Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels

  1. Michaela A Edmond
  2. Andy Hinojo-Perez
  3. Xiaoan Wu
  4. Marta E Perez Rodriguez
  5. Rene Barro-Soria  Is a corresponding author
  1. Department of Medicine, Miller School of Medicine, University of Miami, United States
  2. Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, United States

Abstract

Neuronal KCNQ channels mediate the M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that different mutations have on channel function remains poorly defined, largely because of our limited understanding of the voltage-sensing mechanisms that trigger channel gating. Here, we define the parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-associated mutations using cysteine accessibility and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of eight to nine amino acids in the S4 becomes exposed upon voltage sensing domain activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. VCF reveals different mechanisms by which different epilepsy-associated mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

Editor's evaluation

This study makes an important technical advance with measurements of voltage-dependent conformational changes of KCNQ2/Kv7.2 channels, measurements which are known to be extremely difficult for this biologically important channel. This advance sheds light on the mechanism by which two human mutations act and opens the door to further investigations of voltage sensor movement in these channels.

https://doi.org/10.7554/eLife.77030.sa0

Introduction

Voltage-gated K+ (Kv) channels play a crucial role in regulating excitability, and dysregulation of their function has been associated with several disorders, including cardiac arrhythmias, epilepsy, and autism. Kv channels, including all members of the Kv7 family (Kv7.1–Kv7.5, also known as KCNQ as they are encoded by KCNQ1–5 genes; Jespersen et al., 2005; Jentsch, 2000; Abbott and Pitt, 2014), are highly heterogenous and widely expressed in excitable cells where they regulate resting membrane potential, shape the firing and duration of action potentials, and control rhythmic events (Hille, 2001).

One of the major potassium currents throughout the central and peripheral nervous systems is the M-current. The M-current is mainly conducted by heterotetrameric combinations of KCNQ2/3 and KCNQ3/5 channels (Brown and Adams, 1980; Halliwell and Adams, 1982; Wang et al., 1998; Schroeder et al., 2000), but homotetrameric assemblies of channel subunits have also been shown to generate the M-current in neurons (Schroeder et al., 2000; Soh et al., 2014). KCNQ are non-inactivating channels with slowly activating and deactivating kinetics and a negative voltage for half-activation (Brown and Adams, 1980; Halliwell and Adams, 1982). These biophysical properties make KCNQ channels important regulators of neuronal excitability. For example, the peculiar lack of inactivation at voltages near the threshold for action potential initiation confers KCNQ channel’s dominant role in regulating membrane excitability as one of the main outward sustained currents. Thus, inhibition of the KCNQ channel lowers the action potential threshold and slows excitatory postsynaptic potentials (Adams et al., 1982), resulting in reduced adaptation and prolonged repetitive neuronal firing (Adams et al., 1986). Among the KCNQ family of proteins, KCNQ2 channels have received particular attention because mutations in this channel have been associated with a variety of neurodevelopmental phenotypes (Jentsch, 2000; Greene and Hoshi, 2017; Geisheker et al., 2017), including epileptic encephalopathy (Weckhuysen et al., 2012; Weckhuysen et al., 2013; Orhan et al., 2014; Saitsu et al., 2012; Kato et al., 2013; Rauch et al., 2012), and more recently, autism (Cornet et al., 2018). Since KCNQ2 channels are central to physiological and pathophysiological events, it is important to understand the voltage-dependent mechanisms underlying channel opening and thereby define its role in physiological control of neuronal excitability, as well as providing a better understanding of how specific disease-associated variants alter KCNQ2 channel function.

The recently elucidated cryo-EM structure of human KCNQ2 channels (Li et al., 2021b) shows that, like canonical Kv channels (Long et al., 2005), KCNQ2 has a domain-swapped tetrameric architecture with six transmembrane helices (S1–S6) and cytosolically oriented N-terminal and C-terminal that form functional tetramers. The S5–S6 of the four subunits form a centrally located potassium selective pore that is flanked by the four voltage- sensing domains (VSDs; S1–S4; Long et al., 2005), where the C-terminal end of the S6 segments forms the gate (Long et al., 2005; del Camino and Yellen, 2001). Similar to that seen in other Kv channels, the fourth transmembrane segment contains several highly conserved positively charged amino acid residues that move in response to changes in membrane voltages that function as the voltage sensor (Mannuzzu et al., 1996; Larsson et al., 1996; Seoh et al., 1996; Aggarwal and MacKinnon, 1996). Gating current recordings have not been resolved for KCNQ2 channels, likely due to low channel density within the membrane and/or the slow kinetics of activation compared to other Kv channels (Miceli et al., 2012). Insight into the S4 movement of KCNQ2 has been inferred by previous mutagenesis studies showing that charge neutralization of the arginine residues in S4 altered the voltage sensitivity of channel opening (Soldovieri et al., 2019; Miceli et al., 2008). In addition, a disulfide crosslinking study showed that cysteine-substituted residues in the extracellular end of S4 crosslinked with a cysteine in S1 only in the closed state, further implying S4 movement (Gourgy-Hacohen et al., 2014). Although these studies provided insight into S4 rearrangements, they did not define parameters of S4 movement, such as the dynamic relationship between S4 activation and pore opening during voltage-controlled gating of KCNQ2 channels.

Our understanding of the voltage-controlled activation mechanisms of KCNQ2 channels is limited compared to other Kv channels like the related KCNQ1, whose physiological role in cardiac tissue has been extensively investigated (Nerbonne and Kass, 2005). Kv channel opening can occur either after all four S4 have been activated (Zagotta et al., 1994), or alternatively through independent activation of each S4 (Horrigan et al., 1999), as also reported for KCNQ1(35). Interestingly, pore opening of KCNQ1 channels can occur from two defined S4 conformations involving intermediate and fully activated S4 states (Zaydman et al., 2014; Hou et al., 2017; Hou et al., 2020). This activation scheme in which opening may occur from multiple S4 states, has provided a valuable framework to understand voltage-dependent gating of KCNQ1 with different accessory subunits, thereby allowing interpretation of its versatile physiology. The lack of mechanistic understanding of voltage-dependent gating in neuronal KCNQ2 channels has made it difficult to understand the impact that disease-associated variants will have on channel functionality. We here describe the mechanisms underlying voltage sensor movement in KCNQ2 channels relevant to understand epilepsy-associated KCNQ2 mutations.

We provide an extensive exploration of positions where cysteine could be inserted into the S3-voltage sensor (S4) loop and S4 helix and used with both voltage clamp fluorometry (VCF) (Mannuzzu et al., 1996) and cysteine accessibility (Larsson et al., 1996) to study S4 activation and its influence on disease-causing mutations. Cysteine accessibility reveals that a stretch of eight to nine S4 residues becomes exposed upon VSD activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. In addition, VCF data shows that two epilepsy-associated mutations – R198Q and R214W – perturb channel opening through two distinct mechanisms with R198Q directly altering S4 movement, while R214W uncouples voltage sensor movement and pore opening. These results provide critical information about KCNQ2 channel gating that will aid in future studies on KCNQ2 channelopathies.

Results

State-dependent external S4 modifications consistent with S4 as voltage sensor

The combination of cysteine-scanning mutagenesis and methanethiosulfonate (MTS) derivative modification is a powerful tool to study conformational changes in ion channel gating. This methodology assumes that covalent modification of substituted cysteines leads to functional changes in channel gating (Figure 1A). We test how the fourth transmembrane domain (S4) moves in the KCNQ2 channel by measuring state-dependent accessibility changes of introduced cysteines in the S4 (or in the S3–S4 linker) (Figure 1A–B). We assess the state-dependent modification of substituted cysteines by plotting the membrane-impermeable thiol reagents (MTS)-induced change in current against the cumulative exposure to MTS reagents at either hyperpolarized (closed) or depolarized (open) voltages (See Materials and methods section and voltage protocols on top of Figure 1C). This approach has been previously used to demonstrate that S4 crosses the membrane during gating of the Shaker channel (Larsson et al., 1996) and assumes that changes in state-dependent modification rates of substituted-cysteines by externally applied MTS compounds indicate that some residues in S4 move (outward) across the membrane during channel activation.

Figure 1 with 5 supplements see all
State-dependent modification of KCNQ2-R198C by external methanethiosulfonate (MTSET) is consistent with outward S4 motion.

(A) Cartoons showing cysteine accessibility method with MTSET and two-electrode voltage clamp setup. (B) Sequence alignment of homologous S4 residues in KCNQ2, KCNQ3, KCNQ1, and Shaker channels. (C, E, and G) Currents from oocytes expressing (C) KCNQ2-N190C, (E) R198C, and (G) F202C channels in response to 20 mV voltage steps from –140 mV to +40 mV (left panels) before and after applications of MTSET (after washout, gray) in the closed and (after washout, color-coded) open states. MTSET is first applied (‘closed state’-middle panels) at –80 mV for 5 s in between 25 s washouts for 8–15 cycles and the change in current is measured at +20 mV. On the same cell and after MTSET is washed out of the bath, MTSET is reapplied (‘open state’-middle panels) at +20 mV using a similar protocol. We used 100 μM MTSET in (C) and (G), and 50 μM MTSET in (E). (D, F, and H) Steady-state conductance/voltage relationships, G(V)s, (lines from a Boltzmann fit) of (D) KCNQ2-N190C, (F) R198C, and (H) F202C channels normalized to peak conductance before MTSET application (black). The G(V) relationships after MTSET application in the closed (–80 mV, gray) and open (+20 mV, color-coded) states are obtained from recordings of panels (C), (E), and (G), (‘closed- and open state’-middle panels, respectively); mean ± SEM, n=3–24. (I) The rate of MTSET modification of R198C channels at +20 mV (red squares) or –80 mV (gray squares) was measured using the difference in current amplitudes taken at 400 ms after the start of the +20 mV voltage step, vertical dashed arrows in (E) between the first sweep (before MTSET application, which is represented by #0 along the vertical dashed arrows in (E) and normalized to zero) and the subsequent sweeps (after several MTSET application which are represented by #1, 2, …8–9 along the vertical dashed arrows in (E)) from the ‘closed-state and open-state’-middle panels. The normalized delta current amplitude was plotted versus the cumulative MTSET exposure and fitted with an exponential. The fitted second-order rate constant in the open state protocol is shown in red. kopen = 3230 ± 3.8 M–1 s–1 (n=8). (J) Cartoon representing the voltage-dependent cysteine accessibility data. MTSET modifies N190 in both the closed and open states. While F202 remains unmodified in both states (seemingly buried in the membrane), R198 becomes accessible only in the open state. Dashed line indicates the proposed outer lipid bilayer boundary.

In total, we made eight cysteine mutants within the S4 (or in the S3–S4 linker) of KCNQ2 channels (Figure 1 and Figure 1—figure supplement 1). The cysteine mutants (N190C, A193C, S195C, A196C, R198C, S199C, L200C, and F202C) vary in steady-state conductance/voltage curve (G(V)) when compared to wild-type-KCNQ2 channels (Figure 1—figure supplement 1B, and Supplementary file 1). We express these mutants, one at a time, in Xenopus oocytes and use two-electrode voltage clamp to probe the external accessibility of the substituted cysteines to the MTS reagent (2-[ammonium]ethyl) methanethiosulfonate (MTSET) at both hyperpolarized (closed) and depolarized (open) voltages (Figure 1 and Figure 1—figure supplement 1). As an important control, MTSET does not modify wt-KCNQ2 at either depolarized or hyperpolarized voltages (Figure 1—figure supplement 1A and Supplementary file 1). Note that the perfusion system quickly delivers a 5 s pulse of MTSET to the external surface of oocytes. This ensures perfusion of MTSET only at the indicated voltage as shown by the time course of solution exchange from 100 mM NaCl to 100 mM KCl (Figure 1—figure supplement 2).

For each cysteine mutant, we measure a family of currents in response to 20 mV voltage steps from –140 mV to +40 mV before (Figure 1C, E and G, left panels, and Figure 1—figure supplement 1C-G, left) and after applications of MTSET in both states (Figure 1C, E and G, middle and right panels, and Figure 1—figure supplement 1C-G, middle and right panels). To assess the state-dependent modification of substituted cysteines, we first apply MTSET at hyperpolarized voltages (closed channels) for 5 s in between 25 s washouts for 8–15 cycles and assayed the change in current at +20 mV (Figure 1C, E and G, ‘closed state’-middle panels). On the same cell and after MTSET is washed out of the bath, we repeat a similar protocol but now applying MTSET at +20 mV (Figure 1C, E and G, ‘open state’-right panels). External application of MTSET in the closed state significantly increases the current amplitude and shifts the G(V) relationship of N190C channels to the left (ΔGV1/2 N190C closed = –6.3 ± 1.3 mV, n=11, Figure 1C–D and Figure 1—figure supplement 3A-B, gray). We find that after the second MTSET application (now using the open state protocol) there is no additional increase in the current amplitude, and the G(V) relationship is not shifted further (ΔGV1/2 N190C open = –7.0 ± 1.7 mV, n=9, Figure 1C–D, and Figure 1—figure supplement 3A-B, yellow), as if all N190C channels were fully modified in the closed state. To test whether N190C is also accessible in the open state, we performed a separate experiment in which MTSET is applied at +20 mV (Figure 1—figure supplement 4). Using this protocol, we find that MTSET also increases the current amplitude and shifts the G(V) relationship of N190C channels to negative voltages (ΔGV1/2 N190C open = –12.2 ± 10 mV, n=5, Figure 1—figure supplement 4). Together, these results suggest that N190 is accessible and exposed to the extracellular solution in both the closed and open states (Figure 1J, yellow).

