Domain-domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel.

Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore.

Introduction

Voltage-gated ion channels sense changes in membrane voltage and respond by opening or closing a pore through which selected ions cross the membrane, generating a transmembrane current. These channels consist of four voltage-sensing domains (VSDs) surrounding a central pore. In voltage-gated potassium (Kv) channels, this structure results from the tetrameric assembly of Kv-α subunits, each of which contain six transmembrane-spanning segments (S1–S6). S1–S4 of each subunit forms a voltage-sensing domain (VSD), and S5–S6's from all four subunits form the pore. Sensing of membrane voltage occurs within the VSDs, which contain a mobile S4 segment with several highly conserved basic residues. At depolarized voltages, the forces of the membrane electric field on these positively charged residues promotes the outward displacement of S4 toward its activated state (Papazian et al., 1995; Larsson et al., 1996; Silva et al., 2009; Wu et al., 2010a; Delemotte et al., 2011; Jensen et al., 2012). The pore contains the ion permeation pathway that can be opened and closed by the reorientation of the intracellular portions of the S6 helices (Liu et al., 1997; Webster et al., 2004; del Camino and Yellen, 2001; Jiang et al., 2002). Critical to voltage-dependent gating are the interactions between the VSDs and the pore, which couple the activation of the VSD to the opening of the pore, resulting in a voltage-gated conductance (Chen et al., 2001; Lu et al., 2001, 2002; Long et al., 2005; Lee et al., 2009; Zaydman et al., 2013).

In their pioneering work on the action potential of the squid giant axon, Hodgkin and Huxley empirically derived a model for the K+ conductance in which a transmembrane pathway is gated by four voltage-dependent particles (Hodgkin and Huxley, 1952). The legacy of the Hogdkin and Huxley model can still be found in the current Kv channel models, which assume that (1) VSD activation and pore opening are two-state (i.e., all or none) processes and (2) gating and permeation are independent so that the VSD conformation changes the probability of pore opening, but does not affect the properties of the open pore. Several recent studies call into question the assumption that VSD activation and pore opening are two-state processes. Computational and experimental studies have demonstrated that VSD activation actually occurs in a series of stepwise transitions due to salt bridge interactions between the basic residues on S4 and acidic residues on S1 and S2, which define resting, intermediate, and activated states (Papazian et al., 1995; Tiwari-Woodruff et al., 1997; Wu et al., 2010a; Delemotte et al., 2011; Jensen et al., 2012; Lacroix et al., 2012). With regards to pore opening, recordings of single channel currents from Kv channels revealed multiple open states discernable by their different conductance levels (Chapman et al., 1997), although the identities of these subconductance states remain unclear. In our present study of KCNQ1 (Kv7.1, KvLQT1) channels, we found that both the intermediate and fully-activated states of the VSD yielded robust pore-opening. Remarkably, the intermediate-open and activated-open states had different permeation and pharmacological properties revealing that VSD-pore interactions determine both open probability and open conformation, demonstrating that gating and permeation are not independent.

KCNQ1 channels generate currents with very different properties as a result of tissue specific expression of KCNE family accessory subunits (Abbott, 2014). In the heart, channels formed by KCNQ1 and KCNE1 subunits are responsible for the slow-delayed rectifier potassium current, IKs (Barhanin et al., 1996; Sanguinetti et al., 1996), which plays a critical role in limiting action potential duration when beta-adrenergic tone is elevated. The importance of this response is highlighted by a large set of loss-of-function mutations of KCNQ1 or KCNE1 that have been associated with Long QT Syndrome and result in an elevated risk of fatal arrhythmias during times of stress (Paavonen et al., 2001; Schwartz et al., 2001; Hedley et al., 2009). Although KCNE1 is a small, single-membrane-spanning peptide, its coassembly dramatically alters every physiologically relevant property of the KCNQ1 channel: voltage-dependence, current kinetics, inactivation, current amplitude, single channel conductance, selectivity, and pharmacology (Wrobel et al., 2012; Sun et al., 2012). The mechanism of how KCNE1 modulates KCNQ1 has been a longstanding topic of debate with several groups arguing that KCNE1 alters VSD activation (Nakajo and Kubo, 2007; Ruscic et al., 2013), several other groups arguing that KCNE1 alters pore opening (Rocheleau and Kobertz, 2008; Osteen et al., 2010), and several reports claiming that KCNE1 directly contributes to the inner structure of the pore (Wang et al., 1996; Tai and Goldstein, 1998). However, these mechanisms are not able to simultaneously account for the effects of KCNE1 on gating and the observed changes in permeation and pharmacology.

Here we made three observations regarding the function of homomeric KCNQ1 channels that were not previously reported. First, we found that VSD activation occurs in two resolvable steps through a stable intermediate state. Second, we observed that the intermediate-state of the VSD is sufficient to promote KCNQ1 channel opening, resulting in both intermediate-open and activated-open states. Third, we observed that the intermediate-open and activated-open states have different permeation and pharmacological properties. With these critical observations, we were able to reexamine how KCNE1 affects KCNQ1. We found that coexpression of KCNE1 prevented the intermediate-open state and changed the properties of the activated-open state. The apparent decoupling of pore opening from the resting to intermediate transition of the VSD suggested that KCNE1 changes how the VSD and pore interact. Consistent with this hypothesis, changing the VSD-pore interactions directly via a single point mutation also prevented the intermediate-open state and modified the properties of the activated-open state. Using a kinetic model, we demonstrated that the effects of KCNE1 on VSD-pore interactions, as suggested by our data, are sufficient to simultaneously explain most of the changes in activation gating without any direct impacts on VSD activation or pore opening. Furthermore, as VSD-pore interactions were found to determine the open-pore properties, the effects of KCNE1 on permeation and pharmacology could also be rationalized. Therefore we conclude that altering VSD-pore interactions is likely the primary mechanism through which KCNE1 modulates KCNQ1.

Results

Voltage-clamp fluorometry (VCF) simultaneously monitors VSD-activation and pore opening (Mannuzzu et al., 1996). In our VCF records, a fluorophore attached to the S3–S4 linker of pseudo-WT KCNQ1 channels (with mutations C214A/G219C/C331A) reports on conformational changes associated with VSD activation (Figure 1A–H, green), while the ionic currents report the opening of the pore (Figure 1A–H, black) (Osteen et al., 2010; Zaydman et al., 2013). We observed multiple components of the fluorescence signals for KCNQ1 (Figure 1A–D) and, as recently reported (Barro-Soria et al., 2014), for KCNQ1+KCNE1 (Figure 1E–H). Most of the total change in fluorescence intensity was due to a fast component occurring at hyperpolarized voltages (Fmain), but a small additional change was observed due to a slow component occurring at highly depolarized voltages (Fhigh). Fhigh was more prominently observed when KCNQ1 channels were labeled with a different dye (Figure 1—figure supplement 1A,B), or coexpressed with a mutant KCNE1 (Figure 1—figure supplement 1C,D).

Figure 1.

KCNE1 suppresses the intermediate-open state of KCNQ1.

(A–H) Fluorescence (green) and current (black) signals from Xenopus oocytes injected with cRNA encoding pseudo-WT (C214A/G219C/C331A) KCNQ1 alone (KCNQ1, A–D) or coinjected with cRNAs encoding pseudo-WT KCNQ1 and KCNE1 (KCNQ1+KCNE1, E–H). The cells were labeled with Alexa 488 C5-maleimide. (A and E) GV and FV relationships (solid) with the main and high voltage FV components plotted (dotted lines). (B and F) normalized fluorescence and current responses to a 60 mV pulse shown with fits (thin grey lines) to a single- or bi-exponential function. Averaged fast (C and G) and slow (D and H) tau values of fluorescence and current responses to various voltage pulses. (I) Intermediate- (E1-R2, top) and activated- (E1-R4, bottom) state homology models of KCNQ1 after 100 ns of MD simulation. Side view of one VSD (left) and bottom view of the pore (right). (J) Currents from the cells expressing E160R/R231E (E1R/R2E, top) or E160R/R237E (E1R/R4E, bottom) both alone (−KCNE1, middle) or with KCNE1 (+KCNE1, right).

DOI: http://dx.doi.org/10.7554/eLife.03606.003

(A and B) VCF data from cells expressing pseudo-WT KCNQ1, labeled with Alexa 546 C5-maleimide (KCNQ1 [Alexa 546]). (C and D) VCF data from pseudo-WT KCNQ1+R67E/K69E/K70E KCNE1 labeled with Alexa 488 C5-maleimide (KCNQ1+RKK/EEE [Alexa488]). (A and C) Normalized current and fluorescence responses to a 60 mV pulse. (B and D) GV and FV relationships (solid) with the main and high voltage FV components plotted (dotted lines). Current signals (black), fluorescence signals (color, red = Alexa 546, green = Alexa 488). The RKK/EEE mutation in KCNE1 causes a leftward shift of Fmain, relative to that of WT KCNE1, which increases the separation between Fmain and Fhigh.

DOI: http://dx.doi.org/10.7554/eLife.03606.004

GV (solid black) and FV (solid green) relationships for R243Q/psWT KCNQ1 channels expressed alone (R243Q) (A) or with R67E/K69E/K70E KCNE1 (R243Q+RKK/EEE) (B). Green dotted lines show the main and high voltage FV components. The GV relationship of pseudo-WT channels (black dashed lines) is significantly different from that of R243Q; however, the relationship of the GV to different FV components are preserved.

