1. Structural Biology and Molecular Biophysics

Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex

  1. Go Kasuya  Is a corresponding author
  2. Koichi Nakajo  Is a corresponding author
  1. Division of Integrative Physiology, Department of Physiology, Jichi Medical University, Japan
https://doi.org/10.7554/eLife.81683
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  1. Go Kasuya
  2. Koichi Nakajo
(2022)
Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex
eLife 11:e81683.
https://doi.org/10.7554/eLife.81683
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Abstract

Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues (volume scanning), we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.

Editor's evaluation

This study of physiologically important K+ channel complexes is expected to be important to electrophysiologists and biophysicists. This structure-motivated mutagenesis and biophysical study provide compelling evidence that a structural interface between a K+ channel and a class of modulatory subunits is a critical functional interface that determines the efficacy of the modulatory subunits.

https://doi.org/10.7554/eLife.81683.sa0

Introduction

KCNQ1 (Kv7.1) is a voltage-gated K+ channel. Its gating behavior depends mainly on its auxiliary subunit KCNE proteins (Wang et al., 2020). There are five KCNE genes in the human genome, and all of them are known to modify KCNQ1 channel gating behavior, at least in Xenopus oocytes or mammalian cell lines (Bendahhou et al., 2005). Therefore, the physiological functions of the KCNQ1 channel are determined by the type of KCNE proteins that are co-expressed in a tissue. The most well-studied example is the cardiac KCNQ1-KCNE1 channel, which underlies the slow cardiac delayed-rectifier K+ current (IKs) (Barhanin et al., 1996; Sanguinetti et al., 1996; Takumi et al., 1988). Another example is the KCNQ1-KCNE3 channel, a constitutively open channel expressed in epithelial cells in the trachea, stomach, and intestine. This channel complex couples with some ion transporters to facilitate ion transport by recycling K+ (Abbott, 2016; Grahammer et al., 2001; Preston et al., 2010; Schroeder et al., 2000).

The mechanisms by which KCNE proteins modify KCNQ1 channel gating behavior have been a central question of this ion channel. Since KCNQ1 is a classic shaker-type tetrameric voltage-gated K+ channel, it has four independent voltage sensor domains (VSDs), one from each α subunit (Long et al., 2005). Each VSD consists of four transmembrane segments, S1–S4. Each S4 segment bears several positively charged amino acid residues. When the membrane potential is depolarized, each S4 segment moves upward (toward the extracellular side). That upward movement eventually triggers pore opening (Jensen et al., 2012; Larsson et al., 1996; Mannuzzu et al., 1996). Therefore, the S4 segment is considered to be an essential part of voltage sensing (Aggarwal and MacKinnon, 1996; Catterall, 1988; Fedida and Hesketh, 2001; Liman et al., 1991; Logothetis et al., 1992; Noda et al., 1984; Papazian et al., 1991). As in the Shaker K+ channel, the S4 segment of the KCNQ1 channel also moves upward with depolarization, as proved by scanning cysteine accessibility mutagenesis (Nakajo and Kubo, 2007; Rocheleau and Kobertz, 2008) and voltage-clamp fluorometry (VCF; Barro-Soria et al., 2014; Nakajo and Kubo, 2014; Osteen et al., 2012; Osteen et al., 2010). In those studies, the presence of KCNE proteins was found to affect the voltage sensor movement. These results suggest that KCNE proteins stabilize a specific state during the voltage sensor transition (Barro-Soria et al., 2017; Barro-Soria et al., 2015; Barro-Soria et al., 2014; Nakajo, 2019). There should be at least three positions in the VSDs of KCNQ1: ‘down,’ ‘intermediate,’ and ‘up’ (Hou et al., 2017; Taylor et al., 2020). KCNE1 stabilizes the intermediate position of VSDs along with a direct interaction of the pore domain and inhibits opening of the pore domain (Taylor et al., 2020). In contrast, KCNE3 may stabilize the intermediate or up position of VSDs and indirectly stabilize the channel’s open state (Barro-Soria et al., 2017; Barro-Soria et al., 2015; Taylor et al., 2020). However, it remains unknown how KCNE proteins stabilize VSDs at a specific position.

Early studies by Melman et al. revealed ‘the triplet’ of amino acid residues in the middle of the transmembrane segment (‘FTL’: F57-T58-L59 in KCNE1 and ‘TVG’: T71-V72-G73 in KCNE3) as structural determinants of KCNE modulation properties (Melman et al., 2002; Melman et al., 2001). Exchanging the triplet (or one of the three amino acid residues) between KCNE1 and KCNE3 could introduce the other’s modulation properties, at least partially. For example, introducing ‘FTL’ into KCNE3 transforms it into a KCNE1-like protein. Therefore, it has been long considered that ‘the triplet’ is a functional interaction site between KCNQ1 and KCNE proteins. Possible interaction sites of the KCNQ1 side have also been explored and are believed to be located between the pore domain and the VSD (Chung et al., 2009; Kang et al., 2008; Nakajo and Kubo, 2007; Van Horn et al., 2011; Xu et al., 2008). By utilizing the KCNQ1 ortholog from ascidian Ciona intestinalis, which lacks KCNE genes, we previously found that F127 and F130 of the S1 segment are required for KCNE3 to make KCNQ1 a constitutively open channel (Nakajo et al., 2011). A recent computational model and the cryo-EM structure of the KCNQ1-KCNE3-calmodulin (CaM) complex clearly showed that KCNE3 interacts with the S1 segment and the pore domain (Kroncke et al., 2016; Sun and MacKinnon, 2020). However, the mechanism by which KCNE3 retains the KCNQ1 VSD at a specific position is still not clearly understood.

In this work, by taking advantage of the cryo-EM structure of the KCNQ1-KCNE3-CaM complex (Sun and MacKinnon, 2020), we created a series of the S1 segment and KCNE3 mutants to change the bulkiness of the S1 segment and KCNE3 interface, and we found that the interaction between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of the S1 segment and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests tight binding of the S1 segment and KCNE3 is crucial for controlling the VSDs.

Results

The side-chain volumes of amino acid residues in the S1 segment of KCNQ1 facing KCNE3 are optimized for channel modulation