For R198C, external MTSET application in the open state (at +20 mV), increases the current amplitude and left-shifts the G(V) relationship (Figure 1E–F red and Figure 1—figure supplement 3C-D, red). In contrast, when MTSET is applied in the closed state (at –140 mV), R198C channels are not modified (Figure 1E–F gray and Figure 1—figure supplement 3C-D, gray). Since MTSET modifies residue R198C relatively fast at depolarized potentials (Figure 1E,I, red) but not significantly at hyperpolarized potentials (Figure 1E,I, gray), that suggests that this residue is not accessible (i.e. is buried in the membrane) in the closed state (with S4 down) but becomes accessible in the open state (with S4 up, Figure 1J, red). We find similar state-dependent modifications upon external MTSET perfusion for KCNQ2 channels with cysteine substitutions at residues A193C, S195C, A196C, S199C, and L200C (Figure 1—figure supplement 1C-G). External application of 0.1 mM MTSET (and even up to 1 mM) shows no modification of channels with cysteines introduced further toward the C-terminus of the S4, such as F202C in either the open or closed states (Figure 1G–H and Figure 1—figure supplement 3E-F). F202 is the outermost N-terminal residue in the S4 segment to remain unmodified by external MTSET. This result suggests that either F202C remains buried in the membrane during S4 activation (unmodified), even under conditions of high MTSET concentrations and strong depolarization to +20 mV (Figure 1J, brown), or alternatively that the modification does not significantly alter channel gating. Figure 1J, Figure 1—figure supplement 1H, and Figure 1—figure supplement 5 show cartoons summarizing a map of the voltage-dependent distribution of S4 residues in the resting and activated conformations inferred from the extracellular cysteine accessibility data.

Tracking S4 movement of KCNQ2 channels using voltage-clamp fluorometry

VCF allows simultaneous measurements of S4 movement (by fluorescence) and gate opening (by ionic current) based on the physicochemical properties of fluorescent probes in different environments and on their short half-life once excited (Lakowicz, 2006; Mannuzzu et al., 1996). To identify the best candidate site for fluorescent tracking of S4 movement in KCNQ2 channels using VCF, we first performed, one at a time, cysteine substitution of residues in the extracellular S3-S4 linker (Figure 2A). We find that this region exhibits sensitivity to cysteine mutations (Figure 2B–D), similar to a previous report for homologous cysteine mutations in KCNQ3 channels (Kim et al., 2017). Compared to wt-KCNQ2 channels, the mutants Q188C, G189C, and N190C shift the steady-state conductance/voltage curve, G(V), toward positive voltages (ΔGV1/2 = +9.3 ± 0.7 mV, ΔGV1/2 = +24.2 ±0.7 mV, and ΔGV1/2 = +29.8 ± 0.3 mV, respectively), whereas the mutants V191C and F192C shift the G(V) curves toward negative voltages (ΔGV1/2 = –2 mV ± 0.9 mV and ΔGV1/2 = –12.5 mV ± 1.7 mV, respectively; Figure 2C–D, open symbols and Supplementary file 1). Unlike the F192C mutant, the wt channels and the other cysteine mutants exhibit a sigmoidal time course and appear to have multiple exponential components (Figure 2B), with the F192C mutant generating the fastest time course of current activation (Figure 2—figure supplement 1A). Moreover, all five cysteine substitutions showed a further leftward G(V) shift upon fluorophore labeling (Figure 2D, filled symbols). The mechanisms by which the cysteine substitutions and their dye-conjugated versions may alter some of the gating properties are unknown and were not investigated further.

Figure 2 with 1 supplement see all
Labeled KCNQ2-F192C channels track S4 movement.

(A) Cartoon showing the topology of one KCNQ2 subunit and the residues in the S3–S4 linker that were sequentially mutated to cysteine. (B) Currents from oocytes expressing a series of cysteine mutants in the S3–S4 linker of KCNQ2 channel. Cells are held at –80 mV and stepped to potentials between −140 mV and +40 mV in 20 mV steps for 2 s followed by a tail to –40 mV. (C) Normalized G(V) (lines from a Boltzmann fit) curves from (open symbols) unlabeled and (filled symbols) Alexa-488-maleimide labeled wt and cysteine mutations shown in (B). The midpoints of activation for the fits are shown in Supplementary file 1. Data are mean ± SEM, n=5–24; see Materials and methods. (D) Summary of G(V)1/2 values for the wt and cysteine mutants (open symbols) before and (filled symbols) after Alexa-488-maleimide labeling. (E) Cartoon representing the voltage clamp fluorometry (VCF) technique. A cysteine is introduced at position 192 (close to the voltage sensor [S4]) and labeled with a fluorophore tethered to a maleimide group (Alexa-488–5 maleimide). Upon voltage changes, labeled-S4s move and the environment around the tethered fluorophore changes, altering fluorescence intensity. Both current and fluorescence are recorded simultaneously using a VCF set up. (F) Representative current (black) and fluorescence (cyan) traces from Alexa-488-labeled KCNQ2-F192C channels (KCNQ2*) for the indicated voltage protocol (top). A sweep to 0 mV is depicted in red to facilitate comparison of time courses in (H–I). (G) Normalized G(V) (black solid lines from Boltzmann fit) and F(V) (cyan circles and cyan solid line from a Boltzmann fit) curves from (black circles) F192C-Alexa-488 ‘KCNQ2*’, (black squares) unlabeled F192C, and (gray squares) wt channels. The midpoints of activation for the fits are: GV1/2F192C-Alexa-488 = –77.1 ± 2.7 mV, (n=9), FV1/2F192C-Alexa-488 = –87.1 ± 3.9 mV, (n=8), GV1/2 unlabeled-F192C = –55.8 ± 0.8 mV, (n=9), and GV1/2wt = –43 ± 0.7 mV, (n=21), Supplementary file 1. Data are mean ± SEM; see Materials and methods. (H) Representative current time courses of (gray) wt, (black) F192C, and (red) F192C-Alexa-488 channels in response to the protocol shown on top. The dashed line represents 50% of the maximum current level at the end of the depolarizing pulse. (I) The time courses of current activations are quantified as the time to reach half the maximum current level at the end of the depolarizing pulse in (H, dashed line). Data are presented as mean ± SEM, n=9–21. Statistical significance was determined using ANOVA and Tukey’s post hoc test, and significance level was set at p<0.05. Asterisks denote significance: p<0.05*. V: voltage; PD in this cartoon represents: photodiode photodetector.

Out of the five KCNQ2 substituted cysteines in the S3–S4 linker, the labeled mutant KCNQ2-F192C exhibits the most reliable and robust voltage-dependent fluorescence signals (maximum fluorescence change, dF/F~1%) that saturates well at negative and positive voltages, when either labeled with Alexa488 5-maleimide (Figure 2E–G) or DyLight488-maleimide (Figure 2—figure supplement 1B,D). The time courses of fluorescence signal labeled with either fluorophore are similar (Figure 2—figure supplement 1C, right panel). The fluorescence signals in Figure 2F (and Figure 2—figure supplement 1B) have a non-linear voltage dependence and are much slower than the voltage changes per se, which suggest that the fluorescence changes are not electrochromic responses of the dye to voltage changes. Importantly, the changes in fluorescence signal are likely caused by Alexa488-maleimide (or by Dylight488-maleimide) attached to F192C as oocytes expressing wild-type KCNQ2 channels treated with either fluorophore do not show a voltage-dependent fluorescence signal (Figure 2—figure supplement 1E,F). While KCNQ2-F192C channels labeled with both fluorophores render robust fluorescence signals (Figure 2F and Figure 2—figure supplement 1B), we use Alexa488- maleimide to label KCNQ2-F192C (henceforth called KCNQ2*) throughout the study.

Figure 2F shows VCF from labeled KCNQ2* channels in response to a family of voltage steps (from –160 to +60 mV). The steady-state fluorescence/voltage curve (F[V]), tracks the G(V) of KCNQ2* channels (F(V)1/2 F192C = –87.1 ± 3.9 mV, n=8 and G(V)1/2 F192C = –77.1 ± 2.7 mV, n=9, Figure 2G and Supplementary file 1). The gating properties of KCNQ2* channels (G[V] and the time courses) deviate from that of wt and unlabeled KCNQ2-F192C channels (Figure 2G–I). Labeling F192C with Alexa488-maleimide (or with Dylight488-maleimide) shifts the G(V) relationship to negative voltages relative to unlabeled KCNQ2-F192C and wt channels (ΔGV1/2 = –21.3 ± 0.8 mV and ΔGV1/2 = –35.4 ± 2.2 mV, respectively, Figure 2C, D and G and Figure 2—figure supplement 1D). Moreover, compared to wt KCNQ2 channels, both labeled and unlabeled F192C accelerate the time course of current activation (Figure 2H–I).

Next, we measure the time course of fluorescence signals and ionic currents of KCNQ2* channels during both depolarization-induced activation and repolarization-induced deactivation using VCF (Figure 3). We use a prepulse of −120 mV to completely close the channel before stepping to the test voltages (Figure 3A and C). The fluorescence signal decreases in response to the prepulse to −120 mV (Figure 3A and C, arrow), indicating that not all voltage sensors are in their resting position at the −80 mV holding potential. There is a close correlation between the time course of fluorescence signals and ionic currents at all the voltages tested (Figure 3B and D). The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1; Osteen et al., 2010b) and KCNQ3 channels (Kim et al., 2017; Barro-Soria, 2019).

Fluorescence from KCNQ2* correlates with channel opening.

(A–D) Representative current (black) and fluorescence (cyan) traces from KCNQ2* channels for the activation (A) and deactivation (C) voltage protocols (top). In response to the prepulse to −120 mV, the fluorescence signal decreases (cyan dashed arrow), indicating that not all voltage sensors were in their resting position at the holding potential (−80 mV). Representative experiments showing time courses of (B) activation and (D) deactivation of current (black) and fluorescence (cyan) signals from KCNQ2* channels at different voltages as in (A) and (C), respectively. Note that the current and the fluorescence signals correlate during both channel activation and deactivation.

The voltage dependence of A193C accessibility matches the GV curve of KCNQ2 channels

We use the state dependent modification of A193C by MTSET (Figure 1—figure supplement 1C) to measure the rate of access to MTSET at different voltages as an independent assay of S4 movement in KCNQ2 channels (Figure 4). External MTSET modification speeds up the activation of A193C channels and increases the current amplitude (Figure 4B). While MTSET modifies A193C channels at voltages more positive than –100 mV (Figure 4B and C), the rate of MTSET modification of KCNQ2 A193C was fivefold faster at +20 mV compared to −100 mV (Figure 4C and Supplementary file 1). The modification rate for A193C approaches zero between −140 mV and −160 mV, as if A193C is inaccessible at those voltages (Figure 4D). The voltage dependence of the modification rate by MTSET (mod. rate[V]) follows the G(V) for A193C channels (mod. rate[V] = –72.8 ± 24.5 mV, n=3–8 and G[V] = –70 ± 2.4 mV, n=12, Figure 4D). Because steady-state conductance/voltage curves, G(V)s, of A193C and labeled F192C channels are similar (GV1/2A193C = –70 ± 2.4 mV, [n=12] and GV1/2F192C-Alexa = –77.1 ± 2.7 mV, [n=9], Figure 4E and E’), and under the assumption that these two channels use the same S4 movement to generate these similar G(V)s, we compare the voltage dependence of the modification rate (mod. rate) of A193C with the voltage dependence of the fluorescence of labeled F192C. The mod. rate(V) of A193C has a similar voltage dependence as the F(V), (F[V] = –87.1 ± 3.9 mV, n=8, Figure 4E), as if the fluorescence of KCNQ2* accurately reflects S4 movement.

Accessibility of residue A193C supports voltage-dependent motion of S4 segment.