DOI: http://dx.doi.org/10.7554/eLife.03606.005

(A) Snapshots of one VSD (left, side view) or the pore domain (right, bottom view) following 100 ns of MD simulations. In the resting, intermediate or activated VSD, E160 (E1, red) forms a salt bridge with R228 (R1), R231 (R2) or R237 (R4), respectively. (B) Averaged (over several trajectories) pore radius vs position along the axis normal to the membrane (Z). (C) Snapshots of the PIP2 intrasubunit-binding site in the three states. In the resting/closed state, PIP2 interacts with positive residues of S4 (cyan). When the VSD is intermediate or activated, PIP2 shifts closer to S6 (yellow) and anchors its positive residues (K354 and K358). (D) Probability of salt bridges formation between positive residues of S6 (K354 and K358) and PIP2. The lipid interacts with S6 only when the VSD is intermediate or activated, not when it is resting. Error bars represent SD. K354 and K358 interactions are not statistically different for the E1-R2 and E1-R4 states. Figure 1—figure supplement 3B represents the averaged pore radius profiles along the axis normal to the membrane (Z). In the activated/open and intermediate states, the minimal radiuses of the pore at this level are 3.5 ± 0.4 Å and 2.8 ± 0.6 Å respectively. For comparison, in the Kv1.2 open state, the corresponding radius (pdb 3LUT [Chen et al., 2010]) is 4.2 Å, in the Kv1.2/2.1 paddle chimera open state (pdb 2 R9R [Long et al., 2007]) it is 4.2 Å also, and in the NavMS open state (pdb 3ZJZ [Loussouarn et al., 2003]) it is 2.3 Å. Therefore, the minimal pore radius at the intercellular gate level in the models of the Kv7.1 activated and intermediate states corresponds to the open pore. In the resting/closed state, this radius decreases to 1.5 ± 0.5 Å. This is similar to the closed states of KcsA (pdb 1K4C [Zhou et al., 2001]), NavAB (pdb 4EKW [Payandeh et al., 2012]) and NavAP (pdb 4DXW [Zhang et al., 2012]), where these values are 1.1, 1.2 and 0.9 Å respectively. The activated/open, intermediate and resting/closed states of Kv7.1 differ by their properties as evidenced from the reported experimental data. Taking advantage of our simulations, we attempted to investigate whether the interactions between PIP2 and positive residues of the Kv7.1 intrasubunit binding site are different. Indeed PIP2 interacts preferably with the VSD (S4) when the channel is resting/closed or with the pore (S6) when the channel is activated/open (Kasimova et al., 2014) (Figure 1—figure supplement 3C, top and bottom panels). In the intermediate state, the lipid forms salt bridges with both S4 (R243) and S6 (K354 and K358) simultaneously (Figure 1—figure supplement 3C, middle panel). Its equilibrium position is also between these in the activated/open and resting/closed states. Interestingly, the probability of interaction between PIP2 and S6 (K354 and K358) is rather high (Figure 1—figure supplement 3D). The average values are slightly higher for the intermediate than for the activated/open states: 40 and 26% for K354, 68 and 42% for K358 respectively. However, this difference is statistically insignificant due to the estimated error bars.

DOI: http://dx.doi.org/10.7554/eLife.03606.006

(A) Cartoons illustrating the mutational strategy used to arrest the VSD near the intermediate- and activated-states. The E160R (E1R) mutation was used to disrupt the native electrostatic interactions between the S2 and S4 segments that stabilize the VSD as it undergoes activation. E1R was paired with R231E (R2E) or R237E (R4E) to stabilize the putative intermediate- and activated-states, respectively. (B) Currents from oocytes expressing WT or mutant KCNQ1 subunits alone (−KCNE1, left) or with KCNE1 (+KCNE1, right) in response various voltage test pulses. (C and D) Averaged steady-state current–voltage relationships for cells expressing WT or mutant KCNQ1 subunits alone (C, −KCNE1) or with KCNE1 (D, +KCNE1).

DOI: http://dx.doi.org/10.7554/eLife.03606.007

Biotinylation of intact oocytes allowed separation of membrane proteins from the cell lysate using streptavidin beads. The membrane fraction and the cell lysate were subjected to Western Blot using antibodies against KCNQ1 (Top) or Gβ, a soluble protein not found in the membrane. (A) E1R/R2E and E1R/R4E reached the cell membrane with similar efficiency. (B) KCNE1 did not decrease the expression of E1R/R2E to the membrane.

DOI: http://dx.doi.org/10.7554/eLife.03606.008

Figure 1—figure supplement 1.

Improved resolution of Fhigh.

(A and B) VCF data from cells expressing pseudo-WT KCNQ1, labeled with Alexa 546 C5-maleimide (KCNQ1 [Alexa 546]). (C and D) VCF data from pseudo-WT KCNQ1+R67E/K69E/K70E KCNE1 labeled with Alexa 488 C5-maleimide (KCNQ1+RKK/EEE [Alexa488]). (A and C) Normalized current and fluorescence responses to a 60 mV pulse. (B and D) GV and FV relationships (solid) with the main and high voltage FV components plotted (dotted lines). Current signals (black), fluorescence signals (color, red = Alexa 546, green = Alexa 488). The RKK/EEE mutation in KCNE1 causes a leftward shift of Fmain, relative to that of WT KCNE1, which increases the separation between Fmain and Fhigh.

DOI: http://dx.doi.org/10.7554/eLife.03606.004

Figure 1—figure supplement 3.

MD simulations predict that, unlike the resting-state, both the intermediate- and activated-states of the VSD stabilize pore opening through state-dependent protein and lipid interactions.

(A) Snapshots of one VSD (left, side view) or the pore domain (right, bottom view) following 100 ns of MD simulations. In the resting, intermediate or activated VSD, E160 (E1, red) forms a salt bridge with R228 (R1), R231 (R2) or R237 (R4), respectively. (B) Averaged (over several trajectories) pore radius vs position along the axis normal to the membrane (Z). (C) Snapshots of the PIP2 intrasubunit-binding site in the three states. In the resting/closed state, PIP2 interacts with positive residues of S4 (cyan). When the VSD is intermediate or activated, PIP2 shifts closer to S6 (yellow) and anchors its positive residues (K354 and K358). (D) Probability of salt bridges formation between positive residues of S6 (K354 and K358) and PIP2. The lipid interacts with S6 only when the VSD is intermediate or activated, not when it is resting. Error bars represent SD. K354 and K358 interactions are not statistically different for the E1-R2 and E1-R4 states. Figure 1—figure supplement 3B represents the averaged pore radius profiles along the axis normal to the membrane (Z). In the activated/open and intermediate states, the minimal radiuses of the pore at this level are 3.5 ± 0.4 Å and 2.8 ± 0.6 Å respectively. For comparison, in the Kv1.2 open state, the corresponding radius (pdb 3LUT [Chen et al., 2010]) is 4.2 Å, in the Kv1.2/2.1 paddle chimera open state (pdb 2 R9R [Long et al., 2007]) it is 4.2 Å also, and in the NavMS open state (pdb 3ZJZ [Loussouarn et al., 2003]) it is 2.3 Å. Therefore, the minimal pore radius at the intercellular gate level in the models of the Kv7.1 activated and intermediate states corresponds to the open pore. In the resting/closed state, this radius decreases to 1.5 ± 0.5 Å. This is similar to the closed states of KcsA (pdb 1K4C [Zhou et al., 2001]), NavAB (pdb 4EKW [Payandeh et al., 2012]) and NavAP (pdb 4DXW [Zhang et al., 2012]), where these values are 1.1, 1.2 and 0.9 Å respectively. The activated/open, intermediate and resting/closed states of Kv7.1 differ by their properties as evidenced from the reported experimental data. Taking advantage of our simulations, we attempted to investigate whether the interactions between PIP2 and positive residues of the Kv7.1 intrasubunit binding site are different. Indeed PIP2 interacts preferably with the VSD (S4) when the channel is resting/closed or with the pore (S6) when the channel is activated/open (Kasimova et al., 2014) (Figure 1—figure supplement 3C, top and bottom panels). In the intermediate state, the lipid forms salt bridges with both S4 (R243) and S6 (K354 and K358) simultaneously (Figure 1—figure supplement 3C, middle panel). Its equilibrium position is also between these in the activated/open and resting/closed states. Interestingly, the probability of interaction between PIP2 and S6 (K354 and K358) is rather high (Figure 1—figure supplement 3D). The average values are slightly higher for the intermediate than for the activated/open states: 40 and 26% for K354, 68 and 42% for K358 respectively. However, this difference is statistically insignificant due to the estimated error bars.

DOI: http://dx.doi.org/10.7554/eLife.03606.006

Strikingly, KCNE1 shifted the conductance-voltage (GV) relationship so that it correlated with a different component of the fluorescence-voltage (FV) relationship. In the absence of KCNE1, pore opening (i.e., GV) occurred in a similar voltage range as Fmain (Figure 1A, Figure 1—figure supplement 1B). In contrast, in the presence of KCNE1, pore opening was not observed unless more depolarized voltages were applied, as with Fhigh (Figure 1E, Figure 1—figure supplement 1D). These FV-GV correlations were not likely to be coincidental because they were maintained in the presence of a KCNQ1 mutation, R243Q, that shifted the voltage dependence of channel opening but did not change the correlation of the GV to Fmain or Fhigh in the absence or precense of RKK/EEE KCNE1, respectively (Figure 1—figure supplement 2).

Figure 1—figure supplement 2.

GV/FV relationships are maintained in channels the mutation R243Q in KCNQ1.

GV (solid black) and FV (solid green) relationships for R243Q/psWT KCNQ1 channels expressed alone (R243Q) (A) or with R67E/K69E/K70E KCNE1 (R243Q+RKK/EEE) (B). Green dotted lines show the main and high voltage FV components. The GV relationship of pseudo-WT channels (black dashed lines) is significantly different from that of R243Q; however, the relationship of the GV to different FV components are preserved.

DOI: http://dx.doi.org/10.7554/eLife.03606.005

KCNE1 also altered the time-dependence of pore opening. KCNQ1 current onset had two exponential components following the two timecourses of the fluorescence (Figure 1B–D). With KCNE1, the channels remained closed during the fast fluorescence increase and, after this initial delay, the channels opened with a single timecourse, similar to the slow fluorescence component (Figure 1F–H). Both the steady-state and kinetic VCF data can be easily explained if one VSD transition (Fmain) is sufficient to open KCNQ1, but an additional transition (Fhigh) is required for KCNQ1+KCNE1.