The cryo-EM structure of the KCNQ1-KCNE3-CaM complex revealed that KCNE3 interacts with the S1 segment of KCNQ1 in the transmembrane segment (Sun and MacKinnon, 2020). In the structure, the side chains of seven amino acid residues in the S1 segment of KCNQ1 (F123, F127, F130, L134, I138, L142, and I145) face lining those of six amino acid residues of the KCNE3 transmembrane segment (S57, I61, M65, A69, G73, and I76; Figure 1). We previously reported that F127 and F130 are required to make KCNQ1-KCNE3 a constitutively open channel (Nakajo and Kubo, 2011). Therefore, we hypothesized that the interactions between the S1 segment of KCNQ1 and KCNE3 might be crucial for stabilizing the open states. To confirm the functional roles of these amino acid residues, we first created and tested small alanine and large tryptophan-substituted mutants of the S1 segment of KCNQ1. If the alanine- and/or tryptophan-substituted mutants changed the functional output induced by the KCNQ1-KCNE3 interaction (i.e. did not show the constitutive activity), we also created and tested intermediate-sized hydrophobic residues valine-, leucine-, and phenylalanine-substituted mutants. When expressed alone, most of the S1 segment mutants showed conductance-voltage (G-V) curves similar to that of KCNQ1 WT (Figure 2—figure supplements 1–7). We then tested how these mutations introduced to the S1 segment affect the modulation by KCNE3. When co-expressed with KCNE3 WT, KCNQ1 WT shifted the G-V curve in the far-negative direction, becoming a constitutively open channel for the physiological membrane potential range (Figure 2A, B, G and L; black traces and black G-V curves). Because of that, the relative conductance of KCNQ1 WT-KCNE3 WT at –100 mV (G-100mV/Gmax) was 10-times higher (KCNQ1 WT, 0.08±0.01; KCNQ1 WT-KCNE3 WT, 0.80±0.02; n=10 for each; Figure 2 M-S; black bars). In contrast, when co-expressed with KCNE3 WT, the S1 segment mutants yielded various G-V curves and G−100mV/Gmax values. Since one of the unique features of the KCNE3 modulation is constitutive KCNQ1 activity even in the hyperpolarized voltage range, we then mainly evaluated the effect of each mutation on KCNE3 modulation by the G−100mV/Gmax value for comparison. However, other parameters, such as the midpoint of the G–V curve (V1/2) or effective charge (z), were evaluated when possible (Figure 2—source data 1, Figure 3—source data 1, and Figure 4—source data 1). Both of the F123 mutants (F123A and F123W) shifted the G-V curves in the far-negative direction and kept the G−100mV/Gmax values over 0.5 (F123A, 0.61±0.01; F123W, 0.66±0.01; n=10 for each; Figure 2M and Figure 2—figure supplement 1), suggesting that the F123 residue did not have a large impact on the KCNE3 modulation. Among the F127 mutants (F127A, F127V, F127L, and F127W), F127A, F127V, and F127L mutants failed to shift the G-V curves in the far-negative direction and showed smaller G−100mV/Gmax values depending on the side-chain size (F127A, 0.17±0.02; F127V, 0.25±0.02; F127L, 0.38±0.02; n=10 for each; Figure 2C–G and N and Figure 2—figure supplement 2). It seemed that the modulation depended on the side-chain volume: the more different the size was, the more significant the change of functional output induced by the KCNQ1-KCNE3 interaction was (Figure 2N). The F130 mutants (F130A, F130V, F130L, and F130W) failed to shift the G-V curves in the far-negative direction or even positively shifted the G-V curves. They showed substantially reduced G−100mV/Gmax values (F130A, 0.02±0.00; F130V, 0.10±0.01; F130L, 0.01±0.00; F130W, 0.37±0.02; n=10 for each; Figure 2O and Figure 2—figure supplement 3). Among the L134 mutants (L134A, L134V, L134F, and L134W), the L134A, L134V, and L134F mutants failed to shift the G-V curves in the far-negative direction and reduced the G−100mV/Gmax values (L134A, 0.25±0.01; L134V, 0.08±0.01; L134F, 0.35±0.02; n=10 for each; Figure 2P and Figure 2—figure supplement 4). All the I138 mutants (I138A, I138V, I138F, and I138W) showed relatively mild attenuation in the G-V shift and G−100mV/Gmax values (I138A, 0.36±0.02; I138V, 0.66±0.01; I138F, 0.64±0.01; I138W, 0.36±0.02; n=10 for each; Figure 2Q and Figure 2—figure supplement 5). The I138 mutants showed an explicit size dependency in the KCNE3 modulation with wild-type isoleucine having the largest impact on the KCNE3 modulation, as in the case of the F127 mutants (Figure 2N). The L142 mutants (L142A, L142V, L142F, and L142W) failed to shift the G-V curves in the negative direction and mildly reduced the G−100mV/Gmax values (L142A, 0.24±0.03; L142V, 0.42±0.02; L142F, 0.60±0.01; KCNQ1 L142W-KCNE3 WT, 0.23±0.02; n=10 for each; Figure 2R and Figure 2—figure supplement 6). Again, the L142 mutants showed an explicit size dependency in the KCNE3 modulation with wild-type leucine, as in the case of the F127 and I138 mutants (Figure 2N, Q and R). Among the I145 mutants (I145A, I145V, I145F, and I145W), smaller amino acid substitutions (I145A and I145V) showed similar functional outputs to that of WT. In contrast, the I145F and I145W mutants positively shifted the G-V curves and reduced the G−100mV/Gmax values as the side-chain size was increased (I145F, 0.11±0.01; I145W, 0.03±0.00; n=10 for each; Figure 2H–L and S and Figure 2—figure supplement 7).

Figure 1
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Key residues involved in the interaction between KCNQ1 and KCNE3.

(A) Close-up view of the interface between KCNQ1 and KCNE3 in the KCNQ1-KCNE3-CaM complex structure (PDB: 6V00). Three KCNQ1 subunits are colored in blue, green, and gray. A KCNE3 subunit is colored in red. The residues involved in the KCNQ1-KCNE3 interaction are depicted by stick models. The molecular graphics were illustrated with CueMol (http://www.cuemol.org/). (B and C) Sequence alignment around the S1 segment of KCNQ1 (B) and the TM segments of KCNE3 and KCNE1 (C). Amino acid sequences were aligned using Clustal Omega and are shown using ESPript3 (Robert and Gouet, 2014). KCNQ1 residues focused on in this work are highlighted with blue dots. KCNE3 residues focused on in this work and ‘the triplet’ (Melman et al., 2002; Melman et al., 2001) are highlighted with red dots and an orange square, respectively. For sequence alignment, human KCNQ1 (HsKCNQ1, NCBI Accession Number: NP_000209), mouse KCNQ1 (MmKCNQ1, NP_032460), chicken KCNQ1 (GgKCNQ1, XP_421022), Xenopus KCNQ1 (XlKCNQ1, XP_018111887), human KCNE3 (HsKCNE3, NP_005463), mouse KCNE3 (MmKCNE3, NP_001177798), chicken KCNE3 (GgKCNE3, XP_003640673), Xenopus KCNE3 (XlKCNE3 NP_001082346), and human KCNE1 (HsKCNE1, NP_000210) were used. (D) The sizes of amino acid residues focused on in this work. The numbers are from Tsai et al., 1999.

Figure 2 with 7 supplements see all
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Functional effects of KCNQ1 S1 mutants on KCNQ1 modulation by KCNE3.

(A–G) Representative current traces (A–F) and conductance-voltage (G-V) relationships (G) of KCNQ1 WT with or without KCNE3 WT as well as the F127 mutants with KCNE3 WT. (H–L) Representative current traces (H–K) and G-V relationships (L) of the I145 mutants with KCNE3 WT. (M–S) Ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) of KCNQ1 F123 (M), F127 (N), F130 (O), L134 (P), I138 (Q), L142 (R), and I145 (S) mutants with (filled bars) or without (open bars) KCNE3 WT. Error bars indicate ± SEM for n=10 in (G, L, and M–S).

Figure 2—source data 1

Summary of the electrophysiological properties of KCNQ1 WT and mutants with or without KCNE3 WT.

Maximum tail current amplitudes (Imax), parameters deduced from the Boltzmann fitting (V1/2 and z), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) of KCNQ1 WT and mutants with or without KCNE3 WT. n is the number of experiments. n.d., not determined.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig2-data1-v2.xlsx
Figure 2—source data 2

Excel file with numerical electrophysiology data used for Figure 2.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig2-data2-v2.xlsx

In summary, when mutated, six of seven tested amino acid residues (F127, F130, L134, I138, L142, and I145) in the S1 segment changed the functional output induced by the KCNQ1-KCNE3 interaction. Five of them (F127, F130, I138, L142, and I145) showed some side-chain volume dependency for the modulation by KCNE3: KCNQ1 WT showed the highest modulation effect by KCNE3, and the modulation effects by KCNE3 changed gradually if introduced mutations at the S1 segment became more different from WT in terms of amino acid size. Therefore, a series of side-chain volumes facing KCNE3 in the S1 segment is tightly optimized and vital for proper gating modulation of KCNQ1 induced by KCNE3.

The side-chain volumes of amino acid residues of KCNE3 facing the KCNQ1 S1 segment are also optimized for channel modulation

