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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.


In the presence of KCNE1, S140G slows current deactivation and voltage sensor movement while V141M slows current deactivation without slowing voltage sensor movement.(a–c) Current and fluorescence traces for IKs (a), IKsS140G (b), and IKsV141M (c) in the presence of KCNE1 using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +80 mV followed by a repolarizing step to −40 mV. Cells were held at −110 mV. Arrows indicate effect of mutations on current or fluorescence. (d) Normalized current during deactivation at −100 mV for IKs, IKsS140G, and IKsV141M. Inset shows the first 4 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −120 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −110 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 4 s of deactivation. (f) Time to 75% deactivation (t75%). Data are shown as mean ± SEM (error bars). *P < 0.05.
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f4: In the presence of KCNE1, S140G slows current deactivation and voltage sensor movement while V141M slows current deactivation without slowing voltage sensor movement.(a–c) Current and fluorescence traces for IKs (a), IKsS140G (b), and IKsV141M (c) in the presence of KCNE1 using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +80 mV followed by a repolarizing step to −40 mV. Cells were held at −110 mV. Arrows indicate effect of mutations on current or fluorescence. (d) Normalized current during deactivation at −100 mV for IKs, IKsS140G, and IKsV141M. Inset shows the first 4 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −120 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −110 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 4 s of deactivation. (f) Time to 75% deactivation (t75%). Data are shown as mean ± SEM (error bars). *P < 0.05.

Mentions: To understand the mechanisms underlying the effects of the mutations on the physiologic IKs current, we co-expressed KCNQ1 or KCNQ1 mutants with KCNE1 and used VCF to measure voltage sensor movement simultaneously with current. Figure 4a–c shows currents and fluorescence changes from IKs or mutant channels in response to voltage pulses to +80 mV for 5 s followed by repolarization to −40 mV. For IKs, the fluorescence activation is much faster than the channel’s sigmoidal current activation, as previously reported18. Neither mutant channel appreciably affects the kinetics of current activation. However, whereas IKs deactivates completely at −40 mV, neither IKsS140G nor IKsV141M deactivates appreciably. On the other hand, IKsS140G and IKsV141M exert distinct effects on voltage sensor movement. IKsS140G apparently slows both the activation and deactivation kinetics of voltage sensor movement. On the other hand, IKsV141M does not apparently alter voltage sensor kinetics, but leads to a different steady state level at −40 mV.


Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations
In the presence of KCNE1, S140G slows current deactivation and voltage sensor movement while V141M slows current deactivation without slowing voltage sensor movement.(a–c) Current and fluorescence traces for IKs (a), IKsS140G (b), and IKsV141M (c) in the presence of KCNE1 using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +80 mV followed by a repolarizing step to −40 mV. Cells were held at −110 mV. Arrows indicate effect of mutations on current or fluorescence. (d) Normalized current during deactivation at −100 mV for IKs, IKsS140G, and IKsV141M. Inset shows the first 4 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −120 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −110 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 4 s of deactivation. (f) Time to 75% deactivation (t75%). Data are shown as mean ± SEM (error bars). *P < 0.05.
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f4: In the presence of KCNE1, S140G slows current deactivation and voltage sensor movement while V141M slows current deactivation without slowing voltage sensor movement.(a–c) Current and fluorescence traces for IKs (a), IKsS140G (b), and IKsV141M (c) in the presence of KCNE1 using a single pulse protocol. From a prepulse of −140 mV, a test pulse was applied at +80 mV followed by a repolarizing step to −40 mV. Cells were held at −110 mV. Arrows indicate effect of mutations on current or fluorescence. (d) Normalized current during deactivation at −100 mV for IKs, IKsS140G, and IKsV141M. Inset shows the first 4 s of deactivation. Deactivation was examined using the following voltage protocol: from a prepulse of −120 mV, an activating pulse was applied at +40 mV, followed by a repolarizing step to −100 mV. Channels were held at −110 mV. (e) Normalized percent change in fluorescence during deactivation at −100 mV. Inset shows first 4 s of deactivation. (f) Time to 75% deactivation (t75%). Data are shown as mean ± SEM (error bars). *P < 0.05.
Mentions: To understand the mechanisms underlying the effects of the mutations on the physiologic IKs current, we co-expressed KCNQ1 or KCNQ1 mutants with KCNE1 and used VCF to measure voltage sensor movement simultaneously with current. Figure 4a–c shows currents and fluorescence changes from IKs or mutant channels in response to voltage pulses to +80 mV for 5 s followed by repolarization to −40 mV. For IKs, the fluorescence activation is much faster than the channel’s sigmoidal current activation, as previously reported18. Neither mutant channel appreciably affects the kinetics of current activation. However, whereas IKs deactivates completely at −40 mV, neither IKsS140G nor IKsV141M deactivates appreciably. On the other hand, IKsS140G and IKsV141M exert distinct effects on voltage sensor movement. IKsS140G apparently slows both the activation and deactivation kinetics of voltage sensor movement. On the other hand, IKsV141M does not apparently alter voltage sensor kinetics, but leads to a different steady state level at −40 mV.

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.