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.

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.

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

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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

   "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

   "This ORCID iD identifies the author of this article:" 0000-0003-4804-2739

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