Next, we assessed the functional role of the six amino acid residues of KCNE3 facing the S1 segment (Figure 1). As in the case of evaluating the S1 segment, we created various mutants of these residues and co-expressed them with KCNQ1 WT. Throughout the experiments, 10 ng RNA of KCNQ1 was co-injected with 1 ng RNA of KCNE3 into oocytes since 1 ng RNA of KCNE3 was sufficient to fully modulate KCNQ1 currents (Figure 3—figure supplement 1). In the S57 mutants (S57G, S57A, S57V, S57L, S57F, and S57W), the G−100mV/Gmax values were more gradually reduced when introduced mutations became more different from WT, as seen in some S1 mutants (S57G, 0.32±0.02; S57A, 0.66±0.02; S57V, 0.47±0.01; S57L, 0.20±0.01; S57F, 0.10±0.01; S57W, 0.10±0.01; n=10 for each; Figure 3A–G and N). Among the I61 mutants (I61G, I61A, I61V, I61F, and I61W), the I61V mutant reduced its G−100mV/Gmax value to 0.57±0.02 (n=10), but the value was still relatively high. The other four I61 mutants shifted the G-V curves in the positive direction and greatly reduced their G−100mV/Gmax values (I61G, 0.04±0.01; I61A, 0.03±0.01; I61F, 0.04±0.00; I61W, 0.11±0.01; n=10 for each; Figure 3O and Figure 3—figure supplement 2). The M65 mutants (M65G, M65A, M65V, M65L, M65F, and M65W) showed similar side-chain volume dependency as seen in the S57 mutants and some S1 mutants. They showed less shifted G-V curves and reduced the G−100mV/Gmax values depending on how different the side-chain size was from that of WT, except M65V, which showed a significant change of functional output despite having a size similar to that of WT (M65G, 0.22±0.01; M65A, 0.48±0.01; M65V, 0.16±0.02; M65L, 0.63±0.03; M65F, 0.36±0.02; M65W, 0.07±0.01; n=10 for each; Figure 3P and Figure 3—figure supplement 3). Among the A69 mutants (A69G, A69V, A69L, A69F, and A69W), only the smaller A69G mutant showed a similar functional output to that of WT at this position. A69G shifted the G-V curve in the far-negative direction and kept the G−100mV/Gmax value over 0.5 (0.57±0.03, n=10), while the other four A69 mutants shifted the G-V curve in the positive direction and showed substantially smaller G−100mV/Gmax values (A69V, 0.19±0.02; A69L, 0.17±0.01; A69F, 0.11±0.01; A69W, 0.04±0.01, n=10 for each; Figure 3Q and Figure 3—figure supplement 4). Only the small amino acid alanine showed a similar functional output to that of WT again in the G73 mutants (G73A, G73V, G73L, G73F, and G73W; Figure 3H–M and R). The G73A mutant shifted the G-V curve in the far-negative direction and kept the G−100mV/Gmax value over 0.5 (0.77±0.01, n=10). The other four G73 mutants mildly shifted the G-V curve in the negative direction and reduced the G−100mV/Gmax values to variable extents (G73V, 0.22±0.02; G73L, 0.35±0.02; G73F, 0.43±0.02; G73W, 0.24±0.01; n=10 for each; Figure 3H–M and R). All of the I76 mutants (I76G, I76A, I76V, I76F, and I76W) showed reduced G−100mV/Gmax values, depending on the side-chain volume (I76G, 0.06±0.01, n=10; I76A, 0.14±0.02, n=10; I76V, 0.43±0.02, n=10; I76F, 0.11±0.01, n=10; I76W, 0.16±0.01; n=10 for each; Figure 3S and Figure 3—figure supplement 5).

Figure 3 with 5 supplements see all
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Functional effects of KCNE3 mutants on KCNQ1 modulation by KCNE3.

(A–G) Representative current traces (A–F) and G-V relationships (G) of KCNQ1 WT with the KCNE3 S57 mutants. (H–M) Representative current traces (H–L) and G-V relationships (M) of KCNQ1 WT with the KCNE3 G73 mutants. (N–S) Ratios of conductance at –100 mV (G−100mV) and maximum conductance (Gmax) of KCNQ1 WT with KCNE3 S57 (N), I61 (O), M65 (P), A69 (Q), G73 (R), and I76 (S) mutants. Error bars indicate ± SEM for n=10 in (G, M,N–S).

Figure 3—source data 1

Summary of the electrophysiological properties of KCNQ1 WT with KCNE3 mutants.

Maximum tail current amplitudes (Imax), parameters deduced from the Boltzmann fitting (V1/2 and z), and ratios of conductance at –100 mV (G−100mV) and maximum conductance (Gmax) of KCNQ1 WT with KCNE3 mutants. n is the number of experiments. n.d., not determined.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig3-data1-v2.xlsx
Figure 3—source data 2

Excel file with numerical electrophysiology data used for Figure 3.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig3-data2-v2.xlsx

In summary, when mutated, all of the tested amino acid residues in KCNE3 showed some changes in functional output induced by the KCNQ1-KCNE3 interaction. Furthermore, KCNE3 WT showed the highest ability to modulate the KCNQ1 gating among all of the tested amino acid residues. In most cases, the abilities to modulate the KCNQ1 gating changed more significantly if the size of introduced mutations differed more from WT. As in the case of the S1 segment (Figure 2 and Figure 2—figure supplements 1–7), these results demonstrate that a series of side-chain volumes facing the S1 segment in KCNE3 is tightly optimized for proper gating modulation of KCNQ1 induced by KCNE3.

Functional restoration of the S1 mutants by the KCNE3 mutants

We then examined whether the KCNE3 mutations could restore the functional output of the KCNQ1-KCNE3 interaction distorted by the S1 segment mutations. As guided by the KCNQ1-KCNE3-CaM complex structure (Sun and MacKinnon, 2020), we tested the idea with five candidate pairs positioned to the same layer in the structure (F127[Q1]-G73[E3], F130[Q1]-A69[E3], I138[Q1]-M65[E3], L142[Q1]-I61[E3], and I145[Q1]-S57[E3]; Figure 1). In the F127(Q1)-G73(E3) pairs, we chose the KCNQ1 F127A mutant since it showed the largest change of functional output among the F127 mutants. Among the KCNE3 G73 mutants, the F127A-G73L pair showed the largest restoration of the G−100mV/Gmax value (0.67±0.01, n=10) as compared to that of the F127A-KCNE3 WT pair (0.17±0.02, n=10; Figure 4A–G). Other G73 mutants, G73V (0.36±0.02, n=10), G73F (0.45±0.02, n=10), and G73W (0.38±0.01, n=10), also mildly but significantly restored the modulation (Figure 4A–G). In the F130(Q1)-A69(E3) pairs, we examined whether KCNQ1 F130A and F130V mutants were restored by a series of the KCNE3 A69 mutants since they showed large changes in functional output among the KCNQ1 F130 mutants. Although KCNE3 A69G, A69F, and A69W mutants slightly increased the G−100mV/Gmax value of the KCNQ1 F130A mutant (A69G, 0.10±0.01; A69F, 0.05±0.00; A69W, 0.10±0.01; KCNE3 WT, 0.02±0.00; n=10 for each), most of the KCNE3 A69 mutants failed to restore the KCNQ1 F130A and F130V mutants (Figure 4—figure supplement 1). In the I138(Q1)-M65(E3) pairs, we examined whether KCNQ1 I138A and I138W mutants were restored by a series of the KCNE3 M65 mutants since they showed large changes in functional output among the KCNQ1 I138 mutants. In the KCNQ1 I138A-KCNE3 M65 pairs, the KCNQ1 I138A-KCNE3 M65L and KCNQ1 I138A-KCNE3 M65F pairs showed some restoration of the G−100mV/Gmax values (KCNQ1 I138A-KCNE3 M65L, 0.52±0.02, n=10; KCNQ1 I138A-KCNE3 M65F, 0.53±0.01, n=10) as compared to that of the KCNQ1 I138A-KCNE3 WT pair (0.36±0.02, n=10; Figure 4—figure supplement 2A–H). The KCNQ1 I138W-KCNE3 M65L pair also showed mild restoration of the G−100mV/Gmax value (0.46±0.02, n=10; Figure 4—figure supplement 2I–P), although it is not clear why both I138A and I138W were restored by the same M65L mutant. In the L142(Q1)-I61(E3) pairs, we examined whether KCNQ1 L142A and L142W mutants were restored by a series of the KCNE3 I61 mutants. However, none of the pairs were restored (Figure 4—figure supplement 3). In the I145(Q1)-S57(E3) pairs, we examined whether KCNQ1 I145F and I145W mutants were restored by a series of the KCNE3 S57 mutants. Although the KCNQ1 I145W mutant was slightly restored by the KCNE3 S57 mutants, no KCNE3 S57 mutants showed a G−100mV/Gmax value higher than 0.18 (Figure 4—figure supplement 4). In contrast, KCNQ1 I145F was successfully restored by KCNE3 S57G and S57A mutants with smaller residues than WT (G−100mV/Gmax values of I145F-S57G, 0.43±0.01; I145F-S57A, 0.61±0.02; n=10 for each) as compared to that of the I145F-KCNE3 WT pair (0.11±0.01, n=10; Figure 4H–O).

Figure 4 with 4 supplements see all
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Functional restoration of KCNQ1 mutants by KCNE3 mutants.

(A–G) Representative current traces (A–E), conductance-voltage (G-V) relationships (F), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (G) of the KCNQ1 F127A mutant with the KCNE3 G73 mutants. (H–O) Representative current traces (H–M), G-V relationships (N), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (O) of the KCNQ1 I145F mutant with the KCNE3 S57 mutants. Error bars indicate ± SEM for n=10 in (F, G, N, and O).

Figure 4—source data 1

Summary of the electrophysiological properties of KCNQ1 mutants with KCNE3 mutants.

Maximum tail current amplitudes (Imax), parameters deduced from the Boltzmann fitting (V1/2 and z), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) of KCNQ1 mutants with KCNE3 mutants. n is the number of experiments. n.d., not determined.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig4-data1-v2.xlsx
Figure 4—source data 2

Excel file with numerical electrophysiology data used for Figure 4.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig4-data2-v2.xlsx

These results suggest that at least two of the five pairs of residues tested (F127[Q1]-G73[E3] and I145[Q1]- S57[E3]) closely interact with each other. This is the strong evidence that the specific interaction between the S1 segment and KCNE3 is important.