(A) Cartoon representing extracellular cysteine accessibility of residue A193C as in Figure 1A. (B) Currents in response to +20 mV voltage steps before (gray trace #0) and during several 5 s applications of methanethiosulfonate (MTSET) at +20 mV (green traces #1–7), –100 mV (blue traces #1–12), and –140 mV (black traces #1–19) on A193C channels for the indicated voltage protocol. We repeat MTSET applications (10 μM at +20 and –100 mV, and 20 μM at –140 mV) in between 25 s washouts as shown in each voltage protocol. (C) Normalized current of A193C during MTSET exposure at +20 mV (green), 0 mV (orange), – 80 mV (pink), –100 mV (blue), and – 140 mV (black). (D and E) Normalized G(V) curves (squares and black line from a Boltzmann fit) of A193C channels and voltage dependence of the modification rate (mod. rate [V], green circles and green line from a Boltzmann fit) for MTSET to residue A193C. In (E), dashed lines represent ‘wt’ KCNQ2* (black) G(V) and (cyan) F(V) curves for comparison. (E’) Summary of G(V)1/2 for (open squares) A193C and (open gray circles) labeled F192C-Alexa channels. Data are presented as mean ± SEM, n=9–12. Statistical significance was determined using paired Student t-test and significance level was set at p<0.05, p=0.062. The midpoints of activation for the fits are: GV1/2A193C = – 70 ± 2.4 mV, (n=12) and GV1/2F192C-Alexa = –77.1 ± 2.7 mV, (n=9); Mod. rate V1/2 A193C = –72.8 ± 24.5 mV, (n=3–8); GV1/2A193C = – 70 ± 2.4 mV, (n=12); see Figure 2G for KCNQ2* GV1/2 and FV1/2, values.

Disease-causing mutations differentially affect S4 and gate domains

Next, we investigate the mechanism(s) by which the epilepsy-associated mutations R198Q and R214W (Millichap et al., 2017; Castaldo et al., 2002) alter KCNQ2 channel voltage-dependent activation. R198Q, which neutralizes the first gating charge of S4 in KCNQ2 channels (Figure 5A), was previously shown to shift the G(V) to more hyperpolarized potentials and to slow the kinetics of deactivation (Millichap et al., 2017). To study the effect of the R198Q mutation, we introduce R198Q into the KCNQ2* background and simultaneously monitor S4 movement (by fluorescence) and gate opening (by ionic current) using VCF (Figure 5A–C). In line with a previous report (Millichap et al., 2017), we find that compared to KCNQ2* channels, the (labeled) KCNQ2*-R198Q mutation causes a hyperpolarizing shift in the G(V) curve (Figure 5C, black arrow and Figure 5—figure supplement 1E) and slows the time course of current deactivation (Figure 5—figure supplement 1B,D, red). VCF shows that KCNQ2*-R198Q channels exhibit fluorescence signals and ionic currents that continue to closely follow each other in terms of their time courses and voltage dependence of activation (Figure 5B–B’ and C and Figure 5—figure supplement 1C,E, red). Moreover, compared to KCNQ2*, KCNQ2*-R198Q channels exhibit fluorescence signals that are shifted to negative voltages (ΔF1/2 = −32.7 ± 1.4 mV) similar to its negatively shifted G(V) curve (ΔG1/2 = −33.2 ± 1.3 mV) (Figure 5C and Figure 5—figure supplement 1E). These results suggest that the R198Q mutant, which neutralizes the first gating charge, alters channel function by directly affecting S4 activation.

Figure 5 with 2 supplements see all
Disease-causing mutations in KCNQ2 channels differentially affect S4 and gate domains.

(A) Cartoon representing the voltage-clamp fluorometry (VCF) technique as in Figure 2A. The localization of the two epilepsy-associated mutations - R198Q (red) and R214W (maroon) are shown. (B) Representative (black) current and (red) fluorescence traces from KCNQ2*-R198Q channels for the indicated voltage protocol (top). (B’) Comparison of the (black) time course of current activation and (red) fluorescence signals from KCNQ2*-R198Q channels in response to the voltage protocol shown. (C) Normalized G(V) (black triangles and black solid line from a Boltzmann fit) and F(V) (red triangles and red solid line from a Boltzmann fit) curves from KCNQ2*-R198Q. (D) Representative (black) current and (maroon) fluorescence traces from KCNQ2*-R214W channels for the indicated voltage protocol (top). (D’) Comparison of the (black) time course of current activation and (maroon) fluorescence signals from KCNQ2*- R214W channels in response to the voltage protocol shown. (E) Normalized G(V) (black squares and black solid line from a Boltzmann fit) and F(V) (maroon squares and maroon solid line from a Boltzmann fit) curves from KCNQ2*- R214W. (C and E) Dashed lines represent KCNQ2-F192C labeled with Alexa-488 (KCNQ2*) G(V) (black) and F(V) (cyan) curves for comparison. The same color code for the two KCNQ2 mutations is shown throughout the figure. The midpoints of activation of the fits are (GVR198Q 1/2 = –110.3 ± 3.5 mV, (n=10), FVR198Q 1/2 = –119.8 ± 4.2 mV, [n=4], GV214W 1/2 = –17.1 ± 0.9 mV, [n=8], and FVR214W 1/2 = – 77 ± 0.6 mV, [n=7]) and in Supplementary file 1. Data are mean ± SEM.

In contrast to R198Q, the R214W mutation was previously reported to shift the G(V) relationship to more depolarized voltages, to slow the kinetic of current activation, and to accelerate the kinetic of current deactivation (Castaldo et al., 2002). We also introduce R214W into the KCNQ2* background and perform VCF (Figure 5D–E). Compared to KCNQ2*, KCNQ*-R214W channels display a rightward shifted G(V) curve (ΔG1/2 = +60 mV ± 1.8 mV, Figure 5E, black arrow and Figure 5—figure supplement 1E), slow the time course of current activation, and accelerate the time course of current deactivation (Figure 5—figure supplement 1A-D, maroon), as previously reported for R214W channels (Castaldo et al., 2002). VCF shows that in R214W channels, the time course of fluorescence signal precedes the time course of ionic current (Figure 5D’, and Figure 5—figure supplement 1C, maroon). Interestingly, the F(V) curve of R214W, which is similar to the F(V) curve of KCNQ2* channels, is markedly left shifted compared to its G(V) curve (FV1/2 R214W = –77 ± 0.6 mV, [n=7] and GV1/2 214W = –17.1 ± 0.9 mV, [n=8], Figure 5E and Figure 5—figure supplement 1E). The separation between the F(V) and G(V) curves suggests that R214W dissociates voltage sensor (S4) movement from channel opening. Since R214W is in the loop connecting S4 to the S4–5 linker (not within the voltage sensor itself, Figure 5—figure supplement 2A), our data most likely suggests that this mutation affects activation gating without directly affecting the S4 movement which results in fluorescence change.

Discussion

In this paper, we provide functional data characterizing the voltage sensing mechanism of KCNQ2 channels. We show that during activation, a stretch of S4 residues becomes exposed to the extracellular solution, thereby revealing S4 outward motion. Our fluorescence measurements show a close correspondence between the voltage sensor (S4) movement and channel opening in KCNQ2 channels as both voltage dependence and the time courses of fluorescence and ionic current closely correlate. We find that two epilepsy-associated mutations cause shifts in the voltage dependence of channel opening by two different mechanisms, with the R198Q mutation shifting S4 movement while the R214W mutation uncoupling VSD and channel opening. Our findings shed light on the dynamics and state-dependent molecular rearrangements that lead to channel gating. Since KCNQ2 channels play a pivotal role in controlling neuronal excitability, these results provide critical clues to aid in our understanding of the impact of channelopathies on neuronal function. Understanding how mutations affect channel activity can lead to better ways to correct these mutational defects.

Using a state-dependent cysteine modification approach, we map the extracellular boundaries of S4 residues during membrane depolarization. Our cysteine accessibility data suggests that a stretch of 8–9 amino acids (~193 to 200–201), about half of the 17–19 residues forming the S4(21), moves from a membrane-buried position in the resting state to the extracellular solution during activation gating. Previously, mutagenesis and disulfide crosslinking of substituted cysteines or metal-ion bridge experiments inferred putative closed-resting states of S4 of KCNQ2 channels (Gourgy-Hacohen et al., 2014). In this study, the first and second positively charged residues of S4 (R198 and R201) were assumed to interact with the first and second counter-charge residues (E130 and E140) in the S2 segment. This arrangement positioned the gating charge transfer center in S2 (F137) in between R198 and R201 in what was assumed to be a deep closed-resting state of S4 (Figure 1—figure supplement 5A). More recently, the cryo-EM structure of KCNQ2 channels (Li et al., 2021b; PDB:7CR0) revealed the snapshot of the channel in its activated (S4 up) state and the pore in the closed state. This structure shows that R198 and R201 have moved about three helical turns outward (upward) from F137 into a position close to or within the extracellular space (Li et al., 2021b; Figure 1—figure supplement 5B). These rearrangements are in line with our cysteine accessibility data in which residues N-terminal to residue F202 become exposed in the activated state of S4 at strong depolarizations (Figure 1—figure supplement 5D). Our cysteine accessibility data also provides a snapshot of the resting state of S4 in which residues C-terminal to residue A193 are buried at hyperpolarization (Figure 1—figure supplement 5C), as previously predicted (Li et al., 2021b).

To date, the direct measurement of gating charge movement during channel gating (gating current measurements) has not been resolved in KCNQ2 channels (nor in KCNQ3). Therefore, the VCF signals of the F192C mutant reported here represent a valuable tool to study voltage sensing in human KCNQ2 channels. Our study reveals, however, that both labeled and unlabeled F192C mutants alter the gating properties of KCNQ2 channels (Figure 2 and Figure 2—figure supplement 1). Our results show that the S3–S4 loop is highly sensitive to both mutations and dye-conjugations, with some mutants (and labeled conjugates) generating large shifts of the G(V) relation and accelerating the time course of current activation compared to wt-channels. While not optimal, this is not surprising because similar G(V) shifts of about ~ –10 to –30 mV upon Alexa-488 maleimide labeling have been previously reported in this region for other Kv channels, including KCNQ1(35), KCNQ3(40), BK (Savalli et al., 2006), and Kv1.5 (Vaid et al., 2008). We did not explore these effects any further, but it may be possible that tethering the dyes Alexa/Dylight to F192C in KCNQ2 channels could interfere with S4 such that labeled F192C might prevent complete S4 deactivation or could help stabilize the activated (or help destabilize the resting) conformation of the S4, hence shifting the voltage dependence to hyperpolarizing voltages and promoting a faster channel opening. These limitations should be taken into considerations in future studies aiming to refine our understanding of the KCNQ gating mechanisms.

Our VCF data shows that both the steady-state voltage dependence and the time course of S4 transitions of fluorescent-labeled KCNQ2 channels closely follow those of the ionic currents, which have virtually no delay and no sigmoidal time course. The close correlations in time and voltage dependence of fluorescence and current of KCNQ2 channels resemble the one-to-one relationship between S4 movement and channel opening reported in KCNQ1 (without KCNE1) (Osteen et al., 2012), suggesting that these two homologous channels share similar gating mechanisms. VCF on linked concatemers of KCNQ1 subunits showed that all four voltage sensors can move independently and channel opening can proceed from individual voltage sensor movements (Osteen et al., 2012). These findings indicate that S4 does not necessarily require independent conformational changes in all four KCNQ2 subunits before channel opening, as shown for classical voltage-gated K+ channels (Hodgkin and Huxley, 1990), but this needs to be further tested in linked-subunit experiments. In KCNQ4 channels, in contrast to KCNQ2, the S4 moves (measured by gating current) much faster than the rate of activation (ionic current), as if the S4 movement was poorly coupled to opening/closing (Miceli et al., 2012). Indeed, the gating scheme of KCNQ4 resembles that of the uncoupling R214W mutation (discussed below), whose S4 movement clearly precedes ionic current (Figure 5D–D’). Interestingly, the subunit composition of KCNQ channels in the nervous system seems to exhibit higher flexibility and heterogeneity than previously assumed. For instance, besides the well-characterized KCNQ2/3 and KCNQ3/5 heteromeric channels found in the brain, a recent report shows that KCNQ2 not only forms multimeric assemblies with KCNQ5 in vivo, but intriguingly, is able to form part of a more diverse KCNQ2/3/5 heteromeric complex (Soh et al., 2022). Thus, insight into the subtle differences in voltage-sensing mechanisms among different KCNQ channel family members is important to understand the different physiological functions that these channels play in the nervous (Delmas and Brown, 2005), auditory (Kubisch et al., 1999), and cardiac (Wang et al., 1996) systems.

