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Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations

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.


UCL2077 inhibition confirms that KCNQ1S140G slow voltage sensor deactivation independent of channel opening.The following protocol was used: 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. This protocol was used before and after inhibition of current with 10 μM UCL2077. (a,b) Current before and after inhibition with UCL2077 for KCNQ1 (a) and KCNQ1S140G (b). (c,d) Normalized fluorescence deactivation traces at −100 mV for KCNQ1 (red) and KCNQ1S140G (dark red) in drug-free control (c) and in 10 μM UCL2077 (d). (e) Time to half deactivation (t1/2) of fluorescence. Data are shown as mean ± SEM (error bars). *P < 0.05.
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f3: UCL2077 inhibition confirms that KCNQ1S140G slow voltage sensor deactivation independent of channel opening.The following protocol was used: 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. This protocol was used before and after inhibition of current with 10 μM UCL2077. (a,b) Current before and after inhibition with UCL2077 for KCNQ1 (a) and KCNQ1S140G (b). (c,d) Normalized fluorescence deactivation traces at −100 mV for KCNQ1 (red) and KCNQ1S140G (dark red) in drug-free control (c) and in 10 μM UCL2077 (d). (e) Time to half deactivation (t1/2) of fluorescence. Data are shown as mean ± SEM (error bars). *P < 0.05.

Mentions: In the second approach, we employed UCL2077, an inhibitor of IKs previously shown to prevent channel opening but still allow S4 movement18. Application of 10 μM UCL2077 drastically reduces current of both KCNQ1 and KCNQ1S140G (Fig. 3a,b). When analyzing the kinetics of fluorescence deactivation, we found that both in the control condition and in the presence of 10 μM UCL2077, KCNQ1S140G slows fluorescence deactivation (Control: Ft1/2 = 893 ± 59 ms; UCL2077: Ft1/2 = 602 ± 61 ms) compared with KCNQ1 (Control: Ft1/2 = 109 ± 23 ms; UCL2077: Ft1/2 = 109 ± 7 ms) (Fig. 3c–e). Taken together, these experiments show that KCNQ1S140G slows voltage sensor deactivation even in the absence of channel opening.


Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations
UCL2077 inhibition confirms that KCNQ1S140G slow voltage sensor deactivation independent of channel opening.The following protocol was used: 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. This protocol was used before and after inhibition of current with 10 μM UCL2077. (a,b) Current before and after inhibition with UCL2077 for KCNQ1 (a) and KCNQ1S140G (b). (c,d) Normalized fluorescence deactivation traces at −100 mV for KCNQ1 (red) and KCNQ1S140G (dark red) in drug-free control (c) and in 10 μM UCL2077 (d). (e) Time to half deactivation (t1/2) of fluorescence. Data are shown as mean ± SEM (error bars). *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: UCL2077 inhibition confirms that KCNQ1S140G slow voltage sensor deactivation independent of channel opening.The following protocol was used: 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. This protocol was used before and after inhibition of current with 10 μM UCL2077. (a,b) Current before and after inhibition with UCL2077 for KCNQ1 (a) and KCNQ1S140G (b). (c,d) Normalized fluorescence deactivation traces at −100 mV for KCNQ1 (red) and KCNQ1S140G (dark red) in drug-free control (c) and in 10 μM UCL2077 (d). (e) Time to half deactivation (t1/2) of fluorescence. Data are shown as mean ± SEM (error bars). *P < 0.05.
Mentions: In the second approach, we employed UCL2077, an inhibitor of IKs previously shown to prevent channel opening but still allow S4 movement18. Application of 10 μM UCL2077 drastically reduces current of both KCNQ1 and KCNQ1S140G (Fig. 3a,b). When analyzing the kinetics of fluorescence deactivation, we found that both in the control condition and in the presence of 10 μM UCL2077, KCNQ1S140G slows fluorescence deactivation (Control: Ft1/2 = 893 ± 59 ms; UCL2077: Ft1/2 = 602 ± 61 ms) compared with KCNQ1 (Control: Ft1/2 = 109 ± 23 ms; UCL2077: Ft1/2 = 109 ± 7 ms) (Fig. 3c–e). Taken together, these experiments show that KCNQ1S140G slows voltage sensor deactivation even in the absence of channel opening.

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.