The KCNE3 mutants restored voltage sensor movement of the S1 mutants

Previous electrophysiological studies demonstrated that KCNE3 influences the voltage sensor movement, especially the S4 segment movement (Barro-Soria et al., 2017; Barro-Soria et al., 2015; Nakajo and Kubo, 2007; Rocheleau and Kobertz, 2008). We next performed VCF to monitor the S4 segment to investigate whether the mutations of the S1 segment affect voltage sensor movement and whether it is restored by the identified pairs of mutants (F127A[Q1]-G73L[E3] and I145F[Q1]-S57A[E3]). The KCNQ1 construct for VCF (KCNQ1 C214A/G219C; this construct hereafter being referred to as ‘KCNQ1vcf WT’) was labeled at the introduced cysteine residue (G219C) by Alexa Fluor 488 maleimide (Osteen et al., 2012; Osteen et al., 2010). Currents and fluorescence changes were recorded in response to voltage steps (from 60 to –160 mV) from a holding potential of –100 mV (Figure 5 inset). KCNQ1vcf WT alone showed a fluorescence-voltage (F-V) relationship that mostly fitted to a single Boltzmann function and closely overlapped with its G-V curve (Figure 5—figure supplement 1A, D and E). In contrast, KCNQ1vcf WT co-expressed with KCNE3 WT showed a split F-V relationship. Its fluorescence changes were observed in the far negative and positive voltages, while they were very small and remained almost unchanged within the voltage range between 0 and –100 mV (Figure 5A, F and G). Consequently, the F-V relationship of KCNQ1vcf WT-KCNE3 WT does not fit to a single Boltzmann function but fits to a double Boltzmann function (Figure 5A, F and G), which is consistent with the results of a previous VCF study (Taylor et al., 2020). These results suggest that most of the S4 segments of the channels are at the down position and move to the upper position with depolarization in KCNQ1vcf WT alone and that a substantial number of S4 segments are in the intermediate position and move either with depolarization or deep hyperpolarization in KCNQ1vcf WT-KCNE3 WT. We then assessed the F-V relationships of the KCNQ1vcf F127A mutant co-expressed with the KCNE3 WT or G73L mutant as well as those of the KCNQ1vcf I145F mutant co-expressed with the KCNE3 WT or S57A mutant. The KCNQ1vcf F127A and F145F mutants alone showed G-V and F-V relationships similar to those of KCNQ1vcf WT alone (Figure 5—figure supplement 1B–E). The KCNQ1vcf F127A mutant co-expressed with KCNE3 WT showed an F-V relationship that still fitted to a double Boltzmann function but lost a plateau phase observed in KCNQ1vcf WT-KCNE3 WT (Figure 5B and G), resulting in a shift of the half-activation voltage in the first fluorescence component (V1/2[F1]), which seems to be correlated to pore opening/closure, toward the positive direction (–77.1±3.0 mV, n=5) as compared to that of KCNQ1vcf WT-KCNE3 WT (–141.7±10.8 mV, n=5). In contrast, the KCNQ1vcf F127A mutant co-expressed with the KCNE3 G73L mutant showed an F-V relationship that better fitted to a double Boltzmann function and shifted its V1/2(F1) toward a more negative direction (< –160 mV, n=5) than the KCNQ1vcf F127A mutant co-expressed with KCNE3 WT (Figure 5C and G). These results suggest that the number of S4 segments in the intermediate position were increased in the KCNQ1vcf F127A mutant co-expressed with the KCNE3 G73L mutant.

Figure 5 with 1 supplement see all
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Conductance-voltage (G-V) and fluorescence-voltage (F-V) relationships for KCNQ1 mutants with KCNE3 mutants.

(A–E) Ionic currents (upper row) and fluorescence traces (lower row) of KCNQ1vcf WT-KCNE3 WT (A), KCNQ1vcf F127A-KCNE3 WT (B), KCNQ1vcf F127A-KCNE3 G73L (C), KCNQ1vcf I145F-KCNE3 WT (D), and KCNQ1vcf I145F-KCNE3 S57A (E). (F–I) G-V (F and H) and F-V (G and I) relationships of KCNQ1vcf WT-KCNE3 WT, KCNQ1vcf F127A-KCNE3 WT, KCNQ1vcf F127A-KCNE3 G73L, KCNQ1vcf I145F-KCNE3 WT, and KCNQ1vcf I145F-KCNE3 S57A. Error bars indicate ± SEM for n=5 in (F–I).

Figure 5—source data 1

Summary of the electrophysiological properties of KCNQ1vcf WT and mutants with or without KCNE3 mutants.

Maximum tail current amplitudes (Imax) and parameters deduced from the Boltzmann fitting (V1/2 and z) of KCNQ1vcf WT and mutants with or without KCNE3 mutants. n is the number of experiments. n.d., not determined.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig5-data1-v2.xlsx
Figure 5—source data 2

Summary of the properties of KCNQ1vcf WT and mutants with or without KCNE3 mutants acquired from voltage-clamp fluorometry (VCF) recordings.

Parameters deduced from the double Boltzmann fitting (V1/2[F1] and V1/2[F2]) of KCNQ1vcf WT and mutants with or without KCNE3 mutants. n is the number of experiments. >60 means over 60 mV. < –160 means under –160 mV.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig5-data2-v2.xlsx
Figure 5—source data 3

Excel file with numerical electrophysiology data used for Figure 5.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig5-data3-v2.xlsx

A similar tendency was observed in the KCNQ1vcf I145F mutant co-expressed with the KCNE3 WT or S57A mutant (Figure 5D, E, I). The KCNQ1vcf I145F mutant co-expressed with KCNE3 WT showed an F-V relationship that still fitted to a double Boltzmann function but lost a plateau phase and shifted its V1/2(F1) toward the positive direction (–121.2±11.6 mV, n=5). In contrast, the KCNQ1vcf I145F mutant co-expressed with the KCNE3 S57A mutant showed an F-V relationship that better fitted to a double Boltzmann function and shifted its V1/2(F1) toward a more negative direction (< –160 mV, n=5). These results suggest that the number of S4 segments in the intermediate position were increased in the KCNQ1vcf I145F mutant co-expressed with the KCNE3 S57A mutant. This is the strong evidence that it is an interaction between the S1 segment and KCNE3 that is important for modulating for VSD movement.

The side-chain volumes of amino acid residues in the S1 segment of KCNQ1 are important for channel modulation by KCNE1

We finally examined whether the side-chain volume of amino acid residues in the S1 segment also influences the KCNQ1 modulation by KCNE1. We co-expressed a series of the KCNQ1 F127 mutants (F127A, F127V, F127L, and F127W) with KCNE1 WT. All the F127 mutants shifted the G-V curve in the positive direction by co-expression of KCNE1. Interestingly, the induced shift depended on the side-chain volume again: the more different side-chain sizes from WT (F127) were, the larger V1/2 shifted (Figure 6 and Figure 6—source data 1; Figure 6—source data 2). The G-V curve could not fit to a Boltzmann equation properly in the case of F127A mutant with KCNE1, although it was apparent that V1/2 is larger than +60 mV (Figure 6K inset). While KCNE1 WT and KCNE3 WT shift the G-V curve of KCNQ1 WT in opposing directions, both modulation effects seemed similarly dependent on the tight interaction of the S1 segment. However, since we examined only the F127 mutants in this study, further experiments will be needed to reveal the possible roles of the S1 segment in the modulation by KCNE1.

Figure 6
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Functional effects of KCNQ1 F127 mutants on KCNQ1 modulation by KCNE1.

(A–N) Representative current traces (A–J) and conductance-voltage (G-V) relationships (K–N) of KCNQ1 WT and F127 mutants with or without KCNE1 WT. In panel (A), the current-voltage (I-V) relationship of KCNQ1 F127A with KCNE1 normalized by tail current amplitudes at 60 mV (I60mV) is shown as an inset since its G-V curve shifted in the far-positive direction and could not fit to a single Boltzmann equation properly. (O) The half-activation voltage of KCNQ1 WT and F127 mutants with (filled bars) or without (open bars) KCNE1 WT. ‘>60’ means over 60 mV, as the G-V curve could not properly fit to a single Boltzmann equation. Error bars indicate ± SEM for n=5 in (I–M).

Figure 6—source data 1

Summary of the electrophysiological properties of KCNQ1 F127 mutants with KCNE1 WT.