Our work provides a framework to understand more in-depth pathophysiological mechanisms of KCNQ2 variants. The R198Q mutation in KCNQ2 channels causes infantile spasms with hypsarrhythmia and encephalopathy associated with severe developmental delay (Millichap et al., 2017). Compared to KCNQ2*, KCNQ2*-R198Q channels display left-shifted G(V) and F(V) curves. In addition, KCNQ2*-R198Q channels exhibit fluorescence signals and ionic currents that closely overlap in terms of their time courses and voltage dependence of activation (Figure 5B–B’ and C and Figure 5—figure supplement 1C,E, red). Together, these data suggest that the R198Q mutation alters channel function by directly impacting S4 activation. Conversely, fluorescence data from the epilepsy-associated mutation R214W shows a marked separation between the G(V) and F(V) and a faster fluorescence time course compared to the ionic current time course (Figure 5 and Figure 5—figure supplement 1, brown), suggesting that R214W changes the VSD-PD coupling of KCNQ2. How does R214W dissociate voltage sensor movement from channel opening? Unlike the ILT mutation in the Shaker K+ channels, in which pore opening is dissociated from the first VSD activation but coupled to the second (Pathak et al., 2005), KCNQ2*-R214W fluorescence signals do not show a second fluorescence component associated with channel opening. This suggests that the R214W and the ILT mutants decouple VSD-PD through different mechanisms, or alternatively through similar mechanisms but our labeled F192C is unable to resolve the late component of gating charge movement (fluorescence) associated with pore opening. Moreover, previous studies in the related KCNQ1 channel showed that the F351A mutation separated F(V) from G(V) (Osteen et al., 2010a), similar to what is seen with the R214W variant in KCNQ2. Mechanistically, it was postulated that KCNQ1-F351 may couple S4 to pore opening possibly through a physical interaction of F351 with residues within the S4–S5 linker such that point mutations like F351A would alter the VSD-PD interactions, suggesting that F351A eliminates the intermediate open state (Osteen et al., 2010a; Zaydman et al., 2014; Taylor et al., 2020). However, unlike the F351 residue that is localized in the S6 helix (PD) pointing toward the S4–S5 linker of KCNQ1 channels (Sun and MacKinnon, 2020), the R214 residue of KCNQ2 lies in the loop region that connects the S4 to the S4–S5 linker (Li et al., 2021b; Figure 5—figure supplement 2A), as if F351A and R214W may also decouple VSD-PD through different mechanisms.

The recent cryo-EM structures of KCNQ1, KCNQ4, and KCNQ2 channels have provided insights into how mutations in the N-terminal portion of the S4-S5 linker may alter channel gating (Li et al., 2021b; Sun and MacKinnon, 2020; Li et al., 2021a). The cryo-EM structures of KCNQ1 and KCNQ4 show PIP2 bound to these channels close to the S4/S4–S5 interface, in a positively charged pocket. Superimposing the KCNQ2 structure (Long et al., 2005) with the homologous structure of KCNQ1 bound to PIP2(55) (Figure 5—figure supplement 2B), we noted that residue R214 lies very close to PIP2, suggesting that R214 in the KCNQ2 channel could form part of the positively charged pocket that coordinates PIP2 binding (Figure 5—figure supplement 2B). Previous studies in KCNQ1 and KCNQ3 channels have shown that PIP2 directly affects the VSD-PD coupling (Kim et al., 2017; Zaydman et al., 2013), but the molecular details remain unknown. Based on our VCF results (Figure 5D and E), which suggest that the R214W mutant dissociates S4 movement from channel opening, we hypothesize that the positive charge of residue R214 is crucial for PIP2 binding. We propose that in KCNQ2, PIP2 may act like a molecular ‘glue’ that tightly ties the loop connecting S4 and S4–S5 linker such that during depolarization, the S4 movement effectively pulls S4–S5 away from the pore domain to activate potassium conductance. Therefore, charge-neutralizing mutations like the R214W variant, would affect PIP2 binding and, thereby, weaken the VSD-PD coupling. Supporting this idea, previous studies on KCNQ2 channels bearing the charge neutralizing mutations, R214Q or R214W, found that the loss of the positive charge, and not changes in residue size, was the main functional effect of these disease-associated mutations as both smaller hydrophilic glutamine and bulkier aromatic tryptophan residues at position 214 favored the resting conformation of S4 and, as such, promoted more channel closure (Miceli et al., 2008).

One important goal of modern precision medicine is to develop potent/selective therapeutics targeting voltage-gated ion channels. We show that the KCNQ2 variants R198Q and R214W alter the relationship between VSD conformation and gating through different mechanisms. Understanding the impact of human mutations in key regions of the channel, such as the VSD and the pore, will facilitate the prediction of compounds that most effectively restore functionality to specific channel mutations while minimizing potential off-target effects. Small-molecule modulators of KCNQ2 channels have been identified, including the pore opener retigabine (Maljevic and Lerche, 2014; Brodie et al., 2010; Wuttke and Lerche, 2006) and the VSD-targeting ICA family of compounds (Wulff et al., 2009; Wickenden et al., 2008; Roeloffs et al., 2008). Retigabine exhibits poor specificity between KCNQ channel subunits (except for KCNQ1) possibly due to its binding to the S5 segment of the pore (Wuttke et al., 2005), which in contrast to the highly diverse VSD region, shows a more conserved sequence among Kv channels. Pore openers like retigabine, which cause a hyperpolarizing shift in the voltage dependence of activation, might seem like a suitable choice to effectively target VSD-PD uncoupling mutations like R214W, but off-target effects on other KCNQ subunits would need to be considered. Unlike retigabine, ICA-like compounds act on the VSD, a less conserved region compared to the pore, presumably allowing ICA to distinguish between KCNQ subunits (Wickenden et al., 2008; Padilla et al., 2009). Therefore, ICA-like compounds (but in a manner that rightward shifts its voltage dependence) would be more effective to target mutations like R198Q that disturb the VSD. Studies like our, aiming to understand how disease-associated mutations disrupt channel function, will help laying the groundwork for the development of mutation-specific antiepileptic therapies.

In summary, the results presented in this paper provide a foundation to mechanistically understand the voltage-controlled S4 activation that promotes KCNQ2 channel opening. Our cysteine accessibility and fluorescence data add to the existing biophysical and chemical tools to study how KCNQ2 channels open and close the pore in response to changes in the transmembrane voltage. Our findings provide a mechanistic framework to understand how disease-associated mutations may affect channel gating and how drugs can modulate channel function. Understanding which parameters are affected could provide insight into what region may cause channel dysfunction, as exemplified in the epilepsy-associated uncoupling KCNQ2-R214W mutation.

Materials and methods

Chemicals

(2-[Trimethylammonium]ethyl)methanethiosulfonate bromide (MTSET) and sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) were purchased from Toronto Research Chemicals Inc (Downsview, ON, Canada). Alexa Fluor 488 C5-maleimide and Dylight-488-maleimide were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Molecular biology

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The full-length human KCNQ2 construct (NCBI Reference Sequence: NP_742105.1; GI: 26051264) was synthesized (GenScript USA, Piscataway, NJ) and ligated between the BamHI and XbaI sites in the multiple cloning sites of the pGEM-HE vector. This vector had been previously modified to contain a T7 promoter and 3’ and 5’ untranslated regions from the Xenopus β-globin gene (Barro-Soria, 2019). A BglII restriction site (AGATCT) and a Kozak consensus sequence (GCCACC) were added before the start codon (ATG) of the KCNQ2 gene. Point mutations were made in the KCNQ2 gene using the Quikchange XL site-directed Mutagenesis kit (Agilent) according to the manufacturer’s protocol. The correct incorporation of the specific variant was assessed by Sanger sequencing (sequencing by Genewiz LLC, South Plainfield, NJ). The RNA was synthesized in vitro using the mMessage mMachine T7 RNA transcription kit (ThermoFisher Scientific) from the linearized cDNA. mRNA (40–50 nL) was injected into Xenopus leavis oocytes (purchased from Ecocyte) using a NanojectII nanoinjector (Drummond Scientific), and electrophysiological experiments were performed 2–5 days after injection.

Cysteine accessibility measurements in TEVC recordings

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We performed cysteine accessibility to MTS reagent (2-[ammonium]ethyl) methanethiosulfonate (MTSET) in two-electrode voltage clamp (TEVC) recordings as previously described (Larsson et al., 1996; Barro-Soria, 2019). Regular ND96 solution for TEVC contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES (pH = 7.5). Stock concentrations of 100 mM MTS reagents were prepared in distilled water (prechilled to +4°C) and stored at −20°C until needed. The MTSET was diluted to the appropriate concentration in ND96 solution just prior to each recording (~30 s prior to perfusion) and kept on ice for 30 min maximum. We delivered high K+ solution (100 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5, adjusted with KOH) before each day of experiments (prior to application of MTSET) to check that the rate of washin and washout of solutions was fast enough to deliver short durations of MTS- reagents to the oocyte (Figure 1—figure supplement 2). A computer-driven, valve controlled, home-made perfusion system that allowed for a rapid switching (within 2 s) between ND96 and MTS reagents during either the open or closed protocol.

We adapted the open and closed state protocols (Larsson et al., 1996) to study the solvent exposure of the substituted cysteines in S3–S4 and S4 and test whether these cysteine residues were exposed in open and/or closed channels using irreversible covalent modification by MTSET (Figure 1B). Briefly, cells were held at −80 mV for 1 s before stepping to +20 mV for 12 s, then repolarized for another 12 s to −80 mV (for the open state) or voltages between −80 and −140 mV (for the closed state), before stepping to the test potential (+20 mV) to measure the change in currents induced by several 5 s cycles of MTS reagents (see black rectangles in Figure 1C, top protocol). We repeat 5 s MTSET application in between 25 s washouts for 8–12 cycles, as shown in the open and closed protocols in Figure 1C. MTSET concentrations were between 10 and 100 μM. Ionic currents were recorded in TEVC using an OC-725C oocyte clamp (Warner Instruments), low-pass filtered at 1 kHz and sampled at 5 kHz. Microelectrodes were pulled using borosilicate glass to resistances from 0.3 to 0.5 MΩ when filled with 3 M KCl. Voltage clamp data were digitized at 5 kHz (Axon Digidata 1,440 A; Molecular devices), collected using pClamp 10 (Axon Instruments). The rate of modification was measured by plotting the change in the current by the MTSET as a function of the exposure to the MTSET (exposure = concentration MTSET [mM] × time [s], measured in [M s]) and fitted with an exponential equation of the form (I[exposure] = I0 exp[−exposure/τ]). We then calculated the second-order rate constant from the τ values (in M s) as 1/τ=kopen (M−1s−1) of the MTS reaction. Experiments where MTSET modification occurred too quickly (in less than three sweeps) with too high concentrations of MTSET were not included since they cannot accurately be fit with an exponential function to obtain a reliable rate of modification. Results are presented as mean ± SEM (n=number of measurements).

Voltage clamp fluorometry

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VCF experiments were carried out as previously reported (Barro-Soria, 2019). Briefly, aliquots of 50 ng of mRNA coding for KCNQ2 or the KCNQ2 variant RNA were injected into Xenopus laevis oocytes. At 2–5 days after injection, oocytes were labeled for 30 min with either 100 μM Alexa-488 maleimide or 100 μM DyLight-488 maleimide (Thermo Fisher Scientific) in high (K+) solution (98 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.05) at 4°C, in the dark. The labeled oocytes were then rinsed three to five times in dye-free ND96 and kept on ice before each recording to prevent internalization of labeled channels. Oocytes were placed into a recording chamber animal pole ‘up’ in ND96 solution (pH 7.5 with NaOH), and electrical measurements were carried out in TEVC using an Axoclamp 900 A amplifier (Molecular devices). Microelectrodes were pulled to resistances from 0.3 to 0.5 MΩ when filled with 3 M KCl. Voltage clamp data were digitized at 5 kHz (Axon Digidata 1550B via a digital Axoclamp 900 A commander, Molecular devices) and collected using pClamp 10 (Axon Instruments). Fluorescence recordings were performed using an Olympus BX51WI upright microscope. Light was focused on the top of the oocyte through a 20× water immersion objective after being passed through an Oregon green filter cube (41,026; Chroma). Fluorescence signals were focused on a photodiode and amplified with an Axopatch 200B patch clamp amplifier (Axon Instruments). Fluorescence signals were low-pass Bessel-filtered (Frequency devices) at 100–200 Hz, digitized at 1 kHz, and recorded using pClamp 10. When needed, we added 100 μM LaCl3 to the batch solution to block endogenous hyperpolarization-activated currents. At this concentration, La3+ did not affect G(V) or F(V) curves from KCNQ2 channels.

Modeling

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The homology model of KCNQ2 channels with S4 in the resting (down) state was created using the Swiss-model program (https://swissmodel.expasy.org/) with the model of KCNQ1 in the resting state (Kuenze et al., 2019), as template. All images were created in UCSF ChimeraX, version 1.1 (2020-10-07).

