.0 mV) does not significantly shift either.

View Article: PubMed Central - PubMed

ABSTRACT

KCNQ1 is a voltage-gated potassium channel that is modulated by the beta-subunit KCNE1 to generate IKs, the slow delayed rectifier current, which plays a critical role in repolarizing the cardiac action potential. Two KCNQ1 gain-of-function mutations that cause a genetic form of atrial fibrillation, S140G and V141M, drastically slow IKs deactivation. However, the underlying gating alterations remain unknown. Voltage clamp fluorometry (VCF) allows simultaneous measurement of voltage sensor movement and current through the channel pore. Here, we use VCF and kinetic modeling to determine the effects of mutations on channel voltage-dependent gating. We show that in the absence of KCNE1, S140G, but not V141M, directly slows voltage sensor movement, which indirectly slows current deactivation. In the presence of KCNE1, both S140G and V141M slow pore closing and alter voltage sensor-pore coupling, thereby slowing current deactivation. Our results suggest that KCNE1 can mediate changes in pore movement and voltage sensor-pore coupling to slow IKs deactivation and provide a key step toward developing mechanism-based therapies.

No MeSH data available.


Related in: MedlinePlus