Maximum tail current amplitudes (Imax), parameters deduced from the Boltzmann fitting (V1/2 and z), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) of KCNQ1 F127 mutants with KCNE1 WT. n is the number of experiments. n.d., not determined.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig6-data1-v2.xlsx
Figure 6—source data 2

Excel file with numerical electrophysiology data used for Figure 6.

https://cdn.elifesciences.org/articles/81683/elife-81683-fig6-data2-v2.xlsx

Discussion

In this work, we conducted site-directed mutational analyses using two-electrode voltage clamp (TEVC) and VCF by changing the side-chain bulkiness of these interacting amino acid residues (volume scanning), inspired by the recently determined cryo-EM structures of the KCNQ1-KCNE3-CaM complex (Sun and MacKinnon, 2020). We found that the hydrophobic interface between the S1 segment and KCNE3 is a key component for the channel modulation by KCNE3, which prevents the S4 segment of the VSD from going to the down position at resting membrane potential. Previous studies demonstrated that ‘the triplet’ of amino acid residues in the middle of the transmembrane segment (‘FTL’ for KCNE1 and ‘TVG’ for KCNE3) is a structural determinant of KCNE modulation properties (Barro-Soria et al., 2017; Melman et al., 2002; Melman et al., 2001). However, why the triplets are important for conferring specific gating properties onto KCNQ1 is still not well understood even though the KCNQ1-KCNE3-CaM complex structure revealed that the triplets are located deep inside the membrane and interact with the S1 and S4 segments of KCNQ1 (Sun and MacKinnon, 2020; Figure 1A). Besides ‘the triplet’, our current work showed that a broader range of amino acid residues (S57, I61, M65, A69, G73, and I76), which forms five helical turns in total in the middle of the transmembrane segment of KCNE3, was involved in the interactions between KCNE3 and the S1 segment and was required for maintenance of constitutive activity of the KCNQ1-KCNE3 channel (Figure 3 and Figure 3—figure supplements 1–5).

Another previous study by Barro-Soria et al. suggested that negatively charged KCNE3 residues (D54 and D55) electrostatically interact with the S4 segment of KCNQ1 to induce constitutive activity of the KCNQ1-KCNE3 channel (Barro-Soria et al., 2015). In this work, we did not investigate these negatively charged KCNE3 residues since they are far away from the S1 and S4 segments and do not directly interact with them in the KCNQ1-KCNE3-CaM complex structure (Sun and MacKinnon, 2020). Further mutational analyses can provide insights into the mechanism of how these residues are involved in gating modulation of KCNQ1 induced by KCNE3.

For the KCNQ1 side, we previously demonstrated that two phenylalanine residues, F127 and F130, in the S1 segment are important for the channel modulation by KCNE3, but how these mutations affect the channel modulation has been unknown (Nakajo et al., 2011). The cryo-EM KCNQ1-KCNE3-CaM structure (PDB: 6V00) clearly shows that F127 and F130 of the S1 segment face KCNE3 (G73 and I76). We therefore hypothesized that the interaction of these amino acid residues is essential for the KCNE3 function and found that besides F127 and F130, a broader range of amino acid residues (F127, F130, L134, I138, L142, and I145), which forms five helical turns in total in the middle of the transmembrane segment of the S1 segment, was important for the channel modulation by KCNE3. Furthermore, in most cases, the introduction of larger and smaller amino acid residues at the hydrophobic interface between the S1 segment and KCNE3 disrupted the KCNE3 function to modulate KCNQ1 gating (Figure 2 and Figure 2—figure supplements 1–7). This suggests that the side-chain volume of the amino acid residues in the S1 segment and KCNE3 is optimized for proper gating modulation of KCNQ1 induced by KCNE3 (Figure 2, Figure 2—figure supplements 1–7, Figure 3, and Figure 3—figure supplements 2–5). The restorations of the KCNQ1 F127A mutant by the KCNE3 G73L mutant and the KCNQ1 I145F mutant by the KCNE3 S57A mutant further support this idea (Figure 4). In contrast, three of the five KCNQ1 residues tested (F130, I138, and L142) were not successfully restored by KCNE3 mutants (Figure 4—figure supplements 1–4). They could have even more prominent roles in the KCNE3 modulation than just the tight hydrophobic interaction tested in this study. Further analyses are needed to understand the functions of these residues in gating modulation by KCNE3.

According to previous VCF studies (Barro-Soria et al., 2017; Barro-Soria et al., 2015; Taylor et al., 2020), we further performed VCF analysis to investigate how the S1 segment and KCNE3 interaction influences the voltage sensor movement. Previous VCF analysis (Taylor et al., 2020) revealed that KCNQ1vcf WT co-expressed with KCNE3 WT showed two components (F1 and F2) in the F-V relationship (Figure 5A, F and G). In addition, KCNQ1vcf F127A-KCNE3 WT and KCNQ1vcf I145F-KCNE3 WT pairs showed positively shifted V1/2(F1) and diminished the plateau phase observed in the KCNQ1vcf WT-KCNE3 WT pair, while KCNQ1vcf F127A-KCNE3 G73L and KCNQ1vcf I145F-KCNE3 S57A pairs partially restored V1/2(F1) and the plateau phase (Figure 5). These results suggest that the tight interaction between the S1 segment and KCNE3 is required to keep the S4 segment in the intermediate position at resting membrane potential. Disrupting the interaction between the S1 segment and KCNE3 by mutation allows the S4 segment to move down by hyperpolarization.

How the tight interaction between the S1 segment and KCNE3 identified in the current study that affects the S4 segment is still up in the air. In the KCNQ1-KCNE3-CaM complex structure, the S4 segment directly interacts with KCNE3 and the S1 segment through its lower part (Sun and MacKinnon, 2020). Therefore, it is reasonable to speculate that the tight interaction between the S1 segment and KCNE3 directly affects the S4 movement upon membrane potential change. However, further mutational analyses are needed to understand how the tight interaction between the S1 segment and KCNE3 changes the S4 movement.

It is interesting whether the tight interaction we saw in the KCNQ1-KCNE3 channel also plays a role in the KCNQ1-KCNE1 channel. We examined a series of KCNQ1 F127 mutants co-expressed with KCNE1 and found that the mutant effects also showed size dependency, as in the case of KCNE3 (Figure 6). It is noteworthy, though, that the F127 mutants strengthened the positive shift of the G-V curve in the KCNQ1-KCNE1 channel, while the same mutants reduced the constitutive activity in the KCNQ1-KCNE3 channel. As previously revealed by some VCF experiments, KCNE1 and KCNE3 stabilize the intermediate state of the VSD (Barro-Soria et al., 2015; Barro-Soria et al., 2014; Osteen et al., 2010; Taylor et al., 2020). One possible interpretation of the KCNE1 results could be that the mutations of F127 might destabilize the intermediate state (or stabilize the closed/down state), as seen in KCNE3 (Figure 5). Therefore, the S1 segment might have a similar role in both KCNE1 and KCNE3 to assist in stabilizing the intermediate state. However, further experiments will be needed to find out the possible role of the S1 segment in the KCNE1 modulation.

In conclusion, our results demonstrate that tight interaction between the S1 segment of the KCNQ1 channel and KCNE3 is required for retaining the VSD in the intermediate position, probably by preventing the S4 segment from going to the down position, thereby keeping the KCNQ1-KCNE3 channels constitutively active.

Materials and methods

Expression in Xenopus laevis oocytes

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The human KCNQ1 (NCBI Accession Number NP_000209.2; WT and mutants), human KCNE1 (HsKCNE1, NP_000210), and mouse Kcne3 (NP_001177798; WT and mutants) cDNAs were inserted into the pGEMHE expression vector (Liman et al., 1992). The cRNAs were transcribed using mMESSAGE mMACHINE T7 Transcription Kits (Thermo Fisher Scientific, AM1344). Oocytes were surgically removed from female X. laevis frogs anesthetized in water containing 0.1% tricaine (Sigma-Aldrich, E10521) for 15–30 min. The oocytes were treated with collagenase (Sigma-Aldrich, C0130) for 6–7 hr at room temperature to remove the follicular cell layer. Defolliculated oocytes of similar sizes at stage V or VI were selected, microinjected with 50 nl of cRNA solution (10 ng for KCNQ1 and 1 ng for KCNE3) using a NANOJECT II (Drummond Scientific Co.), and incubated until use at 18°C in Barth’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.3 mM Ca[NO3]2, 0.41 mM CaCl2, and 0.82 mM MgSO4, pH 7.6) supplemented with 0.1% penicillin-streptomycin solution (Sigma-Aldrich, P4333). All experiments were approved by the Animal Care Committee of Jichi Medical University (Japan) under protocol no. 18027–03 and were performed according to guidelines.