Electrophysiology data analysis

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Data were analyzed with Clampfit 10 (Axon Instruments, Inc, Sunnyvale, CA), OriginPro 2021b (OriginLabs Northampton, MA), and Corel-DRAW Graphics Suite 2021 software. To determine the ionic conductance established by a given test voltage, a test voltage pulse was followed by a step to the fixed voltage of –40 mV (tail), and current was recorded following the step. To estimate the conductance G(V) activated at the end of the test pulse to voltage V, the current flowing after the hook was exponentially extrapolated to the time of the step and divided by the offset between –40 mV and the reversal potential. The conductance G(V) associated with different test voltages V in a given experiment was fitted by the relation:

(1) G(V)=A1+(A2A1)/(1+exp(ze(VV1/2)/kBT))

where A1 and A2 are conductances that would be approached at extreme negative or positive voltages, respectively, V1/2 is the voltage that activates the conductance (A1 + A2)/2, and z is an apparent valency describing the voltage sensitivity of activation (e is the electron charge, kB is the Boltzmann constant, and T is the absolute temperature). Due to the generally different numbers of expressed channels in different oocytes, we compare normalized conductance, G(V):

(2) G(V)=G(V)/A2

Fluorescence signals were corrected for bleaching and time-averaged over 10–40 ms intervals for analysis. The voltage dependence of fluorescence F(V) was analyzed and normalized (F[V]) using relations analogous to those for conductance (equations 1 and 2).

Statistics

All experiments were repeated four or more times from at least three batches of oocytes. Pairwise comparisons were achieved using paired Student’s t-test or one-way ANOVA with a Tukey‘s test. Data are represented as mean ± SEM, and ‘n’ represents the number of experiments.

Data availability

All data generated or analyzed during this study are included in the manuscript and supplementary information (all combined in one pdf file).

References

  1. Book
    1. Hille B
    (2001)
    Ion Channels of Excitable Membranes
    Sinauer Associates, INC.

Decision letter

  1. Jon T Sack
    Reviewing Editor; University of California Davis School of Medicine, United States
  2. Kenton J Swartz
    Senior Editor; National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
  3. Jon T Sack
    Reviewer; University of California Davis School of Medicine, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Jon Sack as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kenton Swartz as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The reviewers and editors appreciate the importance of establishing KCNQ2 VCF, the insights it provides into mechanisms of gating, and how this could enable future mechanistic studies. We think such an advance could be appreciated by the readers of eLife. While we are sorry to say the manuscript will not be accepted in current form, it seems possible that substantial revisions might improve the manuscript. If all of the reviewer concerns can be effectively addressed we would be willing to review a revised manuscript. Here we highlight the types of reviewer concerns (details in reviewer comments) that would be the most essential revisions:

1) Improve or remove the kinetic model. The model presented in Figure 6 is insufficiently described and seems to have several flaws including violation of microscopic reversibility. It is not clear if the model effectively builds on or supports the experimental findings. If a model is to be presented, please give a clear description and rationale for its structure and all the choice of all parameters.

2) Improve reporting of MTS modification and interpretation, especially that of N190C.

3) Generally tighten logic of interpretation. The reviewers point out a number of instances where the narrative of the manuscript has an unclear relation to the data presented and data seem over interpreted. Especially concerning were arguments about the number of voltage sensors moving during gating.

In summary, we appreciate the technical achievement of establishing KCNQ2 VCF, and hope for a manuscript where the evidence for every claim is clearly communicated, with limitations, caveats, and alternate interpretations shared as well.

Reviewer #1 (Recommendations for the authors):

I applaud the authors on their thorough characterization of KCNQ2 voltage sensor movements. the distinctions between KCNQ2 and KCNQ1 seem really intriguing. As do the investigations of the gating modulation by R214W.

Suggestions of where the science and its presentation might be strengthened:

A) Address the possibility the only a subset of voltage sensor movements are reported by the fluorophores at position 192. For example, is seems possible that early, independent movements of voltage sensors are totally missed by the fluorescence.

B) Improve the description of how the Markov-chain model is constructed, and the logic of its parameterization. The model seemingly has the form of an MWC gating model, but it is modified strangely and under described. The results describe the gamma and delta transitions as voltage sensor movements and in the model these are combined with a channel opening parameters (L, f) controlling the opening rate. This all seems strange and not well explained. How much of each voltage sensor movement component contributes to fluorescence is also unclear, and hard to make sense of.

C) Make it clearer what data supports each claim in the results. For example, claims are made repeatedly about kinetics while showing only exemplar data and irreversible changes are mentioned but not backed up. There were some seeming discrepancies between the Results narrative and the data (specific instances described below).

D) Further discussion of how understanding the impacts of human mutations on voltage sensor vs pore movements could be valuable. Perhaps this could be in the context of KCNQ drugs that act on the pore, like retigabine, vs other that act on the voltage sensor.

Specific suggestions:

Page 6 "Modification of N190C channels quickly and irreversibly increases the current amplitude, speeds up the kinetic of activation, and changes the voltage dependence of activation in either closed or open N190C channels, albeit to distinct V1/2 values (as also shown earlier for HCN1 channels (40), Figure 1D, E), as if N190C is always accessible and exposed to the extracellular solution (Figure 1K, yellow). "

Quantitation of quickly, irreversibly, current amplitude, kinetic of activation, seem to be lacking, aside from showing exemplar traces. For N190C, evidence of changes in the voltage dependence of activation at -80 mV seem to be lacking: slightly different V1/2 values are in Supplement Table 1, but differences aren't compelling in Fig 1E. Unclear why the -80 mV MTSET differences in V1/2 are considered changes in the voltage dependence of activation for N190C, but not R198C for example. More discussion would be helpful of how modification of N190C at -80 mV could produce a different result than +20 mV, yet still be similarly accessible. It would seem helpful to quantitate the changes in current amplitude in response to the MTSET, for example in the case of N190C at -80 mV and elsewhere that the change in current amplitude is the evidence of modification.

Page 8 "Compared to unlabeled KCNQ2* channels, the time courses of ionic currents of labeled KCNQ2* (labeled with either fluorophore) are similar"

Suggest providing quantitative backing for this claim. The time courses look faster with fluorophore.

Figure 3C Could the Tau on from fluorescence with 20 ms time constants be limited by the 100-200 Hz filtering of the optical signal? Error bars seem to be missing.

Figure 3F The -180 mV fit appears to have a decaying component with a negative amplitude?

Page 8 "signals follow a double exponential time course" debatable, as the fits aren't amazing. Might be better to state "appeared to have multiple exponential components and were fit by a double exponential".

Page 9 "These data also suggests that an individual voltage sensor movement might be sufficient to open the channel." State more clearly what data is suggestive of this?

Fig 5B,D Please more quantitatively analyze the reported similarity and difference in F and I kinetics

Page 10 "the ILT mutation in the Shaker K+ channels "

the ILT mutations dissociate early voltage sensor movement from pore opening, but the ILT pore opening remains coupling to late voltage sensor movements and is detected by extracellular fluorescence measurements similar to those employed in this manuscript (doi: 10.1085/jgp.200409197). The fact that a fluorescence component is not observed with pore opening of KCNQ2*-R214W could suggests a different mechanism of decoupling than ILT, or that the late component of gating charge movement associated with pore opening is not reported by the fluor.

Fig 6B Something seems wrong here: the fit is purported to represent 1/alpha, but with zalpha = 0.43 the tau fast at +40 should be 2.7x faster than -20 mV, and this is not the case.

Fig 6C Tau on F slow as well as Tau on G slow (Fig 3 C) seem to lose their voltage dependence at more positive voltages. This could mean pore opening itself (gamma) has little voltage dependence.

Fig 6F Why connote that f is always to the 1st power? Do L and f only impact the opening rate? The modeling is not described sufficiently to reproduce it. alpha and beta are missing altogether. zdelta/zgamma should also be listed in Supplement Table 2

Page 11 "We find no experimental evidence supporting a constitutive open state (O0 in Figure 6F, gray)" From the model you can calculate the expected Popen at very negative voltages, it could be just a very low Popen.

Page 12 "By decreasing the opening transition (L in Figure 6F) relative to wt KCNQ2 channels, the model also describes the clear separation between F(V) and G(V) curves observed in mutated KCNQ2*-R214W channels (Figure 6H), under the assumption that R214W changes the voltage sensing domain-pore domain (VSD-PD) coupling such that it prevents opening before multiple S4 have activated (Figure 6F, dashed maroon arrow)."

Difficult to parse this sentence. In addition to L, f and gamma are also changed. What changes VSD-PD coupling, isn't that L?

Page 12 "Additionally, data from the R198Q mutation can also be simulated by shifting the voltage dependence to negative voltages. " I imagine this is probably right but the claim is not justified by simulations.

Page 13 Confidence in estimated total gating charge of 7.37 e0 per channel moved during KCNQ2 activation gating is limited due to the issue poorly described parameterization of the gating model.

Page 14 "the overall voltage-dependent gating mechanisms of KCNQ2 is qualitatively similar to that of KCNQ1". To me it seems that the overall voltage-dependent gating mechanism of KCNQ2 is qualitatively distinct from KCNQ1.

Reviewer #2 (Recommendations for the authors):

1. In Abstract and other places, the sentences such as "channel opening does not require multiple VSD movements" are not clear. Do the authors try to say, "the movements of multiple VSDs" or "the movements of VSDs in multiple steps"?

2. Did N190C really modify the channel at both -80 mV and +20 mV? Why would the same covalent modification of the channel at two voltages result in different GV relations (Fig 1E)? Is there another Cys in the channel that was modified differently at the two voltages? Did the authors use different protocols to measure GV in these two conditions? These need to be explained. The authors claimed a similar result in a reference, but it was not obvious if the reference showed the similar result or explained the result. The authors can cite the reasons given by the reference (40) if these can make sense of the results in Fig 1E.

3. Fig 1J at -80 mV: why is the current amplitude 0? It does not seem to be consistent with the description in the legend or J'.

4. Fig 2C and Fig 2-FigS1E: The FV with Alexa labeling increases at voltages >0 mV. Is this real or artifact? If real, does it indicate a second VSD movement?

5. In Fig 4E, the comparison between MTS modification rate of A193C and FV of KCNQ2* makes no sense. These two curves derived from different mutations and modifications may overlap by coincidence.

6. The data in Fig 6B-E seem to differ from the data in Fig 3C,D although the legend of Fig 6 claims that these are the same data. For instance, in Fig 3C the fast component of F does not show a voltage dependence, but in Fig 6B and C the fast and slow components of F show a similar voltage dependence.

7. The model in Fig 6F raises several concerns: (1) Why do the vertical transitions have the rates of VSD activation, while they should represent pore opening/closing? (2) What does f represent in the scheme? Can it be part of L? (3) Detailed balance is violated in the left-most loop connecting C0, O0, O1, and C1.

8. In Fig 6G, which states were used to represent currents? Which states represent fluorescence? Particularly, with both the horizontal and vertical transitions represent VSD activation, the rationale for simulating fluorescence need to be justified and the methods clearly described.

Reviewer #3 (Recommendations for the authors):

I was very happy to read this paper and feel that this work is an important step forward for those working on these channels. A lot of progress has been made with KCNQ1 because of the relative ease of recording VCF signals, whereas similar work in KCNQ2-5 has been difficult. The KCNQ2-5 channels differ significantly from KCNQ1 in terms of their function and auxiliary protein regulation, so the development of useful tools to carry out detailed biophysical studies on these channels is valuable and took quite a heroic effort.

I have quite a few comments, just important things that I would add to the paper in the interest of being thorough and not over-interpreting some of the findings.

1. Page 3. "muscarine-regulated M-current". I would hesitate to call it 'muscarine regulated' as it can be sensitive to a variety of neurotransmitters that signal via Gq (ie. acetylcholine... I agree with the historical perspective of naming the current, but the wording may imply physiological regulation to some readers).

2. Page 9: "These data also suggest that an individual voltage sensor movement might be sufficient to open the channel". The basis for this interpretation is not clear (at least not at this stage of the paper). Also, as mentioned in the public review, a counterpoint to this observation is that the KCNQ2 or KCNQ3 currents typically exhibit a sigmoidal time course (also see Figure 1) to activation which might be accounted for by a requirement for multiple subunits to reach an activated conformation. Could this arise because arise because the dye labeling may prevent complete VSD deactivation or interfere with gating in some other way. This is also brought up at the top of page 14 and I have concerns that this could be a contentious statement. I would suggest more caution when describing and interpreting these properties.

3. One way to potentially address this explicitly (ie. point #2) would be to include a direct comparison of unmodified and modifier I192C (and maybe WT KCNQ2 as well) in Figure 2. It is 100% fine with me that there are differences, but it should be clearly shown and described as a consideration when interpreting data, and some comparison like that would help readers.