The observation of two fluorescence components (Figure 1A–H) is consistent with the suggestion from previous studies (Silva et al., 2009; Wu et al., 2010a) that, in KCNQ1, VSD activation occurs in two sequential transitions due to electrostatic interactions between E160 (E1) in S2 and arginine residues in S4. These interactions stabilize discrete resting, intermediate, and activated states. We built homology models of KCNQ1 with the VSDs in states where E1 forms a salt bridge with R228 (R1), R231 (R2), or R237 (R4) (Figure 1—figure supplement 3), the three S4 arginines that are known to be critical for voltage-sensing in KCNQ1 (Shamgar et al., 2008; Wu et al., 2010b). In KCNQ1 a neutral residue (Q234) is located in the canonical third arginine position (R3) of other Kv channels; therefore, we did not model the E1-R3 state. In Molecular Dynamic (MD) simulations, we found that the pore was more dilated when the VSDs were in the E1-R2 or E1-R4 states than in the E1-R1 state (Figure 1—figure supplement 3). These simulations suggest that KCNQ1 channels can open when the VSDs assume intermediate or activated states (Figure 1I, Figure 1—figure supplement 3). To capture these states experimentally, we engineered pairwise charge reversal mutations to arrest the VSD near the E1-R2 or E1-R4 states (Figure 1J, Figure 1—figure supplement 4). As shown previously (Wu et al., 2010a), for KCNQ1 channels, mutating E1 to arginine (E1R) caused a severe loss of current that was partially rescued by a charge reversing (R to E) mutation at the R2 (E1R/R2E) or R4 (E1R/R4E) positions (Figure 1—figure supplement 4). E1R/R2E and E1R/R4E channels were constitutively open (Figure 1—figure supplement 4C,D), suggesting that the VSD was trapped in states where the paired residues interact. When KCNE1 was coexpressed, the E1R/R2E currents were eliminated and the E1R/R4E currents were increased by nearly 10-fold (Figure 1J, Figure 1—figure supplement 4), suggesting that KCNE1 suppresses the intermediate-open state and potentiates the activated-open state. Importantly, we found that the abundance of E1R/R2E subunits in the cell membrane was similar when expressed alone or coexpressed with KCNE1, indicating that the inhibition of E1R/R2E currents was due to a functional effect on the intermediate-open state, not an effect on surface expression (Figure 1—figure supplement 5B). Altogether, the results in Figure 1 are consistent with a model in which the VSD undergoes two sequential transitions, resting-to-intermediate and intermediate-to-activated. The first transition is sufficient for KCNQ1 to open, resulting in both intermediate-open and activated-open states. With KCNE1, the second transition is required for opening because the intermediate-open state is suppressed.

Figure 1—figure supplement 4.

VSD mutations reveal that KCNE1 suppresses currents from intermediate-open states and increases those from activated-open states.

(A) Cartoons illustrating the mutational strategy used to arrest the VSD near the intermediate- and activated-states. The E160R (E1R) mutation was used to disrupt the native electrostatic interactions between the S2 and S4 segments that stabilize the VSD as it undergoes activation. E1R was paired with R231E (R2E) or R237E (R4E) to stabilize the putative intermediate- and activated-states, respectively. (B) Currents from oocytes expressing WT or mutant KCNQ1 subunits alone (−KCNE1, left) or with KCNE1 (+KCNE1, right) in response various voltage test pulses. (C and D) Averaged steady-state current–voltage relationships for cells expressing WT or mutant KCNQ1 subunits alone (C, −KCNE1) or with KCNE1 (D, +KCNE1).

DOI: http://dx.doi.org/10.7554/eLife.03606.007

Figure 1—figure supplement 5.

Surface membrane expression of E1R/R2E and E1R/R4E.

Biotinylation of intact oocytes allowed separation of membrane proteins from the cell lysate using streptavidin beads. The membrane fraction and the cell lysate were subjected to Western Blot using antibodies against KCNQ1 (Top) or Gβ, a soluble protein not found in the membrane. (A) E1R/R2E and E1R/R4E reached the cell membrane with similar efficiency. (B) KCNE1 did not decrease the expression of E1R/R2E to the membrane.

DOI: http://dx.doi.org/10.7554/eLife.03606.008

In addition to affecting KCNQ1 channel gating, KCNE1 also alters permeation and pharmacology (Sun et al., 2012). For example, as previously demonstrated by Pusch et al. (2000), KCNQ1 channels had a higher Rb+/K+ permeability ratio than KCNQ1+KCNE1 channels (Figure 2A,B) and, as reported by Cohen and colleagues (Wang et al., 2000), KCNQ1 channels were more sensitive than KCNQ1+KCNE1 channels to the inhibitor XE991 when short duration pulses (comparable to the length of the cardiac action potential) were applied (Figure 2—figure supplement 1). We used the E1R/R2E and E1R/R4E mutations to examine if VSD conformation affects permeation and pharmacology. Strikingly, E1R/R4E channels had a significantly lower Rb+/K+ permeability ratio and a significantly lower apparent affinity for XE991 compared to E1R/R2E and WT KCNQ1 channels, which were similar (Figure 2). These results reveal that different VSD conformations yield functionally distinct open states. Furthermore, the similar properties of E1R/R2E and WT KCNQ1 suggest that the intermediate-open state is either the most populated or the most conductive open-state for WT KCNQ1 channels, a notion that is further supported by our observation that E1R/R2E currents are 2–3 times larger than E1R/R4E currents (Figure 1J, Figure 1—figure supplement 4C) despite having similar levels of membrane expression (Figure 1—figure supplement 5A). Relative to E1R/R4E alone, coexpression of KCNE1 with E1R/R4E significantly decreased the Rb+/K+ permeability ratio (Figure 2A,B) and increased the apparent affinity for XE991 (Figure 2C,D) indicating that, in addition to eliminating the intermediate-open state, KCNE1 alters the properties of the activated-open state. Of note, we found that E1R/R4E+KCNE1 and WT KCNQ1+KCNE1 channels displayed similar Rb+/K+ permeability ratios (Figure 2B) and XE991 sensitivities (Figure 2D), further supporting our conclusion from Figure 1 that WT KCNQ1+KCNE1 currents are conducted by channels in the activated-open state.

Figure 2.

Permeation and pharmacological properties depend on VSD conformation.

Currents from cells expressing WT KCNQ1 alone (KCNQ1, black), E160R/R231E (E1R/R2E, red), E160R/R237E alone (E1R/R4E, blue), E160R/R237E+KCNE1 (E1R/R4E+KCNE1, green), or WT KCNQ1+KCNE1 (KCNQ1+KCNE1, grey). (A) Currents from a single cell in external solutions containing 100 mM of Na+, K+, or Rb+. The currents were elicited by first stepping the voltage to +60 mV for 5 s then to −60 mV for 3 s tails. (B) Averaged Rb+/K+ permeability ratios calculated by comparing the tail current amplitudes. (C). Currents before (CTL) and after (XE991) bath application of 5 μM XE991 in the external solution. (D) Fraction of original current inhibited after 2 s of depolarization vs concentration of XE991 applied shown with fits to the hill equation with a hill coefficient of 1. N.S. = not significant.

DOI: http://dx.doi.org/10.7554/eLife.03606.009

Currents in response to a +60 mV test pulse from oocytes expressing KCNQ1 (A) or KCNQ1+KCNE1 (B), recorded in control (CTL) and 5 μM XE991 external solutions. (C) Time dependence of XE991 inhibition—the averaged ratio of the current in 5 μM XE991 to that in control solutions is plotted vs depolarization time. (D) Averaged fraction inhibited after 200 (dashed line), 500 (thin line) or 2000 (thick line) ms of depolarization is plotted vs concentration of XE991 for oocytes expressing WT KCNQ1 (black) or WT KCNQ1+KCNE1 (grey).

DOI: http://dx.doi.org/10.7554/eLife.03606.010

Figure 2—figure supplement 1.

Inhibition of KCNQ1+KCNE1 channels by XE991 develops slowly over time.

Currents in response to a +60 mV test pulse from oocytes expressing KCNQ1 (A) or KCNQ1+KCNE1 (B), recorded in control (CTL) and 5 μM XE991 external solutions. (C) Time dependence of XE991 inhibition—the averaged ratio of the current in 5 μM XE991 to that in control solutions is plotted vs depolarization time. (D) Averaged fraction inhibited after 200 (dashed line), 500 (thin line) or 2000 (thick line) ms of depolarization is plotted vs concentration of XE991 for oocytes expressing WT KCNQ1 (black) or WT KCNQ1+KCNE1 (grey).

DOI: http://dx.doi.org/10.7554/eLife.03606.010

In our previous studies, we have shown that KCNE1 increases the apparent affinity of KCNQ1 for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) (Li et al., 2011) and that, in KCNQ1, PIP2 binding at the VSD-pore interface mediates the VSD-pore interactions that energetically couple VSD activation to pore opening (Zaydman et al., 2013). Taken together these findings suggest that KCNE1 affects the VSD-pore interactions. To test this hypothesis, we studied the impact of changing VSD-pore interactions directly via a point mutation of KCNQ1, F351A. F351 on S6 is highly conserved among Kv channels and participates in interactions with the S4/S5 linker that are known to be critical for VSD-pore interactions and coupling (Lu et al., 2001; Tristani-Firouzi et al., 2002; Long et al., 2005; Haddad and Blunck, 2011). Remarkably, in VCF experiments, we observed that the F351A GV was correlated with Fhigh instead of Fmain (Figure 3A, left), revealing that, similar to KCNE1 (Figure 1E), F351A suppressed the intermediate-open state. As a result, F351A current onset was delayed and slowed (Figure 3A, right), resembling WT KCNQ1+KCNE1 current onset, as reported previously (Boulet et al., 2007). We also observed that, compared to WT KCNQ1, F351A caused significant changes in the Rb+/K+ permeability ratio (Figure 3B) and the timecourse of inhibition by XE991 (Figure 3C, middle, right). Of note, these properties of F351A were not identical to those of WT KCNQ1+KCNE1. However, such differences are not surprising as prior studies (Kang et al., 2008; Xu et al., 2008; Chung et al., 2009; Lvov et al., 2010; Strutz-Seebohm et al., 2011; Wang et al., 2011; Chan et al., 2012; Xu et al., 2013) have located KCNE1 at the VSD-pore interface and have suggested that KCNE1 engages in a very broad and complex set of interactions with KCNQ1 (Sun et al., 2012); therefore, it would be unreasonable to expect that a single point mutation, such as F351A, would alter the VSD-pore interactions in exactly the same way as KCNE1. Nonetheless, our studies of F351A provide evidence that the VSD-pore interactions determine the permeation and pharmacological properties of the pore as well as which VSD transitions are required for the pore to open. Therefore, altering VSD-pore interactions is a single mechanism that can explain all of these effects of KCNE1.

Figure 3.

Altering VSD-pore coupling directly, by the mutation F351A, changes the gating permeation, and pharmacology of KCNQ1 channels.

(A) VCF recordings from oocytes expressing pseudo-WT/F351A (C214A/G219C/C331A/F351A), labeled with Alexa 488 C5-maleimide. Left–GV (red) and FV (solid green) relationships with the main and high voltage FV components plotted (dotted green lines). Right–normalized fluorescence (green) and current (red) responses to a 60 mV pulse, the current from a cell expressing pseudo-WT KCNQ1+KCNE1 is shown for comparison (grey). (B) Left–currents from a single oocyte expressing F351A in external solutions containing 100 mM of Na+, K+, or Rb+. Right–averaged Rb+/K+ permeability ratios for WT KCNQ1 (black) and F351A (red). (C) Left–currents from an oocyte expressing F351A in control and 5 μM XE991 external solutions. Middle–time dependence of XE991 inhibition—the averaged ratio of the current in 5 μM XE991 to that in control solutions is plotted vs depolarization time. Right–averaged fraction inhibited after 200 (dashed line), 500 (thin line) or 2000 (thick line) ms of depolarization is plotted vs concentration of XE991 for oocytes expressing WT KCNQ1 (black), F351A (red), or WT KCNQ1+KCNE1 (grey).