Two-electrode voltage clamp

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cRNA-injected oocytes were incubated for 1–3 days. Ionic currents were recorded with a two-electrode voltage clamp using an OC-725C amplifier (Warner Instruments) at room temperature. The bath chamber was perfused with Ca2+-free ND96 solution (96 mM NaCl, 2 mM KCl, 2.8 mM MgCl2, and 5 mM HEPES, pH 7.6) supplemented with 100 µM LaCl3 to block endogenous hyperpolarization-activated currents (Osteen et al., 2010). The microelectrodes were drawn from borosilicate glass capillaries (Harvard Apparatus, GC150TF-10) using a P-1000 micropipette puller (Sutter Instrument) to a resistance of 0.2–1.0 MΩ and filled with 3 M KCl. Currents were elicited from the holding potential of –90 mV to steps ranging from –100 to +60 mV in +20 mV steps each for 2 s with 7.5 s intervals for the KCNQ1-KCNE3 complex analyses and for 5 s with 15 s intervals for the KCNQ1-KCNE1 complex analyses. Oocytes with a holding current larger than –0.2 µA at –90 mV were excluded from the analysis. Generation of voltage-clamp protocols and data acquisition were performed using a Digidata 1550 interface (Molecular Devices) controlled by pCLAMP 10.7 software (Molecular Devices). Data were sampled at 10 kHz and filtered at 1 kHz.

Voltage dependence analysis

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G-V relationships were taken from tail current amplitude at –30 mV fitted using pCLAMP 10.7 software (Molecular Devices) to a single Boltzmann equation:

G=Gmin+(Gmax−Gmin)/(1+e−zF[V−V1/2]RT),

where Gmax and Gmin are the maximum and minimum tail current amplitudes, respectively, z is the effective charge, V1/2 is the half-activation voltage, T is the temperature in degrees Kelvin, F is Faraday’s constant, and R is the gas constant. G/Gmax, which is the normalized tail current amplitude, was plotted against membrane potential for presentation of the G-V relationships.

Voltage-clamp fluorometry

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Sample preparation, data acquisition, and data analysis were performed similarly as described previously (Nakajo and Kubo, 2014). cRNA-injected oocytes were incubated for 4–5 days, labeled for 30 min with 5 µM Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific, A10254) in high potassium KD98 solution (98 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.6; Nakajo and Kubo, 2014; Osteen et al., 2010), and washed with Ca2+-free ND96 solution to remove unreacted Alexa probes. The bath chamber was filled with Ca2+-free ND96 solution supplemented with 100 µM LaCl3. The microelectrodes were drawn from borosilicate glass capillaries (Harvard Apparatus, GC150TF-15). Currents were elicited from the holding potential of –100 mV to steps ranging from +60 to –160 mV in –20 mV steps each for 2 s with 10 s intervals. Oocytes with a holding current larger than –0.3 µA at –100 mV were excluded from the analysis. Generation of voltage-clamp protocols and data acquisition was performed using a Digidata 1440 A interface (Molecular Devices) controlled by pCLAMP 10.7 software (Molecular Devices). Data were sampled at 10 kHz and filtered at 1 kHz. Fluorescence recordings were performed with a macro zoom microscope MVX10 (Olympus) equipped with a 2× objective lens (MVPLAPO 2XC, NA = 0.5, Olympus), 2× magnification changer (MVX-CA2X, Olympus), GFP filter cube (U-MGFPHQ/XL, Olympus), and an XLED1 LED light source with a BDX (450–495 nm) LED module (Excelitas Technologies). Fluorescence signals were obtained by using a photomultiplier (H10722-110; Hamamatsu Photonics) and digitized at 1 kHz through Digidata1440, filtered at 50 Hz, and recorded using pClamp10 simultaneously with ionic currents. The shutter for the excitation was open during the recording, which induced a continuous decrease of fluorescence due to photobleaching. Therefore, we calculated the bleaching rate for each experiment using the baseline levels of the initial 1100 ms before test pulses of each trace and compensated the fluorescence traces by subtracting the bleached component calculated from each trace’s bleaching rate (R), assuming that the fluorescence was linearly decreased. Arithmetic operations were performed with Clampfit software from pClamp10.

(Compensated trace) = (recorded trace) × (1 – [R × (time)]) (Nakajo and Kubo, 2014), where (time) is the time value of the point given by Clampfit. We then normalized the fluorescence traces by setting each baseline level to 1.

VCF analysis

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F-V relationships were taken from the fluorescence change from the baseline (ΔF) plotted against membrane potential. ΔF values were then normalized by ΔF60mV for the normalized F-V relationships shown in Figure 5 and Figure 5—figure supplement 1. For KCNQ1 alone, F-V relationships were fitted using Igor Pro software (WaveMatrices Co.) to a single Boltzmann equation:

F=Fmin+(Fmax−Fmin)/(1+e−zF[V−V1/2]RT),

where Fmin and Fmax are the maximum and baseline fluorescence components, z is the effective charge for the fluorescence component, V1/2 is the half-activation voltage for the fluorescence component, T is the temperature in degrees Kelvin, F is Faraday’s constant, and R is the gas constant. For KCNQ1 co-expressed with KCNE3, F-V relationships were fitted using Igor Pro software (WaveMatrices Co.) to a double Boltzmann equation:

F=Fmin+(F1−Fmin)/(1+e−z1F[V−V1/2(F1)]RT)+(F2−F1)/(1+e−z2F[V−V1/2(F2)]RT)

where F1, F2, and Fmin are the first, second, and baseline fluorescence components, z1 and z2 are the effective charges for each fluorescence component, V1/2(F1) and V1/2(F2) are the half-activation voltage for each fluorescence component, T is the temperature in degrees Kelvin, F is Faraday’s constant, and R is the gas constant.

Statistical analysis

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The data were expressed as means ± SEM. Statistical analysis was performed with Student’s t-test and one-way ANOVA with Dunnett’s test for single and multiple comparisons, respectively, with EZR software (Kanda, 2013), and significance was assigned at p<0.05 (*p<0.05, **p<0.01, and ***p<0.001).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

References

Decision letter

  1. Jon T Sack
    Reviewing Editor; University of California Davis School of Medicine, United States
  2. Richard W Aldrich
    Senior Editor; The University of Texas at Austin, United States
  3. Jon T Sack
    Reviewer; University of California Davis School of Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Triad interaction stabilizes the voltage sensor domains in a constitutively open KCNQ1-KCNE3 channel" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Jon T Sack as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor.

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further for publication by eLife.

The functional validation of the KCNQ1/KCNE3 structural interface is compelling, yet was not deemed to comprise a conceptual advance sufficient to merit publication in eLife. The reviewers agreed that validation of the interface by KCNQ1 F127A-KCNE3 G73L is an important result. However, this major conclusion could benefit from further experiments. Reviewers all thought that the data analysis needs substantial revision, and other claims should be tempered. If further experiments lead to additional mechanistic insights, the combined results could be submitted as a new manuscript.

Reviewer #1 (Recommendations for the authors):

This study aimed to identify amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity, and identify residues on the S4 segment which convert KCNE effects into voltage sensor movement. The research appears technically sound and expertly performed. The major strength of this work is the molecular redesign of KCNE3 and the S1 segment of KCNQ1 to rescue the functional interaction between KCNE3 and KCNQ1. However, this key result rests on a single pair of mutations. The weaknesses are some of the mechanistic interpretations pertaining to functional coupling between KCNQ1 and KCNE3. The manuscript achieved its aim of identifying amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity. This work also identifies mutations on the S4 segment which diminish KCNE1 effects, but data seems inadequate to support the claim that the residues identified on the S4 are the site of conversion of KCNE effects into voltage sensor movement, as alternate explanations also appear plausible. Importantly, this work presents strong evidence that the KCNE3/S1 interface observed structurally is accurate in its molecular detail, and crucial for inter-protein coupling. This study should cement the functional relevance of the KCNE3 interface with the S1 of KCNQ1 observed in a recent structure. Additionally. this work identifies key sites that perturb the modulation of KCNQ1 by KCNE proteins.

The KCNQ1 F127A-KCNE3 G73L result is compelling, it is strong conceptual evidence that the KCNE3/S1 interface forms as advertised. I think the paper would be better appreciated if it focused almost entirely on this result, and perhaps further investigate this key finding with a few more related experiments. The manuscript states that " Appropriate side-chain volumes at the interaction interface are necessary" yet could other physical/chemical concepts explain the reason for rescue?

