Peng G et al. (2017), Gating mechanisms underlying deactivation slowi...

XB-ART-55545

Sci Rep 2017 Apr 06;7:45911. doi: 10.1038/srep45911.

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Gating mechanisms underlying deactivation slowing by two KCNQ1 atrial fibrillation mutations.

Peng G , Barro-Soria R , Sampson KJ , Larsson HP , Kass RS .

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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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Species referenced: Xenopus laevis
Genes referenced: kcne1 kcnq1

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	Figure 1. 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.
	Figure 2. In the absence of KCNE1, S140G slowing of voltage sensor deactivation is independent of channel opening based on PIP2 depletion.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 PIP2 depletion by ciVSP, which was activated by repeated depolarization. (a,b) Current before (+PIP2) and after PIP2 depletion (−PIP2) for KCNQ1 (a) and KCNQ1S140G (b). (c,d) Normalized fluorescence deactivation traces at −100 mV for KCNQ1 (red) and KCNQ1S140G (dark red) before (c) and after (d) PIP2 depletion. (e) Time to half deactivation (t1/2) of fluorescence. Data are shown as mean ± SEM (error bars). *P < 0.05.
	Figure 3. 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.
	Figure 4. 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.
	Figure 5. In the presence of KCNE1, S140G slowing of voltage sensor deactivation is dependent on 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 −110 mV. (a,b) Current measured before (+PIP2) and after PIP2 depletion (−PIP2) for IKs (a) and IKsS140G (b). (c,d) Current measured in drug-free control and in 10 μM UCL2077 for IKs (c) and IKsS140G (d). (e,f) Normalized fluorescence deactivation traces at −100 mV before (e) and after PIP2 depletion (f) for IKs (red) and IKsS140G (dark red). (g) Time to 75% deactivation (t75%) of fluorescence before and after PIP2 depletion. (h,i) Normalized fluorescence deactivation traces at −100 mV in drug-free control (h) and in 10 μM UCL2077 (i) for IKs (red) and IKsS140G (dark red). (j) Time to 75% deactivation (t75%) of fluorescence in control and in 10 μM UCL2077. Data are shown as mean ± SEM (error bars). *P < 0.05.
	Figure 6. Simulating effects of S140G on KCNQ1 gating in the absence of KCNE1.(a) Model of KCNQ1 gating. In this model, KCNQ1 can exist in distinct channel states. Horizontal transitions represent voltage sensor movement, while vertical transitions represent pore opening/closing. Voltage sensors can exist in the resting (white), intermediate (red), or fully activated state (blue). (b) Comparing experimental with simulated current (black) and fluorescence (red) traces for KCNQ1 and KCNQ1S140G. (c) Descriptions of deactivation pathways at −100 mV for KCNQ1 and KCNQ1S140G based on model rates. Arrows with greater opacity represent higher probability of channels entering the transitions specified. In the absence of KCNE1, the probability of channels existing in fully activated voltage sensor states (transparent) is low. The KCNQ1 channels can open and close in intermediate voltage sensor states during deactivation. KCNQ1S140G slows voltage sensor deactivation, which causes increased channel openings and closings, as indicated by highlighting. KCNQ1S140G thus indirectly slows current deactivation.
	Figure 7. Simulating the gating effects of S140G and V141M in the presence of KCNE1.(a) Comparing experimental with simulated current (black) and fluorescence (red) traces for IKs (top), IKsS140G (middle), and IKsV141M (bottom). (b) Description of deactivation pathways at −100 mV based on model rates. Arrows with greater opacity represent higher probability of channels entering the transitions specified. States that channels rarely or never enter are grayed out. Highlighting illustrates the most probable pathway of channel deactivation based on rates at −100 mV, starting from the fully activated open state. IKs channels opens and closes only when voltage sensors are fully activated (top). IKsS140G alters VSD-pore coupling and slows pore closing, altering the deactivation pathway such that during early steps of voltage sensor deactivation, channels may repeatedly open and close several times (middle). Like IKsS140G, IKsV141M also alters VSD-pore coupling to allow channel opening in intermediate VSD states (bottom). In addition, IKsV141M slows pore closing. However, the deactivation pathway is different from IKsS140G in that voltage sensors deactivate to a greater extent prior to pore closing.
	Figure 8. Proposed molecular mechanisms underlying effects of S140G and V141M on channel gating.(a,b) Homology model of open tetrameric KCNQ1 and KCNE1 from Kang et al.12 from an extracellular (a) or side view (b). On the S1 helix, S140 (yellow) points toward S4, whereas V141 (blue) points toward KCNE1. The extracellular end of the S6 helix is in proximity to KCNE1. (c) Cartoon representation of proposed molecular mechanisms of S140G and V141M. The channel pore is represented by a cylinder with the K+ permeation pathway at its center. For KCNQ1 alone (top row), V141M has minimal effect on channel gating, whereas S140G disrupts S4 and slows its movement. In the presence of KCNE1 (bottom row), both V141M and S140G alter VSD-pore coupling and slow pore closing. V141M may directly disrupt the orientation of KCNE1, impairing motion of the S6 during channel closing. S140G may cause a similar disruption of KCNE1 indirectly through other residues on S1 that face KCNE1.

References [+] :

Abitbol, Stilbenes and fenamates rescue the loss of I(KS) channel function induced by an LQT5 mutation and other IsK mutants. 1999, Pubmed, Xenbase