DOI: http://dx.doi.org/10.7554/eLife.03606.011

The observation that KCNE1 suppressed the intermediate-state opening (Figure 1, Figure 1—figure supplement 4) strongly suggested that KCNE1 affects the interactions between the intermediate-state of the VSD and the pore. Does KCNE1 also affect the interaction between the activated-state of the VSD and the pore to modulate the activated-open state? In order to detect such effects of KCNE1, we used the apparent affinity of E1R/R4E for PIP2 as a proxy for the strength of VSD-pore interactions in the activated-open state of KCNQ1. The rationale behind this approach was that our previous study has demonstrated that the apparent affinity for PIP2 correlates with the strength of VSD-pore interaction (Zaydman et al., 2013). In our experiments, we used CiVSP, a voltage-sensitive lipid phosphatase (Iwasaki et al., 2008), to cause a rapid decrease in membrane PIP2 upon depolarization (Falkenburger et al., 2010) and observed the resulting time-dependent decay in ionic currents resulting from net unbinding of PIP2. We found that the decay of E1R/R4E+CiVSP currents were significantly slower and less severe when KCNE1 was present (Figure 4A), suggesting that KCNE1 increases the affinity of the activated-open state of KCNQ1 for PIP2. Consistent with this idea, WT KCNQ1+KCNE1 currents, which come from activated-open states exclusively (Figure 1), were observed to be relatively insensitive to the activity of CiVSP as long as they were maintained in the activated-open state by sustained membrane depolarization (Figure 4B,C). When the channels were permitted to close, by hyperpolarizing the membrane potential, PIP2 unbinding was facilitated as evidenced by decreased current amplitude observed with application of a subsequent depolarizing pulse (Figure 4B,C). Altogether these results reveal that KCNE1 causes the activated-open state to have a high apparent affinity for PIP2, suggesting that KCNE1 may increase the strength of VSD-pore interactions in the activated-open state.

Figure 4.

KCNE1 increases the apparent affinity of the activated-open state for PIP2.

(A) Responses of currents from oocytes expressing E1R/R4E alone (left) or E1R/R4E+KCNE1 (middle) to rapid depletion of PIP2 by CiVSP (VSP, blue). The membrane voltage was pulsed to +60 mV to activate CiVSP. Currents were normalized to the value 200 ms after depolarization for comparison with currents from oocytes not expressing CiVSP (black = channel subunits alone, blue = channel subunits + CiVSP). Right–averaged current responses for oocytes expressing E1R/R4E+CiVSP (−KCNE1) or E1R/R4E+KCNE1+CiVSP (+KCNE1). (B) CiVSP responses (VSP, blue) of currents from oocytes expressing WT KCNQ1 or WT KCNQ1+KCNE1. Two +80 mV depolarizing pulses were applied, spaced 30 s apart (note: time scale is broken). The currents were normalized to the value 200 ms into the first depolarizing pulse for comparison with currents from an oocyte expressing channel subunits alone (black). (C) Left–double pulse protocol in which two 25 s depolarizing pulses were applied. In between the two pulses the membrane potential was set to various voltages for 10 s. After each sweep, the membrane potential was held at −80 mV for 300 s to deactivate CiVSP and allow for PIP2 regeneration by the endogenous lipid kinases. Middle–currents from an oocyte expressing KCNQ1+KCNE1+CiVSP subjected to the voltage protocol shown and normalized to the value at the end of the first pulse (I1). Right–fraction of the first pulse current available on the second pulse (I2/I1) is plotted vs voltage of intervening 10 s (blue). The voltage-dependence of I2/I1 (solid blue line) is similar to that of the GV relationship for WT KCNQ1+KCNE1 (dotted black line) suggesting that the open probability during the 10 second interpulse determines the unbinding of PIP2.

DOI: http://dx.doi.org/10.7554/eLife.03606.012

Discussion

The basic experimental phenomena revealed in our study are summarized in Figure 5A. We observed that, in KCNQ1, VSD activation occurred in two steps, with and without KCNE1 (Figure 1A–H). The first step, leading to an intermediate-state, promoted the opening of homomeric KCNQ1 channels causing the GV to overlap with the first component of the FV (Figure 1A). KCNE1 prevented the channel from opening while the VSD is in the intermediate-state and potentiated opening while the VSD is in the activated state resulting in a GV relationship with a similar voltage-dependence as the second component of the FV (Figure 1E). We observed that different states of the VSD yielded functionally different open states (Figure 2) of the pore indicating that the unique sets of interactions with different conformations of the VSD determine the probability of pore opening and selects for different open-pore conformations. In agreement with this interpretation, changing the VSD-pore interactions directly, via the mutation F351A, suppressed the intermediate-open state and changed the properties of the open channel (Figure 3). The ability of this single point mutation at the S4–S5/S6 interface to change the same variety of properties as KCNE1 suggests that a single mechanism for the effects of KCNE1 could be to alter the VSD-pore interactions.

Figure 5.

Modeling the effects of KCNE1.

(A) Cartoon illustrating the observed effects of KCNE1 on KCNQ1. The VSDs transit between resting (R, grey patterned), intermediate (I, red patterned), and activated (A, blue patterned) states. Each VSD conformation has unique interactions (double arrow) with the closed (C) and open (O) conformation of the pore. KCNE1 suppresses the intermediate-open (OI) state and modulates the activated-open (OA) states. (B) Kinetic model of KCNQ1 channel gating where the k parameters are the intrinsic transition rates of the VSD and the pore, and the θ parameters explicitly represent VSD-pore interactions. Fourth power notation ([ ]4) indicates that the model includes four VSDs. (C and D) Experimental data and model simulations with (+KCNE1) and without KCNE1. Steady-state current- (C, top), conductance- (C, middle), and fluorescence- (C, bottom) voltage relationships. (D) Current (black) and fluorescence (green) responses at various voltages.

DOI: http://dx.doi.org/10.7554/eLife.03606.013

The channel is modeled as a pore coupled to four voltage-sensing domains. VSD activation occurs in two transitions, resting (R) to intermediate (I) along the horizontal and intermediate to activated (A) along the vertical. The pore can be fully closed (C, back face, grey) or fully open (O, front face, black), with any combination of VSD-states. The state-notation indicates the conformation of the pore and the combination of the four VSDs (subscripted). For example CRRRR indicates the state where the pore is closed and all four VSDs are resting.

DOI: http://dx.doi.org/10.7554/eLife.03606.014

As described in the text the k parameters represent the intrinsic rates of the two domains and the θ parameters represent the effect of VSD-pore interactions at different states. The closed to open transitions dependent on the intrinsic rate of the pore (kco) divided by the closed state interactions with the VSDs. The open to closed transitions depend on the koc divided by the open state interactions the VSDs. n(X) indiciated the number of VSDs in state X.

DOI: http://dx.doi.org/10.7554/eLife.03606.015

The values represent those used in the kinetic modeling of KCNQ1 gating. ΔKCNE1 column indicates the changes used to simulate the effects of KCNE1 on KCNQ1 channel gating.

DOI: http://dx.doi.org/10.7554/eLife.03606.016

DOI: http://dx.doi.org/10.7554/eLife.03606.017

Descriptions, values, and units for constant parameters used in gating model simulation.

Experimentally, two effects of KCNE1 on VSD-pore interactions were observed. (1) KCNE1 prevented the channel from opening when the VSD was in the intermediate-state suggesting that KCNE1 changes the interactions between the intermediate-state of the VSD and the pore (Figure 1). (2) KCNE1 greatly increased the apparent affinity of the activated-open state for PIP2 suggesting that KCNE1 stabilized the activated-open state interactions (Figure 4). Using kinetic modeling, we sought to test if these experimentally observed effects of KCNE1 on VSD-pore interactions are sufficient to explain the many effects of KCNE1 on KCNQ1 activation gating. Previous Kv channel gating models assume that all four VSDs (Zagotta et al., 1994) or each VSD (Horrigan et al., 1999) must fully activate before the pore can open. Therefore, we could not use these established models as they are not able to represent the coupling between three different states of the VSD and the pore. We developed a new gating model that better reflects the understanding that the VSD and pore can fold and function independently (discussed in our previous review [Zaydman and Cui, 2014]), but couple their motions through state-dependent interactions.

In our gating model, the VSDs can occupy resting (R), intermediate (I), or activated (A) states, and the pore can occupy closed (C) or open (O) states (Figure 5B, Figure 5—figure supplement 1). Assuming that the intrinsic activation of each VSD is identical and non-cooperative, as assumed in the ZHA model (Zagotta et al., 1994), the different combinations of the states of the four VSDs and the pore give rise to only 30 possible channel states. Two sets of parameters, k's and θ's, determine the transitions among these channel states (Figure 5B, Figure 5—figure supplement 2,3). The ‘k’ parameters represent the intrinsic tendencies of the pore to open and close and a VSD to undergo its transitions, and would be measured directly if the VSD and pore could be decoupled entirely. The k parameters concerning VSD transitions (kRI, kIR, kIA, kAI) are assumed to be exponentially dependent on voltage according to Equation 1. On the other hand, the k parameters concerning pore transitions (kCO and kOC) are assumed to be voltage-independent (i.e., constant).where: kij is the voltage-dependent rate of transition from VSD state i to VSD state j, kij0 is the rate of the ij transition at 0 mV, zij is the valence of the ij transition, F is the faraday constant, V is voltage, R is the universal gas constant, T is the absolute temperature.

Figure 5—figure supplement 1.

Scheme for voltage-depedendent gating.

The channel is modeled as a pore coupled to four voltage-sensing domains. VSD activation occurs in two transitions, resting (R) to intermediate (I) along the horizontal and intermediate to activated (A) along the vertical. The pore can be fully closed (C, back face, grey) or fully open (O, front face, black), with any combination of VSD-states. The state-notation indicates the conformation of the pore and the combination of the four VSDs (subscripted). For example CRRRR indicates the state where the pore is closed and all four VSDs are resting.