Testing further sets of double mutations at positions 127 and 73 could test whether this apparent rescue by the swap of side chain bulk is more than just a happy coincidence, and whether bulk inversions at positions 127 and 73 could be found that produce channels even more functionally similar to WT KCNE/KCNQ

I wonder if this KCNQ1 F127A-KCNE3 G73L protein engineering could be used to knock in KCNE-resistant channels that will only respond when a mutant KCNE is expressed…possibly useful for experiments conceptually similar to DREADDs or Kevan Shokat's knob-in-hole chemical genetics.

line 134

"For the KCNQ1 F127A mutant, co-expression with KCNE3 F68A or V72A yielded G-V curves similar to that for KCNE3 WT, implying that F127 of the S1 segment and F68/V72 of KCNE3 are functionally coupled."

I disagree. these results just mean that all of the mutations eliminate the E3 effect. The functional coupling analysis here and in the following sentences (up to line 144) seems flawed, or maybe I am failing to understand it. What basis is there for "additive" shifting?

line 182

"Therefore, we concluded that M238 and V241 are the sites where the binding of KCNE3 is converted to the VSD movement's modulation."

I disagree, and suggest softening this conclusion. In combination with the structure, the possibility of this being a site of conversion does seems plausible, but these results do not make this certain. This result merely means that perturbation of these sites perturbs the coupling. This is consistent with being a site of physical coupling, or conversion, but based on this result alone, other physical possibilities also seem plausible. For example, this could be a site needed to align the site of conversion or There could be multiple sites of conversion.

Figure Supplement 1D

The confocal images do clearly show surface expression, but provide little information about the density of surface KCNE1, nor association with KCNQ1. Quantitation across multiple oocytes could strengthen this control.

Figure 3F, purple traces appear to be mislabeled…should be F130A +A69F?

table 1:

reporting the midpoints of the E1 mutations on their own (no KCNE), even if from another publication, would be helpful

G is conductance, but in table 1 current amplitudes are given (minor semantic issue)

Exclusion criteria for oocyte leak should be in the methods section

Reviewer #2 (Recommendations for the authors):

In this manuscript the authors inspected the cryo-EM structure of KCNQ1 and KCNE1 and made mutations to KCNQ1 and KCNE3 residues that appear to interact in the structure. These mutated residues are located in the transmembrane segment S1 and S4 of KCNQ1 and of KCNE3. The authors found that the KCNE4 mutations F68A, V72A and I 76 A abolished the ability of KCNE3 to shift GV relation to voltages more negative than -160 mV, which is similar to the findings of their previous study that the S1 mutations F127A and F130A also abolished the ability of KCNE3 to shift GV to negative voltages. A double mutation Q1 F127A + E3 G73L rescued a large part of the GV shift, supporting the observation from the structure that KCNQ1 S1 and KCNE3 may interact among these residues for the modulatory effects of KCNE3 on channel gating. The authors also measured VSD movements in the WT and these mutant channels using VCF, and concluded that the S1-E3 interactions at the S1 site are important for the KCNE3 induced shift of VSD activation to the negative voltages. The authors then found that mutations of S4, M238A and M241A, also reduced the ability of KCNE3 to shift the GV relation, and reduce VSD activation at negative voltages. Based on these results, the authors conclude that the interactions among S1, S4 and KCNE3 at the location of these residues modulate VSD activation and stabilize the open state of the channel. The manuscript presents some interesting data, including the Q1 F127A + E3 G73L results as evidence for S1-KCNE3 interaction. However, some of the conclusions made in the manuscript seem to be based on incomplete experiments or analyses of the data.

Some questions and comments are as follows.

1) Although mutant KCNE3 was shown to express in the surface membrane, it is not known if E3 association with Q1 is reduced by the mutations. It is known that Q1:E1 at different ratio can have different stoichiometry (as shown by the author of this manuscript) and the channels with different stoichiometry show different function including G-V and kinetics. Does KCNE3 share similar properties? Additional experiments may be needed to address this concern.

2) The authors used DF-160/DF60 to measure VSD activation. In VCF measurements it is not clear how VCF amplitude is related to VSD movements. This method seems to be flawed.

3) It is not clear why the authors did not analyze the VCF data to show FV. The relationship between FV and GV is not analyzed either. The authors made conclusions in reference of intermediate and activated states of the VSD, but these conclusions were not backed up by data of fluorescence or current measurements.

4) KCNE3 may also contact with S3 and S5 of KCNQ1 in the cryo-EM structure. Why did not the authors inspect the residues in these motifs? In addition, the cryo-EM structure shows the conformation of only one state, but the KCNE3-KCNQ1 interactions in other states are not known. These need to be considered in the structure motivated mutagenesis study.

5) Related to comment 4, how does this study reconcile with the studies by the McDonald lab (Melman et al. 2004 Neuron), which concluded that KCNE1 (and KCNE3) interacted with the pore domain of KCNQ1 to modulate channel activation?

6) Figure 2. What does "functionally coupled" mean? How is this defined? The interpretation of Figure 2 data is hard to follow and seems to be arbitrary. For example, for F127A the E3 mutations are "functionally coupled" when the GV is the same to E3 WT (Figure 2A & B), but for F130A, the E3 mutations are "functionally coupled" when the GV differs from E3 WT (Figure 2C & D).

7) Figure 4. The FV of the mutant channels + E3 WT shifts to negative voltages and shows two components. The shifts need to be characterized and compared to WT KCNQ1+E3. The ratio of DF-160/DF60 in Figure 4F does not reflect the shift and is misleading (see comment 2).

8) E1 differs from E3 in sequence and function but the S4 mutations are shown to affect E1 and E3 similarly, please explain. In addition, previous studies (Melman et al. 2001 JBC) showed that the triplet amino acids in the middle of the transmembrane segment of KCNE1 and 3 can switch the function between KCNE1 and KCNE3. How does this study reconcile with the previous results?

Reviewer #3 (Recommendations for the authors):

This is an interesting manuscript dealing with the functional effect of KCNE3 on KCNQ1 function. Using the latest KCNQ1/KCNE3 CryoEM structures, they test different residues for functional effects on S4 movement or gate opening. They identify some interactions between S1 and KCNE3 and that these interactions affect the voltage sensor S4 movement via two residues in S4. This is a timely study on a physiologically important K channel involved in many diseases. My main concern is how the analysis of the data was done. But if it stands up to more rigorous analysis, I think the findings are very important and would be interesting to a general audience.

1. Pg. 7. Line 108. Is ΔF-160mV/ΔF60mV a convincing measure for determining the voltage dependence? Can authors explain more about the parameter and why they choose this one? To me, it is not clear that this parameter will give unambiguous conclusions. For example, If F1 is shifted to more depolarized potentials, then the ratio will be bigger. But if F2 is shifted to the right instead, then the ratio will also be bigger. So how distinguish between these two cases with this parameter? Why didn't the authors use even more negative voltages (-180 mV) or positive voltages (+80 mV) to get complete FVs?

2. Also, normalizing the FVs at +60mV if they are not saturated at +60mV will distort the FVs. Not sure how to get around this problem though?

3. Pg. 9. Line 167. M238 and V241 are important for KCNE3 binding and modulation of KCNQ1, but no evidence shows that M238 and V241 interact with S1 of KCNQ1, even though they seem face to each other. In other words, the authors have not identified the functional coupling between S1 and S4. Therefore, the subtitle here is not appropriate.

4. Admittedly, KCNE3 interacts with S1 and S4, but the authors have not shown any evidence that S4 and S1 interact, and that these interactions are important for VSD modulation of KCNE1 or KCNE3. The title stating the triad interaction need to be revised. Any statement talking about triad interaction, such as line 73 and 278, should be avoided.

5. Pg. 8. Line 134-138. More explanation is needed for the functional couple between KCNQ1 and KCNE3. Why similar GV curves suggested they are functionally coupled? Why the additive GV curves suggested no interaction? Is this mutant cycle analysis?

6. Pg. 9. Line 151. I understand why the authors used mutation G73L, but wouldn't it be better to switch the two residues at F127-KNCQ1 and G73-KCNE3 (F127A-G73F) and see the role of Phe in the KCNQ1-KCNE3 interaction as they did with KCNQ1-F130A-A69F-KCNE3?

7. Pg. 11. Line 199. I wonder if the mutations affect the kinetics of the S4 movement? This would be helpful understanding how mutations in S4 affect the VSD of KCNQ1 by KCNE1 and KCNE3 modulation.