4. The other technical concern that I had was about fitting the fluorescence traces and perhaps adding complexity where it is not needed and not necessarily supported by data (perhaps this is being done due to analogy to prior work in KCNQ1). Based on the sample sweeps, there does not usually seem to be a great reason to fit with 2 components (eg. Figure 3) - is it really necessary in this case (ie. would it make a difference in terms of the predicted currents, especially given the uncertainty about sigmoid character of current activation)? A few other issues with the description of the model are that some parameters appear to be missing from Supplemental Figure 2 (ie. Alpha and Beta rates, and z for gamma and delta rates). In the text it seems that the gamma and delta rates are meant to be associated with channel opening, but the large amplitude fast component (alpha+beta rates) seem to correlate with the early stages of channel opening, it seems. Perhaps clarifying this by showing individual fit components, or simplifying the fitting/model would be helpful.

https://doi.org/10.7554/eLife.77030.sa1

Author response

Essential revisions:

1) Improve or remove the kinetic model. The model presented in Figure 6 is insufficiently described and seems to have several flaws including violation of microscopic reversibility. It is not clear if the model effectively builds on or supports the experimental findings. If a model is to be presented, please give a clear description and rationale for its structure and all the choice of all parameters.

2) Improve reporting of MTS modification and interpretation, especially that of N190C.

3) Generally tighten logic of interpretation. The reviewers point out a number of instances where the narrative of the manuscript has an unclear relation to the data presented and data seem over interpreted. Especially concerning were arguments about the number of voltage sensors moving during gating.

We thank the editor for their helpful comments. In response, we have removed the kinetic model as suggested by all three reviewers. We agree that the model presented in the original Figure 6 was underdeveloped and would need more experimental data to better describe KCNQ2 channel gating. This kinetic model is deleted in the revised version. (2) We have also added new data to improve our understanding and interpretation of MTSET modification data, including MTSET modification of the N190C mutant in both closed and open states, addressing the comments of Reviewer 1 and 2. (3) We tightened our conclusions to the experimental findings by thoroughly and clearly communicating limitations, caveats, and alternative interpretations, particularly to those concerning labeled and unlabeled F192C and restraining from informing about the number of S4 moving during gating in the results, as suggested by reviewers. Specifics on all these points are discussed below in greater detail.

Reviewer #1 (Recommendations for the authors):

I applaud the authors on their thorough characterization of KCNQ2 voltage sensor movements. the distinctions between KCNQ2 and KCNQ1 seem really intriguing. As do the investigations of the gating modulation by R214W.

Suggestions of where the science and its presentation might be strengthened:

A) Address the possibility the only a subset of voltage sensor movements are reported by the fluorophores at position 192. For example, is seems possible that early, independent movements of voltage sensors are totally missed by the fluorescence.

We thank the reviewer for this observation. We agree with the reviewer that early, independent movements of voltage sensors might have been missed by the fluorescence. We have now re-analyzed the data and determined that while the time course of the fluorescence appeared to have multiple exponentials, our fluorescence data lacked sufficient resolution to reliably estimate in detail the first (fast) component. This might be because of the low signal-to-noise ratio of our VCF or/and as correctly noted by reviewer# 1 below, because the filtering might have limited the tau-on from the optical signal (shown to be 20 ms in Figure 3C of the original submission).

That is why, as suggested by reviewers # 3, we have removed the kinetics comparison of fluorescence and current from the revised version of Figure 3 and do not claim the existence of fast and slow fluorescence components. This comparison on the original submission primarily served to support the Markov kinetic model, which has been removed from the revised manuscript. We now simply state: …” There is a close correlation between the time course of fluorescence signals and ionic currents at all the voltages tested (Figure 3B, D). The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1)(42) and KCNQ3 channels(41, 43).”

B) Improve the description of how the Markov-chain model is constructed, and the logic of its parameterization. The model seemingly has the form of an MWC gating model, but it is modified strangely and under described. The results describe the gamma and delta transitions as voltage sensor movements and in the model these are combined with a channel opening parameters (L, f) controlling the opening rate. This all seems strange and not well explained. How much of each voltage sensor movement component contributes to fluorescence is also unclear, and hard to make sense of.

We have removed the kinetic model as suggested by all three reviewers. We apologize for the flaws shown in the old Figure 6F regarding the violation of reversibility and for the poor descriptions of its logic and parametrization. We agree that gathering more fluorescence and current data with different protocols to extract all the parameters are needed to better describe KCNQ2 gating.

C) Make it clearer what data supports each claim in the results. For example, claims are made repeatedly about kinetics while showing only exemplar data and irreversible changes are mentioned but not backed up. There were some seeming discrepancies between the Results narrative and the data (specific instances described below).

Thanks! In the revised version, we have backed up exemplar data with appropriate statistical analysis (please see statistics in new Figure 1-figures supplement 1, 3, and 4; Figure 2, Figure 2-figure supplement 1; and Figure 5-figure supplement 1). We have been careful to avoid any unclear/imprecise text and made sure to more closely relate the claims being made to specific experimental observations (Please, see below response with specific suggestions).

D) Further discussion of how understanding the impacts of human mutations on voltage sensor vs pore movements could be valuable. Perhaps this could be in the context of KCNQ drugs that act on the pore, like retigabine, vs other that act on the voltage sensor.

We thank the reviewer for this suggestion. We have provided more context to the proposed distinctive gating mechanisms of mutations affecting either voltage sensors or pore movement. We have now expanded on this idea on pages 15 and 16, last and first paragraphs, respectively of the discussion

Specific suggestions:

Page 6 "Modification of N190C channels quickly and irreversibly increases the current amplitude, speeds up the kinetic of activation, and changes the voltage dependence of activation in either closed or open N190C channels, albeit to distinct V1/2 values (as also shown earlier for HCN1 channels (40), Figure 1D, E), as if N190C is always accessible and exposed to the extracellular solution (Figure 1K, yellow). "

Quantitation of quickly, irreversibly, current amplitude, kinetic of activation, seem to be lacking, aside from showing exemplar traces. For N190C, evidence of changes in the voltage dependence of activation at -80 mV seem to be lacking: slightly different V1/2 values are in Supplement Table 1, but differences aren't compelling in Fig 1E. Unclear why the -80 mV MTSET differences in V1/2 are considered changes in the voltage dependence of activation for N190C, but not R198C for example. More discussion would be helpful of how modification of N190C at -80 mV could produce a different result than +20 mV, yet still be similarly accessible. It would seem helpful to quantitate the changes in current amplitude in response to the MTSET, for example in the case of N190C at -80 mV and elsewhere that the change in current amplitude is the evidence of modification.

In the first submission, the term “irreversible” was used to describe those MTSET-modified currents measured after extensive washout of MTSET from the bath (referring to permanently modified channels). We apologize for any confusion caused by this statement. In the revised version we have removed imprecise wording like quickly and irreversible. We have also performed additional experiments and the requested analysis of current amplitude and G(V) shifts from wt and the cysteine substitutions at – 80 mV and + 20 mV. This new data is presented in the new Figure 1 and new Figure 1- figure supplements 1, 3 and 4 of the revised manuscript.

In the first submission, we claimed that MTSET modified N190C channels as the current amplitude in both closed and open states increased after MTSET application (old Figure 1D). We also used the MTSETmediated G(V)1/2 shifts in the open and closed states to support that claim. However, as correctly noted by the reviewer, these G(V)1/2 values shown in the original Table 1 (and depicted in the original Figure 1E as G(V) curves) appeared to show different MTSETmediated shifts in the G(V) relationship for closed and open sates. These G(V)1/2 differences raised the concern that N190C might not be accessible in the closed state.

To address the reviewer’s concern about the extent in MTSET-mediated modification of N190C, we repeated the cysteine accessibility experiments in both the closed and open states. We use the same protocol as shown in the original submission to increase the number of trials (which in the original version was n = 3, highlighted in red in the summary table, Author response image 1e, provided for the reviewers but not included in the revised manuscript). While this new set of data shows the same trend (more negative MTSET-mediated G(V)1/2 shift in the open state than in the closed state) as also seen in the original data (in red), the extent of this change shows no statistical significance between both states (Author response image 1)

Author response image 1

(a) Currents from oocytes expressing KCNQ2-N190C channels in response to 20-mV voltage steps from – 140-mV to + 40-mV (left) before and (right) after application of MTSET in the open state. (middle) currents in response to a + 20-mV voltage steps during MTSET application on N190C channels in the open state for the indicated voltage protocol. MTSET is applied at + 20-mV for 5-s in between 25-s washouts for 8-15 cycles and the change in current is measured at + 20-mV. (b) Normalized steady-state conductance/voltage relationships, G(V), (lines from a Boltzmann fit) of N190C channels (black) before and (gray ‘closed state’ and yellow ‘open state’) after MTSET application. Summary of (c) relative change in current amplitude and (d) voltage dependence shift of MTSET-mediated modification of N190C channels in the open state. (e) Summary table of the G(V) values from each experiment. Values from the original submission are shown in red. (e’) Statistical analysis from red values. Significance was determined using the paired Student’s t-test and significance level was set at P < 0.05. Asterisks denote significance: p < 0.01**.

However, based on the reviewer’s concern, we adopted a new protocol that more rigorously tested whether MTSET modifies (and modifies to completion) N190C in the closed state. This protocol contains the following modifications (performed for each cysteine mutants in ‘new Figure1 of the manuscript’, but only shown here for N190C for the benefit of the reviewers):

We measured a family of currents in response to 20-mV voltage steps from –140 mV to +40 mV before (Author response image 2a) and after applications of MTSET in the closed (Author response image 2b’) and open (Author response image 2c’) states. To assess the state-dependent modification of substituted cysteines, we first applied MTSET at – 80-mV for 5 s in between 25-s washouts for 8-15 cycles and assayed the change in current at + 20-mV (Author response image 2b). On the same cell and after MTSET is washed out of the bath, we repeated a similar protocol but instead applied MTSET at + 20-mV and again assayed the change in current at + 20-mV (Author response image 2c). We reason that if MTSET only modifies N190C in the open state, but not in the closed state as it was suggested by the reviewer, the second application of MTSET at depolarized (open) voltages will cause an additional increase in the current amplitude and will shift the G(V) relationship to more negative voltages.

External application of MTSET in the closed state increased the current amplitude and left-shifts the G(V) relationship of N190C channels (ΔGV1/2 N190C closed = – 6.3 ± 1.3 mV, n = 11, Author response image 2b-b’, d-f, gray). We found that after the second MTSET application (now using the open state protocol), there was no additional increase in the current amplitude and the G(V) relationship did not shift further (ΔGV1/2 N190C open = – 7.0 ± 1.7 mV, n = 9, Author response image 1c-c’, d-f, yellow), as would be expected if all N190C channels were fully modified in the closed state. These results (increased in current amplitude and G(V) shift) strongly support our previous conclusion that N190C was accessible in the closed state (S4 in the resting state).

Author response image 2
(a, b’, and c’) Currents from oocytes expressing KCNQ2-N190C channels in response to 20-mV voltage steps from – 140-mV to + 40-mV (a) before and after application of MTSET in the (b’) closed and (c’) open states.

(b and c) currents in response to a + 20-mV voltage step during MTSET application on N190C channels in the (b) closed and (c) open states for the indicated voltage protocols. MTSET is first applied at (b) – 80 mV for 5-s in between 25-s washouts for 8-15 cycles and the change in current is measured at + 20-mV. On the same cell and after MTSET is washed out of the bath, MTSET is re-applied at (c) + 20-mV using a similar protocol as in (b). (d) Normalized steady-state conductance/voltage relationships, G(V), (lines from a Boltzmann fit) of N190C channels normalized to peak conductance before MTSET application (black). The G(V) relationships of N190C channels before and after MTSET application in the closed (-80-mV, gray) and open (+ 20-mV, yellow) states are obtained from recordings of panels (a), (b’), and (c’), respectively. Summary of (e) relative change in current amplitude and (f) voltage dependence shift of MTSET-mediated modification of N190C channels in the (gray) closed and (yellow) open states. Mean ± SEM, n=9-24. Statistical significance was determined using the Student’s t-test and significance level was set at P < 0.05. Asterisks denote significance: p < 0.001***.

To test whether N190C is also accessible in the open state, we performed a separate experiment in which MTSET is applied at + 20-mV (open) and the change in current is measured at + 20-mV (Figure 1-figure supplement 4). Using this protocol, we found that MTSET also increased the current amplitude and shifted the G(V) relationship of N190C channels (ΔGV1/2 N190C open = – 12.2 ± 10 mV, n = 5, Figure 1-figure supplement 4). Together, these results suggest that N190 was indeed always accessible and exposed to the extracellular solution in both the closed and open states.

Page 8 "Compared to unlabeled KCNQ2* channels, the time courses of ionic currents of labeled KCNQ2* (labeled with either fluorophore) are similar"

Suggest providing quantitative backing for this claim. The time courses look faster with fluorophore.

We thank reviewer #1 for this helpful suggestion. Our statement was wrong. In the revised version, we have rewritten this subsection almost in its entirety and show statistics on new panels in Figure 2 and Figure 2-figure supplement 1. We have now added a comprehensive comparison of the time courses between wt, unlabeled KCNQ2-F192C, and labeled-KCNQ2-F192C channels in the new (Figure 2 and Figure 2-figure supplement 1C, and referenced appropriately in the text on pages 8-9 of the revised manuscript).