DOI: http://dx.doi.org/10.7554/eLife.03606.014

The ‘θ’ terms explicitly represent the net effect of all VSD-pore interactions within each channel state. For example, θΙC, represents the net stabilization of the intermediate-closed state due to all interactions between the intermediate-state of the VSD and the closed-state of the pore. If θIC is greater than 1, these interactions will slow transitions away from the intermediate-closed state. The full model is shown in Figure 5—figure supplement 2 and a shorthand version appears in Figure 5B. The main difference between our model and previous allosteric gating models is that the reference state is an imaginary state in which the two domains are completely isolated (i.e., decoupled) from each other, rather than using transitions among the resting states as a reference. The advantage of our approach is that each parameter has an intuitive physical meaning: the k parameters represent the net effect of all interactions within a state of the VSD or the pore, and the θ parameters represent the net effect of all interactions between the VSD and the pore in a specific channel state.

Figure 5—figure supplement 2.

Balanced gating model showing transition rates.

As described in the text the k parameters represent the intrinsic rates of the two domains and the θ parameters represent the effect of VSD-pore interactions at different states. The closed to open transitions dependent on the intrinsic rate of the pore (kco) divided by the closed state interactions with the VSDs. The open to closed transitions depend on the koc divided by the open state interactions the VSDs. n(X) indiciated the number of VSDs in state X.

DOI: http://dx.doi.org/10.7554/eLife.03606.015

In order to parameterize our model, we started with parameter values from two previous KCNQ1 channel gating models (Silva and Rudy, 2005; Zaydman et al., 2013) and were able to reasonably replicate the experimentally observed KCNQ1 channel gating behavior (Figure 5C,D) with some adjustments to the parameter values to account for the differences in the schemes of these models (Figure 5—figure supplement 3). In order to simulate the effects of KCNE1 on VSD-pore interactions, we made only two changes to the VSD-pore interactions, as suggested by experimental results (Figure 5—figure supplement 3). (1) Experimentally, when KCNE1 was coexpressed, the pore remained closed while the VSD was in the intermediate state (Figure 1E, Figure 1—figure supplement 4). Thus, to model KCNE1, we strengthened the intermediate-closed state interaction, θIC. (2) Also, in experiments, KCNE1 increased the activated-open state affinity for PIP2 (Figure 4). Accordingly, we strengthened the activated-open state interaction, θAO. Making only these two changes mimicked all of the effects of KCNE1 on KCNQ1 activation gating (Figure 5C,D). The GV shifted to more depolarized voltages correlating with Fhigh because the intermediate-closed state interactions became more energetically favorable than the intermediate-open state interactions, preventing pore opening until a more depolarized voltage-range where the intermediate-to-activated transition occurred. In the absence of intermediate-state opening, the resting-to-intermediate transition occurred among closed states and introduced the characteristic delay of current onset of several hundred milliseconds. The maximal current amplitude was increased several fold due to stabilization of the activated-open state. Furthermore, the stabilization of the intermediate-closed state reduced the current near physiological resting voltages and caused a left shift in main component of the FV curve, two phenomena observed experimentally (Figure 5C). The kinetics and steady-state gating behavior predicted by our model were not quantitatively identical to those in experiments; such discrepancies were expected due to several overly simplistic assumptions that we used to limit the number of states in our model. Particularly, we assumed that PIP2 binding was saturated, that all open states had identical conductance, and that inactivated states did not exist. However, previous studies highlight that KCNQ1 is not saturated with PIP2 and that KCNE1 dramatically increases PIP2 binding (Li et al., 2011), that KCNE1 increases the apparent single channel conductance (Sesti and Goldstein, 1998; Yang and Sigworth, 1998), and that KCNE1 prevents the observation of a partially inactivated state (Pusch et al., 1998; Tristani-Firouzi and Sanguinetti, 1998), all of which may contribute to gating and macroscopic current amplitude. Revision of our model to include the influence of PIP2 binding and the different properties of intermediate- and activated-open states will require additional studies to better define these properties. Despite these limitations, it is remarkable that we were able to capture all the major effects of KCNE1 on the activation gating of KCNQ1. This model illustrates that the effects of KCNE1 on VSD-pore interactions are sufficient to explain how KCNE1 affects the activation gating of KCNQ1 without any additional effects on VSD activation or pore opening. Furthermore, as our experimental data demonstrate that VSD-pore interactions determine the permeation and pharmacological properties (Figures 2,3), our proposed mechanism, that KCNE1 affects VSD-pore interactions, provides a relatively complete explanation for how KCNE1 affects KCNQ1.

Figure 5—figure supplement 3.

Desriptions, values and units for free parameters of gating model.

The values represent those used in the kinetic modeling of KCNQ1 gating. ΔKCNE1 column indicates the changes used to simulate the effects of KCNE1 on KCNQ1 channel gating.

DOI: http://dx.doi.org/10.7554/eLife.03606.016

Central to understanding the modulation of KCNQ1 by KCNE1 is a longstanding controversy regarding which stoichiometries of KCNQ1:KCNE1 may exist in the fully assembled channel. Several groups have argued that association of KCNE1 with KCNQ1 dimers during an early stage of biogenesis leads to a fixed 4:2 KCNQ1:KCNE1 stoichiometry and breaks the fourfold symmetry of the channel (Wang and Goldstein, 1995; Chen et al., 2003; Morin and Kobertz, 2008; Plant et al.,2014). Other groups have argued that various stoichiometries, 4:0–4 KCNQ1:KCNE1, are possible, depending on the relative levels of subunit expression (Blumenthal and Kaczmarek, 1994; Cui et al., 1994; Wang et al., 1998; Nakajo et al., 2010; Li et al., 2011). In the present study, we coinjected oocytes with KCNQ1 and KCNE1 transcripts at a 1:1 ratio because previous work, from our lab (Li et al., 2011) and others (Nakajo et al., 2010), demonstrated that this ratio is sufficient to saturate the functional effects of KCNE1 on KCNQ1. In our modeling, we assume that coexpression of KCNE1 affects all four subunits identically as if the channel were saturated by KCNE1, that is, with a 4:4 stoichiometry. This assumption may represent yet another reason why the simulated and experimental gating behavior are not quantitatively identical. Furthermore, it is important to ask, given the ongoing controversy regarding stoichiometry, if the presence of multiple populations of channels with different stoichiometries could complicate our interpretation of our data. Fortunately, the F351A mutation demonstrates that the major finding of our study—the ability to couple pore opening to different transitions within the VSD—is intrinsic to the KCNQ1 subunit and observed even in the absence of the KCNE1 subunit.

In the voltage-gated ion channel field, VSD-pore coupling, aka electromechanical coupling, has been a loosely defined term referring to the experimental observation that pore opening is more likely when the VSDs are activated at depolarized voltages. In our model of voltage-dependent gating, coupling is represented in a very different way than in the previously established models. The landmark linear models of Shaker Kv channels (Schoppa et al., 1992; Zagotta et al., 1994), which require that all four VSDs be activated before the pore can open, do an excellent job replicating the gating of Shaker Kv channels, but do not explicitly define coupling. Another landmark model, the Horrigan-Cui-Aldrich (HCA) model (Horrigan et al., 1999), permits pore-opening without prior activation of all VSDs. In the HCA model, coupling is quantitatively defined by a single term, D, representing how much the closed-open equilibrium of the pore is biased towards open when a single VSD is transitioned from its fully resting to its fully activated state. Our model further develops from these two previous models. Like the shaker model, we assumed two transitions occurring sequentially and independently within each VSD. Similar to the HCA model, we assumed that pore opening can occur with the four VSDs in any combination of states. In our model, we decomposed D into its elementary components, that is, VSD-pore interactions at each channel state. As a result, each parameter (k, θ) in our model represents a specific set of interactions that exist at the same time in the physical world. The relative differences in the strengths of all these state-dependent VSD-pore interactions lead to the experimental observation that a change in VSD conformation leads to a change in the probability of pore opening, that is, coupling. It is important to understand that coupling is a result of all of these state-dependent interactions and a perturbation of any of these interactions may alter the coupling. These points are illustrated by our modeling of the KCNE1 effect on KCNQ1 channel gating where strengthening the intermediate-closed interactions caused an apparent decoupling of pore-opening from the resting-to-intermediate transition of the VSD, that is, the open probability was no longer increased by the transition of the VSD to the intermediate-state at intermediate voltages. Alternatively, weakening the intermediate-open state interactions would also decouple opening from the resting-to-intermediate state of the VSD; however, this would not reproduce the leftward shift of the first FV component (Fmain) that we observed when KCNE1 was expressed (Figure 5C).

The topic of how KCNE1 modulates KCNQ1 has been studied for many years. Early studies by Goldstein and colleagues argued that KCNE1 intercalates deeply into the pore and directly lines the permeation pathway (Goldstein and Miller, 1991; Wang et al., 1996; Tai and Goldstein, 1998). Later, the Kass and George labs argued that KCNE1 associates outside the pore and modulates the pore properties through an allosteric mechanism (Kurokawa et al., 2001; Tapper and George, 2001). Then, the efforts of several labs have detected probable interactions between the extracellular (Xu et al., 2008; Chan et al., 2012), transmembrane (Tapper and George, 2001; Chung et al., 2009; Lvov et al., 2010), and intracellular (Haitin et al., 2009) regions of KCNQ1 and KCNE1. These data have been used to build homology models of the KCNQ1+KCNE1 channel (Kang et al., 2008; Lundby et al., 2010; Xu et al., 2013), all of which place KCNE1 in a cleft between a VSD and the pore where it participates in a broad set of interactions with KCNQ1. Even after all of these excellent studies, there was a lack of a biophysical mechanism to explain how these interactions alter the gating, permeation, and pharmacology of KCNQ1. Prior biophysical studies, which have focused on how KCNE1 slows current onset, have come to conflicting conclusions that KCNE1 either slows VSD activation (Nakajo and Kubo, 2007; Ruscic et al., 2013) or pore opening (Rocheleau and Kobertz, 2008; Osteen et al., 2010). We believe that the interpretations of these data were limited by several missing pieces of information revealed in the present study: (1) VSD activation occurs through a stable intermediate state, (2) full activation of the VSD is not necessarily required to open the pore, and (3) the distinct interactions between different states of the VSD and the pore determine both the probability of opening and the open-state conformation. Also, it is likely that these previous studies did not consider an effect on the state-dependent VSD-pore interactions because such interactions are not explicitly represented in previously established gating models.

Recently, the Larsson and Kass labs reported the presence of a second fluorescence transition in KCNQ1+KCNE1 channels, which they attributed to pore opening (Osteen et al., 2010, 2012) or a concerted step in which pore opening and further S4 movement occur simultaneously (Barro-Soria et al., 2014). From these observations, they concluded that KCNE1 changes the number of subunits that must be activated before pore opening can occur leading to a right shifted GV and a delay in current onset. This model is most similar to ours in that KCNE1 is changing the relationship between VSD activation and pore opening; however, our model is different in several regards. We believe that the second FV component reports on an intrinsic transition of the VSD rather than the pore opening step. This assumption was based on our observation that homomeric KCNQ1 channels also exhibited a second FV component, with similar properties to that of KCNQ1+KCNE1, which occurred in a range of voltages that was more positive than required for pore opening (Figure 1A–H, Figure 1—figure supplement 1). Thus, in our model, channel opening requires an additional transition within a single VSD, rather than the activation of additional subunits. A natural consequence of such gating behavior is the existence of both intermediate-open and activated-open states, which could be detected experimentally and are of great functional importance as the different open states have different conductive and pharmacological properties (Figure 2).

Materials and methods

Mutagenesis

Point mutations were engineered using overlap extension and high-fidelity PCR. Each mutation was verified by DNA sequencing. cRNA was synthesized using the mMessage T7 polymerase kit (Applied Biosystems).

Oocytes expression

Pieces of ovarian lobes were excised from Xenopus laevis by laparotomy. Stage V or VI oocytes from X. laevis were isolated by collagenase (Sigma Aldrich, St Louis, MO) digestion. 9.2 ng of KCNQ1 cRNA was microinjected with or without 2.3 ng of KCNE1 cRNA (using the Drummond Nanoject, Broomall, PA) into each oocyte. For CiVSP expression, 2.3 ng of CiVSP cRNA was coinjected. Injected cells were incubated at 18°C for up to 7 days before recording in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.6).

Electrophysiology

Two-electrode voltage clamp

Microelectrodes were pulled with resistances between 0.3 and 3 MΩ and filled with 3 M KCl solution. Recordings were performed in ND96 solutions unless otherwise indicated. Whole-oocyte currents in response to applied voltage steps were amplified using the CA-1B (Dagan, Minneapolis, MN) amplifier in two-electrode voltage clamp mode and digitized using the HEKA EPC10 (HEKA, Germany) AD/DA board, sampled at 1 KHz and recorded using the Patchmaster (HEKA) software.

Voltage clamp fluorometry

Oocytes were labeled with 10 μM Alexa 488 C5-maleimide or Alexa 546 C5-maleimide (Molecular Probes, Eugene, OR) in high K+ solution (98 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.6) for 45 min on ice. After labeling, the cells were washed with ND96 and kept on ice until recording. Recordings were performed in ND96 solution. Fluorescence emission from the sample was focused onto a Pin20A photodiode (OSI Optoelectronics), amplified by an EPC10 (HEKA) patch amplifier, analog filtered at 200 Hz, sampled at 1 KHz, and recorded simultaneously with whole oocyte currents. A FITC filter cube (Leica, Germany) was used for Alexa 488 labeled cells and a rhodamine cube (Leica) was used for cells labeled with Alexa 546.

Voltage-protocols

Holding potential was set to −80 mV throughout. Voltage steps were applied to elicit current and fluorescence signals. Tail potentials were +60 mV for VCF experiments and measuring IV curves, −60 mV for Rb+/K+ permeability experiments, and −40 mV for XE991 experiments.

Molecular dynamics (MD) simulations

In our previous work (Kasimova et al.,), we have built models of the Kv7.1 activated/open and resting/closed states using homology modeling (Eswar et al., 2007) with the Kv1.2 crystal structure in its activated/open state (pdb code 3LUT [Chen et al., 2010]), α, and in its δ conformational state (Delemotte et al., 2011) as templates. Here, we applied a similar protocol to prepare a model of the Kv7.1 intermediate state. Each state is characterized by a unique set of interactions within the VSD. In particular, E160 (E1) forms a salt bridge with R237 (R4) in the activated/open or with R228 (R1) in the resting/closed states (Figure 1I, S3a). We assumed that, when the VSD is intermediate, E1 interacts with the residue located in between of R4 and R1, namely R231 (R2). Based on this assumption, the γ conformation of Kv1.2 (Delemotte et al., 2011) with the E1-R2, was considered as a template for the Kv7.1 intermediate state model. This model was further embedded in a palmitoyl-oleyl-phosphatidylcholine (POPC) hydrated bilayer and immersed in a 150 mM K+Cl− solution. Due to the importance of PIP2 for Kv7.1 function (Loussouarn et al., 2003; Eswar et al., 2007), four molecules of this lipid were placed at the channel's intrasubunit sites located at the interface between the voltage sensor and the pore (Zaydman et al., 2013; Kasimova et al.,).

The MD simulations were performed using NAMD (Smart et al., 1996). Langevin dynamics was applied to keep the temperature (300 K) and the pressure (1 atm) constant. The time-step of the simulations was 2.0 fs. The equations of motion were integrated using a multiple time-step algorithm. Short- and long-range forces were calculated every 1 and 2 time-steps respectively. Long-range electrostatics was calculated using Particle Mesh Ewald (PME). The cutoff distance of short-range electrostatics was taken to be 11 Å. A switching function was used between 8 and 11 Å to smoothly bring the vdW forces and energies to 0 at 11 Å. During the calculations, chemical bonds between hydrogen and heavy atoms were constrained to their equilibrium values. Periodic boundary conditions were applied.

The protein backbone was constrained during 100 ns allowing PIP2 to sample possible interactions with the channel's positive residues. Based on this trajectory, the time evolution of salt bridges formation was monitored. Several residues of Kv7.1 interacted with the lipid headgroups temporarily, revealing different configurations of the system where corresponding salt bridges were either formed or broken. In total, for the activated/open, intermediate and resting/closed states we have identified 9, 8 and 8 the most frequent configurations respectively. These were considered as starting points for the final equilibration step, involving gradual release of the protein backbone and subsequent relaxation of the entire system during 100 ns for each. For all the trajectories, the root mean square deviation (RMSD) from the initial structure reached a plateau starting from ∼50 ns. The simulation stretch from 50 to 100 ns was used for further analysis.

In order to estimate a degree of the Kv7.1 pore dilation at the intercellular gate level, we applied HOLE (Smart et al., 1996). 50 conformations of Kv7.1 spread equidistantly along the last 50 ns were extracted from each MD trajectory. For these conformations, the pore radius along the axis normal to the membrane (Z) was calculated. The obtained profiles were considered to estimate an average profile and error bars (SD) for each of the channel states.

To analyze the salt bridge formation between PIP2 and Kv7.1, we measured the minimal distance between the nitrogen atoms of arginine and lysine charged groups and the oxygen atoms of the PIP2 phosphates. The salt bridges were assumed formed if the calculated distance was less than 3.2 Å. The probabilities of salt bridge formation were simultaneously estimated for four subunits of the channel as a ratio between the number of frames with a formed salt bridge to its total number. The error bars correspond to a standard deviation (SD) calculated between values obtained from several MD runs.

Data analysis

Relative conductance-voltage (GV) relationships were generated by estimating the instantaneous tail current values following test pulses to various voltages and normalizing to the value following the highest voltage test pulse. For calculation of relative fluorescence changes, a baseline fluorescence was extrapolated by fitting a line to the fluorescence at the holding potential during the 2 s preceding application of the voltage pulse. ΔF/F was calculated as (F(t)-baseline(t))/baseline(t), where F(t) is the raw fluorescence intensity at time t and baseline(t) is the extrapolated baseline value at time t. Fluorescence voltage-relationships were derived by normalizing the ΔF/F value at the end of a four second test pulse to various voltages to the value of the highest voltage test-pulse. FV and GV curves were fits with one or the sum of two Boltzmann equations in the form 1/(1 + exp(−z*F*(V − V1/2)/RT)) where z is the equivelant valence of the transition, V1/2 is the voltage at which the transition is half maximal, R is the gas constant, T is absolute temperature, F is the Faraday constant and V is the voltage. FV curves were derived from the value at the end of the test pulse, GV curved were derived from estimating the instantaneous tail current amplitude.

Statistics

All averaged data reflects n = 6 or more from at least two batches of oocytes. Pairwise comparisons were achieved using Student's t test, multiple comparisons were performed using an ANOVA with Tukey's Post-Hoc Test. All error bars represent standard error mean.

Solutions and chemicals

100 mM Na+ (96 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES) 100 mM K+(100 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES) 100 mM Rb (96 mM RbCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES) ND96 (96 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES) XE991 from Sigma Aldrich. Alexa fluors from Molecular Probes.

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “KCNE1 modulates KCNQ1 channel gating, permeation and pharmacology through a single mechanism” for consideration at eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor), Richard Aldrich (Reviewing editor), and 3 reviewers.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The biophysical and pharmacological properties of KCNQ1 K+ channels are radically altered by co-assembly with KCNE1 accessory beta-subunits. In this study, the authors use voltage-clamp fluorometry (VCF) to show that activation of the voltage sensor domain (VSD) of KCNQ1 channels occurs in two resolvable steps. Both the intermediate and fully-activated states of the VSD result in KCNQ1 channel opening, and these states have different permeation and pharmacological properties. The intermediate-open state was absent when KCNQ1 was co-expressed with its beta-subunit KCNE1, suggesting that accessory subunits changes how the VSD and pore interact. The gating of F351A KCNQ1 channels in many ways mimics KCNQ1/KCNE1 channel gating, supporting the main hypothesis. Finally, a kinetic model was developed that can account for many of the effects of KCNE1 on VSD-pore interactions. These findings provide important and novel insights into the mechanism of IKs channel gating as well as a framework for interpreting KCNQ effects that is novel, well substantiated, conceptually simple, and thermodynamically plausible.

The authors need to more clearly delineate and critically evaluate which aspects of the gating and permeation/conductance differences between the two channels their model does or does not fully describe.

The following issues must be addressed in a revision:

1) The main conclusion, that KCNE1 alters KCNQ1 gating by a single mechanism (altered VSD-pore coupling; modeled by only two changes to a kinetic model) is attractive due to its apparent simplicity; however in reality the effects of the accessory subunit are more complex than just shifting the G-V and slowing activation kinetics. KCNQ1 channels inactivate, whereas KCNQ1/KCNE1 channels do not. Some of the deviation between the data and the predictions of the model may be due to KCNQ1 inactivation, which is not detectable using voltage-activation protocols when KCNE1 is present – something the authors might want to consider in the interpretation of their data.