We now state: “The gating properties of KCNQ2* channels (G(V) and kinetics) deviate from that of wt and unlabeled KCNQ2-F192C channels (Figure 2G-I). Labeling F192C with Alexa488-maleimide (or with Dylight488-maleimide) shifts the G(V) relationship to negative voltages relative to unlabeled KCNQ2-F192C and wt channels (ΔGV1/2 = – 21.3 ± 0.8 mV and ΔGV1/2 = – 35.4 ± 2.2 mV, respectively, Figure 2C, D, G and Figure 2-figure supplement 1D). Moreover, compared to wt KCNQ2 channels, both labeled and unlabeled F192C accelerate the time course of current activation (Figure 2H-I).”

As also suggested for Reviewer# 3, we also show in the revised version a comparison of the time courses of current activation for wt and all scanned cysteine mutants in the extracellular S3-S4 linker (Figure 2-figure supplement 1A and Page 8 of the revised manuscript).

Figure 3C Could the Tau on from fluorescence with 20 ms time constants be limited by the 100-200 Hz filtering of the optical signal? Error bars seem to be missing.

Thanks! Very good point. It might be possible that this is the case, and we acknowledged this technical limitation. We have now re-analyzed the data and concluded that while the time course of the fluorescence appeared to have multiple exponentials, our fluorescence data lacked sufficient resolution to reliably estimate the first (fast) component. This might be because of the low signal-tonoise ratio of our VCF or/and as pointed by the reviewer, because the filtering might have limited the tau-on from the optical signal (shown to be 20 ms in Figure 3C of the original submission).

That is why, as suggested by reviewers # 3, we have removed the kinetics comparison of fluorescence and current from the revised version of Figure 3. This comparison on the original submission primarily served to support the Markov kinetic model, which has been removed from the revised manuscript. We now simply state:

…” There is a close correlation between the time course of fluorescence signals and ionic currents at all the voltages tested (Figure 3B, D). The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1)(42) and KCNQ3 channels(41, 43).”

In the original submission, error bars were shown but these were small and difficult to see since the graph was plotted on a log scale. In any case, this analysis has been removed from the revised version.

Page 8 "signals follow a double exponential time course" debatable, as the fits aren't amazing. Might be better to state "appeared to have multiple exponential components and were fit by a double exponential".

Thanks, we agree! Please see response above.

Figure 3F The -180 mV fit appears to have a decaying component with a negative amplitude?

We do not see a decaying component in the other traces we examined at – 180mV. This might be a clamping artifact at this extremely negative voltage. It is very difficult to hold the cell at voltages more negative than – 140 mV for longer than 1 second. In this case where we showed Fluorescence changes at – 180 mV, it is possible that by the end of the pulse some artifacts produced this apparently decaying component.

Page 9 "These data also suggests that an individual voltage sensor movement might be sufficient to open the channel." State more clearly what data is suggestive of this?

We apologize for the inaccuracy of the claim that an individual voltage sensor movement might be sufficient to open the KCNQ2 channel based on the VCF data provided in Figures 2 and 3. We have clarified this in the revised version of the manuscript and this sentence (and its implications) has been removed from the Abstract and Results sections. We only discussed that these alternatives, concerted or independent S4 movement, might equally well explain our VCF data which shows that both the steady-state voltage dependence of S4 transitions and the kinetics closely follow those of ionic currents.

Fig 5B,D Please more quantitatively analyze the reported similarity and difference in F and I kinetics

We thank the reviewer for this helpful comment. We have performed the requested analysis, added new data, and presented the results in Figure 5-figure supplement 1 (which now contains statistical analysis in panels C, D, and E), and, accordingly, amended the text: see Results, subsection “Disease-causing mutations differentially affect S4 and gate domains”

Page 10 "the ILT mutation in the Shaker K+ channels "

the ILT mutations dissociate early voltage sensor movement from pore opening, but the ILT pore opening remains coupling to late voltage sensor movements and is detected by extracellular fluorescence measurements similar to those employed in this manuscript (doi: 10.1085/jgp.200409197). The fact that a fluorescence component is not observed with pore opening of KCNQ2*-R214W could suggests a different mechanism of decoupling than ILT, or that the late component of gating charge movement associated with pore opening is not reported by the fluor.

We thank the reviewer #1 for pointing out this difference that was also brought up by Reviewer#2. We agree that the decoupling mechanism induced by KCNQ2-R214W seems to be different from that seen with the ILT mutation in Shaker and from the KCNQ1-F351A mutant, as also pointed out by reviewer#2. We have performed more experiments with the R214W mutant and did not see a second fluorescence component. Therefore, in the revised version we have removed from Results: “The separation between F(V) and G(V) suggests that, like the uncoupling mutation F351A in KCNQ1 channels(36, 37, 45) or the ILT mutation in the Shaker K+ channels(46), R214W dissociates voltage sensor movement from channel opening.”

In the revised version of Result we simply say that: “The separation between the F(V) and G(V) curves suggest that R214W dissociates voltage sensor (S4) movement from channel opening. We hypothesize that, since R214W is in the loop connecting S4 to the S4-5 linker (not within the voltage sensor itself, Figure 5-figure supplement 2A), it most likely affects activation gating without directly affecting S4 movement.”

Specifically, we discuss these mechanisms in pages 14-15 of the revised manuscript. “…Unlike the ILT mutation in the Shaker K+ channels, in which pore opening is dissociated from the first VSD activation but coupled to the second (54), KCNQ2*-R214W fluorescence signals do not show a second fluorescence component associated with channel opening. This suggests that the R214W and the ILT mutants decouple VSD-PD pore through different mechanisms, or alternatively through similar mechanisms but our labeled F192C is unable to resolve the late component of gating charge movement (fluorescence) associated with pore opening.

Fig 6B Something seems wrong here: the fit is purported to represent 1/alpha, but with zalpha = 0.43 the tau fast at +40 should be 2.7x faster than -20 mV, and this is not the case.

Fig 6C Tau on F slow as well as Tau on G slow (Fig 3 C) seem to lose their voltage dependence at more positive voltages. This could mean pore opening itself (gamma) has little voltage dependence.

Fig 6F Why connote that f is always to the 1st power? Do L and f only impact the opening rate? The modeling is not described sufficiently to reproduce it. alpha and beta are missing altogether. zdelta/zgamma should also be listed in Supplement Table 2

Page 11 "We find no experimental evidence supporting a constitutive open state (O0 in Figure 6F, gray)" From the model you can calculate the expected Popen at very negative voltages, it could be just a very low Popen.

Page 12 "By decreasing the opening transition (L in Figure 6F) relative to wt KCNQ2 channels, the model also describes the clear separation between F(V) and G(V) curves observed in mutated KCNQ2*-R214W channels (Figure 6H), under the assumption that R214W changes the voltage sensing domain-pore domain (VSD-PD) coupling such that it prevents opening before multiple S4 have activated (Figure 6F, dashed maroon arrow)."

Difficult to parse this sentence. In addition to L, f and gamma are also changed. What changes VSD-PD coupling, isn't that L?

Page 12 "Additionally, data from the R198Q mutation can also be simulated by shifting the voltage dependence to negative voltages. " I imagine this is probably right but the claim is not justified by simulations.

Page 13 Confidence in estimated total gating charge of 7.37 e0 per channel moved during KCNQ2 activation gating is limited due to the issue poorly described parameterization of the gating model.

The above 8 points and concerns raised by reviewer#1 are related to the Markov kinetic model shown in Figure 6 from the original manuscript. As stated above in Recommendations for the Authors, this model has been removed from the revised version of the manuscript, as also suggested by all three reviewers.

Page 14 "the overall voltage-dependent gating mechanisms of KCNQ2 is qualitatively similar to that of KCNQ1". To me it seems that the overall voltage-dependent gating mechanism of KCNQ2 is qualitatively distinct from KCNQ1.

This statement was primarily inferred from the kinetic model and the Rb/K experiments presented in the original submission, which have been removed in the revised version. Our data shows that both the steady-state voltage dependence of S4 transitions and the kinetics closely follow those of ionic currents. This result, in part, lead us to conclude that the gating of KCNQ2 resembles that of KCNQ1 (although we do not show evidence of intermediate open state, which remains to be tested in future studies) and KCNQ3 channels, but differed from homologous KCNQ4 channels. In addition, whether KCNQ2 channels need one (like KCNQ1) or multiple S4 movements to open requires additional support and further experimentation in future studies. We have revised and considered the reviewer’s comment in the discussion of this resubmitted manuscript (page 13).

Reviewer #2 (Recommendations for the authors):

1. In Abstract and other places, the sentences such as "channel opening does not require multiple VSD movements" are not clear. Do the authors try to say, "the movements of multiple VSDs" or "the movements of VSDs in multiple steps"?

Thank you for this important observation, the wording we used was clumsy. Since we removed the kinetic model (Figure 6 in the original manuscript), we have also deleted any sentences that discuss concerted or independent S4 movement in the Abstract and Result sections. We only discussed that these alternatives, concerted or independent S4 movement, might explain our VCF data which shows that both the steady-state voltage dependence of S4 transitions and the kinetics closely follow those of ionic currents. Both references – Osteen et al PNAS 2010 and Westhoff et al PNAS 2019 have also been added – as recommended by the reviewer and apologize for overlooking these references in the original manuscript.

2. Did N190C really modify the channel at both -80 mV and +20 mV? Why would the same covalent modification of the channel at two voltages result in different GV relations (Fig 1E)? Is there another Cys in the channel that was modified differently at the two voltages? Did the authors use different protocols to measure GV in these two conditions? These need to be explained. The authors claimed a similar result in a reference, but it was not obvious if the reference showed the similar result or explained the result. The authors can cite the reasons given by the reference (40) if these can make sense of the results in Fig 1E.

Thank you for pointing out this important point. We have spent a good deal of time since we received the reviews answering this important point that was also raised as a concern by Revewer# 1. To that end, we have included additional data that support the idea that N190C channels are accessible in both the open and closed states. This is now clearly addressed in Recommendations for the Authors, first Specific Suggestions from Reviewer #1. See above Response to the first Specific suggestions from Reviewer# 1 on Pages 2-5.

In the original submission, we only used the protocols shown old Figure 1. We applied MTSET only at +20-mV for the open state and – 80-mV for the closed state. We used – 100-mV and – 120 mV for the closed state of A193C and S199C, respectively, because compared to the wt channels, these cysteine mutants shifted the GV relationship to negative voltages.

In the revised version, to further strengthen our conclusions, we have used a new protocol: For each cysteine mutant, we have designed a protocol in which we first apply MTSET at hyperpolarized voltages (closed) before switching to depolarized voltages (open) on the same cell, in a pairwise manner.

This is now described in the Result subsection “State-dependent external S4 modifications consistent with S4 as voltage sensor”, Pages 6-8 of the revised manuscript and new Figure 1 and Figure 1-figures supplement 3 and 4.

We also apologize for the lack of clarity in citing reference 40 in the original submission. This reference is deleted in the revised version, in light of our new data on N190C (new Figure 1 and Figure 1-figures supplement 3 and 4), which strengthen our claims that N190C modification occurs in in both states (open and closed).

3. Fig 1J at -80 mV: why is the current amplitude 0? It does not seem to be consistent with the description in the legend or J'.

We apologize for this confusion. The amplitude of the current 400 ms after the start of the + 20-mV voltage step (dashed vertical arrows in old Figure 1J’ and J’’) is not zero. What it is zero is the delta current measured between the first sweep (before MTSET, sweep #0) and the subsequent sweeps (after several MTSET application at – 80 mV, which are represented by #1, 2, …9).

In the revised version, we have now changed the Y axes as “Normalized D current”, and corrected the new Figure 1E and I as follow:

(I) The rate of MTSET modification of R198C channels at + 20-mV (red squares) or – 80-mV (gray squares) was measured using the difference in current amplitudes (taken at 400 ms after the start of the +20-mV voltage step, vertical dashed arrows in E) between the first sweep (before MTSET application, which is represented by #0 along the vertical dashed arrows in E, and normalized to zero) and the subsequent sweeps (after several MTSET application, which are represented by #1, 2…8-9 along the vertical dashed arrows in E) from the ‘closed- and open- state’-middle panels. The normalized delta current amplitude was plotted versus the cumulative MTSET exposure and fitted with an exponential. The fitted second-order rate constant in the open state protocol is shown in red. kopen = 3,230 ± 3.8 M–1 s–1 (n = 3).

4. Fig 2C and Fig 2-FigS1E: The FV with Alexa labeling increases at voltages >0 mV. Is this real or artifact? If real, does it indicate a second VSD movement?