2) The single channel properties of KCNQ1 channels differ from KCNQ1/KCNE1 channels. The model should be modified to account for these important differences in channel properties. One would expect for example two discernible channel open states for KCNQ1 (corresponding to the intermediate-open (O1) and activated-open (O2) states), whereas KCNQ1/KCNE1 channels would have only one open state, with properties similar to the O2 state for KCNQ1 channels. It is stated that “The maximal current amplitude was increased several fold due to stabilization of the activated-open state”. Presumably, this means only a change in Po, whereas experimental data indicate that KCNQ1/KCNE1 currents are larger mainly due to an increase in single channel conductance (Yang and Sigworth, 1998. J Gen Physiol 112:665).

3) The data presented in Figure 3 and corresponding text are incomplete and their interpretation (that it indicates increased “strength” of VSD-pore coupling in presence of KCNE1) is not convincing. F351A channels have many properties similar to KCNQ1/KCNE1 channels (especially G-V matching Fmain-V relationship), but the relative Rb/K ion permeation and pharmacology are not the same. The authors state that F351A like KCNE1 cause significant changes in the Rb/K permeability ratio (Figure 3f), however this change is in the opposite direction from KCNE1 (Figure 2b). This KCNQ1 mutant with KCNE1-like activation kinetics should have a significantly lower (not a higher) permeability ratio if it is mimicking KCNQ1/KCNE1 channels. This should be made clear. Similarly, F351A affects XE991 pharmacology, but this is unlike the effect of KCNE1. These experiments need to be expanded (e.g., dose-response to XE991; differences in compound sensitivity requires more than a single concentration) to more quantitatively describe the effects of KCNE1. In addition, the rubidium data and pore dilation hypothesis might be better resolved using external cesium, which is larger, yet permeates homotetrameric KCNQ1 channels.

4) Several figures use whole cell current amplitude as a proxy for channel gating. Authors should address the possibility of surface expression level differences with different proteins, KCNQ mutations and plus/minus KCNE.

5) CiVSP experiments need to be better explained and analyzed. The protocol for CiVSP activation is not explained. There is no analysis or evidence of repeated trials, and the logic is confusing. In the text the claim is made that Figure 3b demonstrates that KCNE1 increases the open state affinity for PIP2. This claim is not well backed up. Explaining why a lack of effect of CiVSP demonstrates high affinity rather than no interaction with PIP2 could be helpful.

6) The modeling could be explained better. The modeled effect of KCNQ invokes a 10 fold stabilization of IC and AO states with no alteration of transition barrier heights. In effect, this results in decreased coupling to the IO state (because the RO is rarely occupied). The implied change in coupling could be more thoroughly discussed, as this is the major intellectual contribution of this study.

7) While it is mentioned that the kinetics of the model don't entirely match the data, it appears that a major feature of KCNQ gating is missed by the model. The “tau slow” of KCNQ (quantified and made note of in the discussion surrounding Figure 1) is proposed to be the transition from an IO state to an AO state. The existence of this slow component is a pillar of their mechanism involving multiple classes of open states. This slow component seems to be missing from the KCNQ1 modeled kinetics.

8) Statistics. Students T test was used without explanation of why such a parametric test is appropriate.

9) Figure 1–figure supplement 4. Analyzing normalized GVs here would be more informative than just the IVs in panels (c),(d).

10) Figure 2a: Explain voltage protocols used more completely. If KCNQ is slowly transiting from an IO state to an AO state wouldn't the Rb/K permeability ratio be time and voltage dependent?

11) The authors should discuss/hypothesize how two KCNE1 subunits fit into their modeling (Figure 4) given the recent paper by the Goldstein lab unequivocally showing that the stoichiometry of the complex is 4:2 Q1:E1.

12) XE991 Pharmacology: The Wang et al. paper showed that KCNQ1/KCNE1 channels are 10-fold less sensitive than KCNQ1 channels; however, in Figure 2C wild type KCNQ1/KCNE1 channels do not appear to be 10-fold less sensitive to the drug. Of all the currents shown, these are the smallest, which makes us wonder if these are endogenous xKCNQ1 channels assembling with exogenously expressed KCNE1 subunits. Besides the inconsistency with wild type, the E1R/R4E+KCNE1 mutant should have similar “inhibition” to E1R/R4E. It appears that the E1R/R4E mutant is modestly activated by XE991.

1) The main conclusion, that KCNE1 alters KCNQ1 gating by a single mechanism (altered VSD-pore coupling; modeled by only two changes to a kinetic model) is attractive due to its apparent simplicity; however in reality the effects of the accessory subunit are more complex than just shifting the G-V and slowing activation kinetics. KCNQ1 channels inactivate, whereas KCNQ1/KCNE1 channels do not. Some of the deviation between the data and the predictions of the model may be due to KCNQ1 inactivation, which is not detectable using voltage-activation protocols when KCNE1 is present – something the authors might want to consider in the interpretation of their data.

The reviewers are right that besides the reasons that we have described, the presence of inactivation gating likely also contributes to the discrepancy between the model kinetics and the experimental data. We have added the following to the manuscript to acknowledge this important point.

“The kinetics and steady-state gating behavior predicted by our model were not quantitatively identical to those in experiments; such discrepancies were expected due to several overly simplistic assumptions that we used to limit the number of states in our model. […] Revision of our model to include the influence of PIP2 binding and the different properties of intermediate- and activated-open states will require additional studies to better define these properties.”

In response to this interesting point, Seebohm et al. interpret KCNQ1 channel inactivation using two open states with different conductances that are occupied sequentially. This idea is consistent with our conceptual model provided the activated open state of KCNQ1 has a lower conductance or lower open probability than that of the intermediate open state. However, as we cannot determine which subset of open-states contribute to the apparent inactivation, we cannot yet use our kinetic model to fit inactivation. Future studies may provide the necessary data to do so and make this mechanism clearer.

2) The single channel properties of KCNQ1 channels differ from KCNQ1/KCNE1 channels. The model should be modified to account for these important differences in channel properties. One would expect for example two discernible channel open states for KCNQ1 (corresponding to the intermediate-open (O1) and activated-open (O2) states), whereas KCNQ1/KCNE1 channels would have only one open state, with properties similar to the O2 state for KCNQ1 channels. It is stated that “The maximal current amplitude was increased several fold due to stabilization of the activated-open state”. Presumably, this means only a change in Po, whereas experimental data indicate that KCNQ1/KCNE1 currents are larger mainly due to an increase in single channel conductance (Yang and Sigworth, 1998. J Gen Physiol 112:665).

The reviewers are right that we did not clearly address the possible contribution of changes in single channel conductance to the change in macroscopic current amplitude when KCNE1 is expressed. Now we acknowledge previous observations that provide additional explanations for how current amplitude is augmented by KCNE1. (See the response to the above question.)

We would like to address the statement that a change in single channel conductance has been already shown to be the main contributor to the difference in KCNQ1 and KCNQ1+KCNE1 macroscopic current amplitude. The difference in single channel conductance between KCNQ1 and KCNQ1+KCNE1 was established in two papers using non-stationary noise analysis (Sesti & Goldstein, 1998; Youshan Yang, 1998). While these data are enlightening, the assertion that the apparent difference in single channel conductance quantitatively accounts for all or most of the difference in current amplitude is uncertain. Current knowledge reveals that there are multiple open states (current manuscript; Pusch, Ferrera, & Friedrich, 2001; Werry, Eldstrom, Wang, & Fedida, n.d.) and that there is a fast flickery process in the KCNQ1 pore that is associated with inactivation and removed by expression of KCNE1 (Pusch et al., 1998; Seebohm, Sanguinetti, & Pusch, 2003). These data were not available at the time of the studies using noise analysis, which assumes only a single open state and that the bandwidth of recording is sufficient to capture all relevant timescales.

Finally, while it may seem simple to define different properties for intermediate-open and activated-open states, doing so is actually complicated and nontrivial. First, the single conductance of the intermediate-open and activated-open states is unknown and exceedingly difficult to measure experimentally. Second, because there are four VSDs, there are open states with different combinations of VSD states. A fascinating, and unanswered, question is what properties an open state with a mixed set (for example 2 intermediate and 2 activated) of VSDs will exhibit. Finally, our data indicate that KCNE1 changes the properties of the O2 (AKA activated-open) state; therefore, it would not be accurate to assume in the model that the open state of KCNQ1+KCNE1 would have the same properties as the O2 state of KCNQ1.

3) The data presented in and corresponding text are incomplete and their interpretation (that it indicates increased “strength” of VSD-pore coupling in presence of KCNE1) is not convincing. F351A channels have many properties similar to KCNQ1/KCNE1 channels (especially G-V matching Fmain-V relationship), but the relative Rb/K ion permeation and pharmacology are not the same. The authors state that F351A like KCNE1 cause significant changes in the Rb/K permeability ratio (), however this change is in the opposite direction from KCNE1 (). This KCNQ1 mutant with KCNE1-like activation kinetics should have a significantly lower (not a higher) permeability ratio if it is mimicking KCNQ1/KCNE1 channels. This should be made clear. Similarly, F351A affects XE991 pharmacology, but this is unlike the effect of KCNE1. These experiments need to be expanded (e.g., dose-response to XE991; differences in compound sensitivity requires more than a single concentration) to more quantitatively describe the effects of KCNE1. In addition, the rubidium data and pore dilation hypothesis might be better resolved using external cesium, which is larger, yet permeates homotetrameric KCNQ1 channels.

1. Increased strength of coupling

The reviewers are right that in the previous version of our manuscript we did not provide a careful explanation of how the CiVSP experiments substantiate our statement that KCNE1 strengthens the VSD-pore interaction in the activated-open state. We have now included additional data to provide evidence of an open-state with high affinity for PIP2 that is observed only when KCNE1 is coexpressed (Figure 4) and we have revised the text to more clearly explain how we interpret the data. Paragraph starting, “Does KCNE1 also affect the interaction between the activated-state of the VSD and the pore to modulate the activated-open state? […] Altogether these results reveal that KCNE1 causes the activated-open state to have a high apparent affinity for PIP2, suggesting that KCNE1 may increase the strength of VSD-pore interactions in the activated open state.”

2. F351A properties are not perfectly matching KCNQ1+KCNE1

This difference was acknowledged in our previous version, although it may not have come across clearly enough. We do not expect that F351A and KCNE1 would cause exactly the same changes because F351A causes only a focal change in the VSD-pore interactions while KCNE1 may cause a more broad change due to its larger interaction surface. We think that the difference between F351A and KCNE1 actually substantiates a central element of our manuscript, i.e. that the pore conformation is sensitive to the VSD-pore interactions. Subjecting these interactions to different perturbations results in different conformations of the open-pore with various functional consequences. We now discuss these aspects in two places in the revised manuscript, at the end of the Results and in the Discussion.

“Of note, these properties of F351A were not identical to those of WT KCNQ1+KCNE1. However, such differences are not surprising as prior studies(Chan et al., 2012; Chung et al., 2009; Kang et al., 2008; Lvov, Gage, Berrios, & Kobertz, 2010; Strutz-Seebohm et al., 2011; Y. H. Wang et al., 2011; X. Xu, Jiang, Hsu, Zhang, & Tseng, 2008; Y. Xu et al., 2013) have located KCNE1 at the VSD-pore interface and have suggested that KCNE1 engages in a very broad and complex set of interactions with KCNQ1(Xiaohui Sun, 2012); therefore, it would be unreasonable to expect that a single point mutation, such as F351A, would alter the VSD-pore interactions in exactly the same way as KCNE1.”

3. Completion of dose response curve

We thank the reviewers for this suggestion and have added the data as requested (Figure 2, Figure 2–figure supplement 1, Figure 3).

4. Cesium experiment and diameter of the permeation pathway

It is not our understanding that the change in relative permeatility toward Rb, K, or perhaps other ions, simply reflects a change in the diameter of the permeation pathway. Here we used the well-studied relative Rb/K permeabilities to probe for different open states associated with the intermediate and activated conformations of the VSD. The Cesium study, suggested by the reviewer, may provide additional mechanistic insights of how ion selectivity is changed amongst different open states in a future study. However, we believe that it is beyond the scope of this present manuscript.

4) Several figures use whole cell current amplitude as a proxy for channel gating. Authors should address the possibility of surface expression level differences with different proteins, KCNQ mutations and plus/minus KCNE.

We thank the reviewers for this excellent and important point. We have now included data (Figure 1–figure supplement 5) to estimate the surface expression of the different KCNQ1 mutations +/- KCNE1 and show that the surface expression is similar for the different mutant and is not significantly altered by KCNE1. Therefore, the differences in current amplitude do not originate from changes in surface expression, and more likely reflect changes in the gating or permeation.

5) CiVSP experiments need to be better explained and analyzed. The protocol for CiVSP activation is not explained. There is no analysis or evidence of repeated trials, and the logic is confusing. In the text the claim is made that demonstrates that KCNE1 increases the open state affinity for PIP2. This claim is not well backed up. Explaining why a lack of effect of CiVSP demonstrates high affinity rather than no interaction with PIP2 could be helpful.

We have revised this section to clarify our logic, and we have included additional data to support the existence of an activated-open state, observed with KCNE1, that has such high affinity for PIP2 and that PIP2 unbinding is minimal until the channels are closed (Figure 4b, c). We have also revised the figures to demonstrate the reproducibility of the response to CiVSP with repeated trials (Figure 4a).

6) The modeling could be explained better. The modeled effect of KCNQ invokes a 10 fold stabilization of IC and AO states with no alteration of transition barrier heights. In effect, this results in decreased coupling to the IO state (because the RO is rarely occupied). The implied change in coupling could be more thoroughly discussed, as this is the major intellectual contribution of this study.

We thank the reviewers for their comments. We have expanded the discussion of the model to illustrate its origins, in which way it differs from previous models, and how a change in the VSD-pore interactions leads to an apparent change in coupling. We would also like to mention that, although some previous work has conceptualized coupling as a barrier between activated and open states in a linear gating scheme, our model describes coupling as net energy of VSD-pore interactions at different states of these two structural domains.

“In the voltage-gated ion channel field, VSD-pore coupling, aka electromechanical coupling, has been a loosely defined term referring to the experimental observation that pore opening is more likely when the VSDs are activated at depolarized voltages. In our model of voltage-dependent gating, coupling is represented in a very different way than in the previously established models. […] It is important to understand that coupling is a result of all of these state-dependent interactions and a perturbation of any of these interactions may alter the coupling. These points are illustrated by our modeling of the KCNE1 effect on KCNQ1 channel gating where strengthening the intermediate-closed interactions caused an apparent decoupling of pore-opening from the resting-to-intermediate transition of the VSD, i.e. the open probability was no longer increased by the transition of the VSD to the intermediate-state at intermediate voltages. Alternatively, weakening the intermediate-open state interactions would also decouple opening from the resting-to-intermediate state of the VSD; however, this would not reproduce the leftward shift of the first FV component that we observed when KCNE1 was expressed (Figure 5c).”

7) While it is mentioned that the kinetics of the model don't entirely match the data, it appears that a major feature of KCNQ gating is missed by the model. The “tau slow” of KCNQ (quantified and made note of in the discussion surrounding ) is proposed to be the transition from an IO state to an AO state. The existence of this slow component is a pillar of their mechanism involving multiple classes of open states. This slow component seems to be missing from the KCNQ1 modeled kinetics.

We thank the reviewers for their thoughtful review. The second transition is indeed present, but it is occurring too quickly to be resolved in the simulated kinetics of KCNQ1. However, it is readily apparent in the steady-state behavior of KCNQ1, and it can be more readily appreciated by altering the parameter values to slow the intermediate-to-activated transition. However, because the parameter space is so vast, we have not yet identified a solution that provides as clear of a demonstration of the steady-state behavior when we make such changes. We have not pursued such a solution further for several reasons. Fist, as described already, our model makes several simplistic assumptions (PIP2 saturated, all open states have equal conductance, no inactivation) that make a perfect fit of our model to the experimental data unlikely. Additionally, we assumed that KCNE1 only increases the intermediate-closed and activated-open interaction because we have experimental evidence that leads us to that such changes occur and are critical to the mechanism of KCNE1. However, it is possible that KCNE1 also affects VSD-pore interactions in other states and those interactions contribute to gating behavior. Finally, it is important to acknowledge that, because our model represents the elementary interactions underlying coupling, it is too underdetermined to identify a single “correct” solution of parameter values that may be expected to represent the “real” solution. As we do not intend for our model to be used as a robust gating model that can be plugged into a cell or tissue model, such a solution may be irrelevant in any case. The true power and utility of our model is to provide the conceptual framework to explore how state-dependent interactions contribute to channel gating. For this purpose, we believe that it does a remarkable job and hope that with additional work in future studies we can continue to refine the model so that the kinetics, inactivation, and PIP2 regulation can be better recapitulated by such simple changes in VSD-pore interactions.

8) Statistics. Students T test was used without explanation of why such a parametric test is appropriate.

The Methods section now better explains which tests were chosen for each data set.

“All averaged data reflects n=6 or more from at least two batches of oocytes. Pairwise comparisons were achieved using Students T test, multiple comparisons were performed using an Anova with Tukeys Post-Hoc Test. All error bars represent standard error mean.”

9) . Analyzing normalized GVs here would be more informative than just the IVs in panels (c), (d).

The purpose of this figure is to compare the current level of different constructs and to demonstrate the ohmic nature of E1R/R2E and E1R/R4E with and without KCNE1. Therefore, we believe that IV is the appropriate format and GV is impossible to construct.

10) : Explain voltage protocols used more completely. If KCNQ is slowly transiting from an IO state to an AO state wouldn't the Rb/K permeability ratio be time and voltage dependent?

The reviewers are right that Rb/K permeability should be time and voltage-dependence for KCNQ1; however, the extent of such effects will depend both on the relative occupancies and conductances of the different states. Along these lines, previous studies have described time dependent changes in the sensitivity to intracellular Na ions(Pusch et al., 2001), which may be related to changes in Rb and K permeation as well. In this initial description, we used a single voltage protocol to demonstrate the differences in the conductances of the different states. We now explain this protocol more clearly in the text.

“Currents from a single cell in external solutions containing 100 mM of Na+, K+, or Rb+. The currents were elicited by first stepping the voltage to +60 mV for 5 s then to -60 mV for 3 seconds tails. (b) Averaged Rb+/K+ permeability ratios calculated by comparing the tail current amplitudes.”

11) The authors should discuss/hypothesize how two KCNE1 subunits fit into their modeling () given the recent paper by the Goldstein lab unequivocally showing that the stoichiometry of the complex is 4:2 Q1:E1.

Discussion of controversy over stoichiometry and how it relates to our modeling has been added to the discussion section.

“Central to understanding the modulation of KCNQ1 by KCNE1 is a longstanding controversy regarding what stoichiometries of KCNQ1:KCNE1 may exist in the fully assembled channel. Several groups have argued that association of KCNE1 with KCNQ1 dimers during an early stage of biogenesis leads to a fixed 4:2 KCNQ1:KCNE1 stoichiometry and breaks the fourfold symmetry of the channel(Chen, Kim, Rajan, Xu, & Goldstein, 2003; Morin & Kobertz, 2008; Plant, Xiong, Dai, & Goldstein, n.d.; K. W. Wang & Goldstein, 1995). […] Fortunately, the F351A mutation demonstrates that the major finding of our study – the ability to couple pore opening to different transitions within the VSD – is intrinsic to the KCNQ1 subunit and observed even in the absence of the KCNE1 subunit.”

12) XE991 Pharmacology: The Wang et al. paper showed that KCNQ1/KCNE1 channels are 10-fold less sensitive than KCNQ1 channels; however, in wild type KCNQ1/KCNE1 channels do not appear to be 10-fold less sensitive to the drug. Of all the currents shown, these are the smallest, which makes us wonder if these are endogenous xKCNQ1 channels assembling with exogenously expressed KCNE1 subunits. Besides the inconsistency with wild type, the E1R/R4E+KCNE1 mutant should have similar “inhibition” to E1R/R4E. It appears that the E1R/R4E mutant is modestly activated by XE991.

We have added additional data (Figure 2–figure supplement 1) and discussion demonstrating that, similar to the report by Wang et al., we observed that KCNQ1/KCNE1 channels are less sensitive than KCNQ1 alone when short pulse durations are considered. These differences become insignificant at longer pulse durations, which is why we did not show a difference in the original version of the manuscript. As described, we did not expect KCNQ1+KCNE1 to have similar properties as E1R/R4E because KCNE1 alters the activated open state. However, based on our hypothesis that KCNQ1+KCNE1 currents come from channels in the activated-open state, we did expect KCNQ1+KCNE1 to have similar properties as E1R/R4E+KCNE1, which is what we observed (Figure 2).