We agree with the reviewer that the claim of a lack of an intermediate open state in KCNQ2 channels based on the Rb/K data provided in the original submission assumed that the pore properties of KCNQ2 are the same as those seen in KCNQ1 channels. Since we did not show sufficient experimental evidence to prove this point, we have removed Figure 6 (the model) from the revised manuscript. In the future, we will provide more evidence to build stronger support for the potential existence of intermediate and active open states in KCNQ2 channels. As such, we have removed the model shown in the original manuscript. Future studies will be performed to refine the KCNQ2 model, including the use of mutations that can lock the S4 in the intermediate or activated states in KCNQ2, as has been performed in the KCNQ1 channel by Zaydman et al; (PMID: 25535795). These experiments will provide more conclusive results regarding the different S4 states.

We have now re-analyzed the data and concluded that while the time course of the fluorescence appeared to have multiple exponentials, our fluorescence data lacked sufficient resolution to reliably estimate the first (fast) component. This might be because of the low signal-to-noise ratio of our VCF or/and because the filtering might have limited the tau-on from the optical signal (shown to be 20 ms in Figure 3C of the original submission). Please, also see above response to Reviewer #1, page 6.

As suggested by reviewers # 3, we have removed the kinetics comparison of fluorescence and current in the revised version of Figure 3, and simply state: …” There is a close correlation between the time course of fluorescence signals and ionic currents at all the voltages tested (Figure 3B, D). The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1)(42) and KCNQ3 channels(41, 43).”

As for the last part of the reviewer comments, the apparent increase in fluorescence after a plateau at voltages > 0mV has now also been revised. We have attempted new VCF at voltages more positive than + 40 mV to probe if a putative second fluorescence component after the plateau phase develops or if it is just artifacts of the experimental system. To get reliably fluorescence signals, we need a huge expression of labeled KCNQ2* channels (often producing currents larger than 100uA). It is considerably more difficult to properly clamp these high expressing cells, especially at extreme voltages. This experimental limitation makes it challenging to draw conclusions about the occurrence of a second fluorescent component. It may be possible to perform the cut—open technique coupled with VCF in order to shed light on this issue, but these experiments would require significant upgrade of the set up that we currently do not have this in place.

5. In Fig 4E, the comparison between MTS modification rate of A193C and FV of KCNQ2* makes no sense. These two curves derived from different mutations and modifications may overlap by coincidence.

We thank the reviewer for this comment. However, we believe that the comparison is still useful. To add more context explaining the rationale of this comparison, in the revised version (Pages 9-10) we now state: …“Because steady-state conductance/voltage curve, G(V), of A193C and labeled F192C channels are similar (G1/2 A193C = – 70 ± 2.4 mV, (n = 12) and G1/2 F192C-Alexa = –77.1 ± 2.7 mV, (n = 9), Figure 4B and B’), and under the assumption that these two channels use the same S4 movement to generate these similar G(V)s, we will compare the voltage dependence of the modification rate (mod. rate) of A193C with the voltage dependence of the fluorescence of labeled F192C…”

Similar comparisons between the voltage dependence of modification rate by MTSET and S4 movements (by fluorescence of gating currents) have been previously used to independently assess the voltage range of S4 movement in different channels (PMID 9655514: Fig. 5A and PMID: 24769622: Fig 2C-D).

6. The data in Fig 6B-E seem to differ from the data in Fig 3C,D although the legend of Fig 6 claims that these are the same data. For instance, in Fig 3C the fast component of F does not show a voltage dependence, but in Fig 6B and C the fast and slow components of F show a similar voltage dependence.

7. The model in Fig 6F raises several concerns: (1) Why do the vertical transitions have the rates of VSD activation, while they should represent pore opening/closing? (2) What does f represent in the scheme? Can it be part of L? (3) Detailed balance is violated in the left-most loop connecting C0, O0, O1, and C1.

8. In Fig 6G, which states were used to represent currents? Which states represent fluorescence? Particularly, with both the horizontal and vertical transitions represent VSD activation, the rationale for simulating fluorescence need to be justified and the methods clearly described.

The reviewer raises an important concern in our original Figure 6F (model). Based on the Editors and reviewers comments, we have removed Figure 6 from the original manuscript to eliminate any of potential misunderstanding about the data presented. In future studies, we will gather additional fluorescence and current data using different protocols and dimer constructs to provide a more in depth description of KCNQ2 gating.

Reviewer #3 (Recommendations for the authors):

I was very happy to read this paper and feel that this work is an important step forward for those working on these channels. A lot of progress has been made with KCNQ1 because of the relative ease of recording VCF signals, whereas similar work in KCNQ2-5 has been difficult. The KCNQ2-5 channels differ significantly from KCNQ1 in terms of their function and auxiliary protein regulation, so the development of useful tools to carry out detailed biophysical studies on these channels is valuable and took quite a heroic effort.

I have quite a few comments, just important things that I would add to the paper in the interest of being thorough and not over-interpreting some of the findings.

1. Page 3. "muscarine-regulated M-current". I would hesitate to call it 'muscarine regulated' as it can be sensitive to a variety of neurotransmitters that signal via Gq (ie. acetylcholine... I agree with the historical perspective of naming the current, but the wording may imply physiological regulation to some readers).

2. Page 9: "These data also suggest that an individual voltage sensor movement might be sufficient to open the channel". The basis for this interpretation is not clear (at least not at this stage of the paper).

Thank you for the helpful comment. We agree that this was brought up very early in the paper and without robust support by the current set of data. This concern was also shared by Reviewers #1 and 2. We have revised the abstract and Results sections to more clearly describe the results presented. In the revised version, we only discuss that either concerted or independent S4 movement, might explain our VCF data which shows that both the steady-state voltage dependence of S4 transitions and the kinetics closely follow that of ionic currents.

Subsequent studies using one, two, or three concatemeric constructs containing mutations that prevent voltage sensors from moving into activated conformations, like those shown in Westhoff M et al., PMID: 30918124, could shed light on the concerted or independent nature of S4 movement in KCNQ2 channels in the future.

Also, as mentioned in the public review, a counterpoint to this observation is that the KCNQ2 or KCNQ3 currents typically exhibit a sigmoidal time course (also see Figure 1) to activation which might be accounted for by a requirement for multiple subunits to reach an activated conformation. Could this arise because arise because the dye labeling may prevent complete VSD deactivation or interfere with gating in some other way. This is also brought up at the top of page 14 and I have concerns that this could be a contentious statement. I would suggest more caution when describing and interpreting these properties.

3. One way to potentially address this explicitly (ie. point #2) would be to include a direct comparison of unmodified and modifier I192C (and maybe WT KCNQ2 as well) in Figure 2. It is 100% fine with me that there are differences, but it should be clearly shown and described as a consideration when interpreting data, and some comparison like that would help readers.

We appreciate this suggestion. We agree with the review on this point. We have now modified the language used in both the Result and Discussion section of the revised manuscript. This is now also addressed in detail in response to the public review above. Please, see also new Figure 2 and Figure 2-figure supplement 1.

We now state on page 8 of the revised manuscript:

…“We find that this region exhibits sensitivity to cysteine mutations (Figure 2B-D), similar to a previous report for homologous cysteine mutations in KCNQ3 channels(41). Compared to wt-KCNQ2 channels, the mutants Q188C, G189C, and N190C shift the steady-state conductance/voltage curve, G(V), towards positive voltages (ΔGV1/2 = + 9.3 ± 0.7 mV, ΔGV1/2 = + 24.2 ± 0.7 mV, and ΔGV1/2 = + 29.8 ± 0.3 mV, respectively), whereas the mutants V191C and F192C shift the G(V) curves towards negative voltages (ΔGV1/2 = – 2 mV ± 0.9 mV and ΔGV1/2 = – 12.5 mV ± 1.7 mV, respectively) (Figure 2C-D, open symbols and supplement Table 1). Unlike the F192C mutant, the wt channels and the other cysteine mutants exhibit a sigmoidal time constant of activation that appear to have multiple exponential components (Figure 2B), with the F192C mutant generating the fastest time course of current activation (Figure 2-figure supplement 1A). Moreover, all five cysteine substitutions showed a further leftward G(V) shift upon fluorophore labeling (Figure 2D, filled symbols). The mechanisms by which the cysteine substitutions and their dye-conjugated versions may alter some of the gating properties are unknown and were not investigated further”.

And later on Page 9:

…”The gating properties of KCNQ2* channels (G(V) and kinetics) deviate from that of wt and unlabeled KCNQ2-F192C channels (Figure 2G-I). Labeling F192C with Alexa488-maleimide (or with Dylight488maleimide) shifts the G(V) relationship to negative voltages relative to unlabeled KCNQ2-F192C and wt channels (ΔGV1/2 = – 21.3 ± 0.8 mV and ΔGV1/2 = – 35.4 ± 2.2 mV, respectively, Figure 2C, D, G and Figure 2-figure supplement 1D). Moreover, compared to wt KCNQ2 channels, both labeled and unlabeled F192C accelerate the time course of current activation (Figure 2H-I)”.

Please, see also discussion of these issues on Pages 12-13 of the revised manuscript.

4. The other technical concern that I had was about fitting the fluorescence traces and perhaps adding complexity where it is not needed and not necessarily supported by data (perhaps this is being done due to analogy to prior work in KCNQ1). Based on the sample sweeps, there does not usually seem to be a great reason to fit with 2 components (eg. Figure 3) - is it really necessary in this case (ie. would it make a difference in terms of the predicted currents, especially given the uncertainty about sigmoid character of current activation)? A few other issues with the description of the model are that some parameters appear to be missing from Supplemental Figure 2 (ie. Alpha and Beta rates, and z for gamma and delta rates). In the text it seems that the gamma and delta rates are meant to be associated with channel opening, but the large amplitude fast component (alpha+beta rates) seem to correlate with the early stages of channel opening, it seems. Perhaps clarifying this by showing individual fit components, or simplifying the fitting/model would be helpful.

We agree with the reviewer’s comment. In terms of the predicted currents and considering the gating changes induced by both the F192C mutation and the dye conjugation together with the low resolution of reliable estimates of the first component, it might not be necessary, nor appropriate, to fit with two exponentials.

As also suggested by reviewer#1, we have removed the kinetics comparison of fluorescence and current in the revised version of Figure 3, and simply state: …” There is a close correlation between the time course of fluorescence signals and ionic currents at all the voltages tested (Figure 3B, D). The close correlations in time (Figure 3) and voltage dependences (Figure 2G) of S4 motion (fluorescence) and activation gate (ionic current) resemble those observed for homologous KCNQ1 (without KCNE1)(42) and KCNQ3 channels(41, 43).”

As for the last part of the reviewer comments, we have removed the kinetic model as suggested by all three reviewers. We agree that the model presented in Figure 6 of the original submission was underdeveloped. We will aim to gathering more fluorescence and current data using additional approaches and concatemeric constructs to provide a more in-depth description of KCNQ2 gating in the future.

https://doi.org/10.7554/eLife.77030.sa2

Article and author information

Author details

  1. Michaela A Edmond

    Department of Medicine, Miller School of Medicine, University of Miami, Miami, United States
    Contribution
    Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  2. Andy Hinojo-Perez

    Department of Medicine, Miller School of Medicine, University of Miami, Miami, United States
    Contribution
    Data curation, Formal analysis, Visualization
    Competing interests
    No competing interests declared
  3. Xiaoan Wu

    Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, United States
    Contribution
    Data curation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2098-7298
  4. Marta E Perez Rodriguez

    Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, United States
    Contribution
    Data curation, Resources
    Competing interests
    No competing interests declared
  5. Rene Barro-Soria

    Department of Medicine, Miller School of Medicine, University of Miami, Miami, United States
    Contribution
    Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing
    For correspondence
    rbarro@miami.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4804-2739

Funding

National Institute of Neurological Disorders and Stroke (R01NS110847)

  • Rene Barro-Soria

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs. Derek Dykxhoorn and Hans Peter Larsson for helpful comments on the manuscript. This work was supported by the National Institutes of Health (1R01NS110847) to Rene Barro-Soria.

Senior Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Reviewing Editor

  1. Jon T Sack, University of California Davis School of Medicine, United States

Reviewer

  1. Jon T Sack, University of California Davis School of Medicine, United States

Publication history

  1. Received: January 13, 2022
  2. Preprint posted: January 21, 2022 (view preprint)
  3. Accepted: May 31, 2022
  4. Accepted Manuscript published: June 1, 2022 (version 1)
  5. Accepted Manuscript updated: June 13, 2022 (version 2)
  6. Version of Record published: June 14, 2022 (version 3)

Copyright

© 2022, Edmond et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Michaela A Edmond
  2. Andy Hinojo-Perez
  3. Xiaoan Wu
  4. Marta E Perez Rodriguez
  5. Rene Barro-Soria
(2022)
Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels
eLife 11:e77030.
https://doi.org/10.7554/eLife.77030
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