8. Figure 1D and 1F. Any evidence showing that KCNE3 is indeed co-expressed with KCNQ1? For me, some mutations, such as Q1-WT-E3-I76A, show very similar current and fluorescence traces with the WT Q1-E3. This concern is also raised for Q1-F127A-E3-G73L in Figure 3B and 3D. Some conclusions in the manuscript couldn't be made without showing KCNE3 or its mutations is present in the KCNQ1 channels.

9. Figure 1D and 1F, for the trace of -160 mV in Q1-WT-E3-WT, the fluorescence signal is lower at the tail voltage of -30 mV than at the resting voltage of -90 mV, which seems incorrect. I would expect S4 would move up higher during -30 mV than -90 mV, which means the fluorescence signal would be bigger at -30 mV than -90 mV. Please show better recordings or explain why. Please also check with Q1-F127A-E3-G73L in Figure 3D, Q1-M238A+E3 WT in Figure 4D.

10. Figure 5B. More positive voltages, at least +80 mV, should be used as in Figure 2D. The three GV curves are not even close to getting saturated at positive voltages. Please also provide GV at + 80 mV and +100 mV for Figure supplement 4B.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex" for further consideration by eLife. Your revised article has been evaluated by Richard Aldrich (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. Provide data assessing whether similar interactions proposed for KCNE3 function are important for KCNE1 function. We imagine only a few key experiments will be needed and not an extensive study. (see Reviewer #3 comment 1)

2. Provide data testing whether the co-expression method allows a full association of KCNE3 with KCNQ1, by testing the dose-dependence of KCNE3 RNA for key KCNE3 mutations. (see Reviewer #3 comment 2)

3. Where fits of the FVs are not constrained enough, just report an upward bound for the V0.5. (see Reviewer #2 comment)

Reviewer #1 (Recommendations for the authors):

This manuscript is a transformed version of the one submitted prior. The new data and improved presentation provide very strong evidence that the KCNQ1 S1 – KCNE3 interface observed in a cryo-EM structure is critical for KCNE3 to exert its proper functional effect on KCNQ1. The volume scanning of each residue on both sides of a double mutant cycle analysis that resulted in rescue by multiple sets of interacting pairs makes it difficult to imagine that the binding interface observed in the structure is not an interface that enables KCNE3 function.

General suggestion:

The language in the paper refers to mutants "impairing" the modulation or being "tolerated". It seemed to me that the mutations may not impair the interaction so much as change the functional output of the interaction.

Can any conclusions be reached or compelling speculations made about whether the mutations prevent E3 from binding to all or a proportion of channels vs E3 binding well to all channels, but in a distorted conformation that induces a different GV?

It looked to me in Figures2L, 3G, 4N that bulkier residues consistently positively shifted the GV, suggesting the latter in those cases. I might speculate that the exact conformation of KCNE3, which is determined by the S1-E3 interface, determines the functional impact of E3 and that could at least partially explain the functional differences between KCNEs. Such speculation is not necessary but is my conceptual takeaway from this study.

Specific suggestions:

Line 112

" It seemed that the modulation depended on the side-chain volume: the more different the size was, the more significant was the impairment of modulation (Figure 2N). "

It could be helpful to quantitate what the side chain volumes are for G, A, V, L, F, W.

line 220

"which further strengthens the importance of the tight interaction between the S1 segment and KCNE3. "

I think it would be more appropriate to state that this is the strong evidence that the specific interaction between the S1 segment and KCNE3 is important. None of the single mutations really show that this precise interface is functionally critical.

line 265

"Overall, the results indicate that KCNE3 residues in the middle of the transmembrane segment are important for channel modulation, probably by preventing the S4 segment from going to the down position. "

Similarly I think it would be more appropriate to state that this is the strong evidence that it is an interaction between the S1 segment and KCNE3 that is important for modulating for VSD movement.

Reviewer #2 (Recommendations for the authors):

The authors have responded well to my critique and made several improvements of the manuscript. I now have only one comment that should be addressed:

The fits of the FVs are in some cases not constrained enough by the data, e.g. when the data do not tend to saturate at either the negative or positive range of voltages. In those cases, it is much better to just report an upward bound for the V0.5. For example, one can state that the V0.5>+60 mV for FVs that do tend to saturate at positive voltages and V0.5<-160 mV for FVs that do tend to saturate at negative voltages. The conclusions will still be the same and valid even without an exact V0.5 number, since for the single mutants you have a V0.5 that is different from +60mV or -160 mV.

Reviewer #3 (Recommendations for the authors):

In this interesting study the interaction between the S1 segment of KCNQ1 and the transmembrane segment of KCNE3 was shown to determine the shift of voltage dependence of activation of both the voltage sensor and the conductance. Guided by the KCNQ1-KCNE3 structure that was previously solved using cryo-EM, extensive mutations suggested that the S1-KCNE3 interaction depended on the size of the side chain of residues in these segments. Changing side chain sizes in either S1 or KCNE3 altered the conductance shifts, and at two pairs of S1-KCNE3 interaction residues, the complementary size changes rescued voltage shifts for both conductance and voltage sensor activation. The data are clearly presented with compelling conclusions. These results provide molecular mechanism for subunit modulation of a physiologically important ion channel. Some comments are as follows.

1. It is known that KCNE1, which is an important KCNQ1 regulatory subunit in the heart, also shifts voltage sensor activation to more negative voltages similarly as KCNE3. This study only focuses on KCNE3. While the study is well done with interesting results, the results would be more significant for publishing in eLife if some of the KCNQ1 mutations can be coexpressed with KCNE1 to examine if similar interactions proposed here are important for KCNE1 function. Such experiments do not have to be extensive as for the KCNE3 study. The insights can be very valuable. It seems that the previous version of the manuscript included some KCNE1 results. The authors should not cut out these results in the revised manuscript.

2. The authors addressed the comments from previous reviews on the issue of association of mutant KCNE3 with KCNQ1. 10 ng KCNQ1 was used to express the channel complex with 1 ng mutant KCNE3. To support that this method allows a full association of KCNE3 with KCNQ1, the functional dependence on different concentrations of KCNE3 RNA, a high and a low concentration, should be shown for at least a couple of KCNE3 mutations.

3. Line 98 and afterward: "∆G", should it be "G"? Please explain in the text that it is conductance.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex" for further consideration by eLife. Your revised article has been evaluated by Richard Aldrich (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Please include the KCNE1 results shown in the rebuttal in the manuscript, and address the mechanistic implications of these results. As reviewer #3 noted, the sequence of F127 mutations in KCNQ1 seem to shift the GV of both +KCNE1 and +KCNE3 in a similar way, even though KCNE1 and KCNE3 shift the GV of KCNQ1 in opposing fashions. This is very interesting, broadens the significance of the findings to the KCNE family generally, and deserves thoughtful mechanistic discussion. Also, please show data quantifying the reproducibility of the Cover Letter Figure 1D results show to back the claim that the F127A+E1 GV is shifted far in the positive direction.

Reviewer #3 (Recommendations for the authors):

The authors have responded to my comments. Yes, please include the KCNE1 results shown in the rebuttal in the manuscript, and a discussion of the comparison between KCNE1 and KCNE3 results will be significant since KCNE1 and KCNE3 modulate channel function with drastic differences. These results, on the other hand, do not show mechanistic differences. What would this comparison mean to the mechanism studied in this manuscript?

https://doi.org/10.7554/eLife.81683.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further for publication by eLife.

The functional validation of the KCNQ1/KCNE3 structural interface is compelling, yet was not deemed to comprise a conceptual advance sufficient to merit publication in eLife. The reviewers agreed that validation of the interface by KCNQ1 F127A-KCNE3 G73L is an important result. However, this major conclusion could benefit from further experiments. Reviewers all thought that the data analysis needs substantial revision, and other claims should be tempered. If further experiments lead to additional mechanistic insights, the combined results could be submitted as a new manuscript.

Reviewer #1 (Recommendations for the authors):

This study aimed to identify amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity, and identify residues on the S4 segment which convert KCNE effects into voltage sensor movement. The research appears technically sound and expertly performed. The major strength of this work is the molecular redesign of KCNE3 and the S1 segment of KCNQ1 to rescue the functional interaction between KCNE3 and KCNQ1. However, this key result rests on a single pair of mutations. The weaknesses are some of the mechanistic interpretations pertaining to functional coupling between KCNQ1 and KCNE3. The manuscript achieved its aim of identifying amino acid residues on KCNE3 facing the S1 segment of KCNQ1 that are required for constitutive activity. This work also identifies mutations on the S4 segment which diminish KCNE1 effects, but data seems inadequate to support the claim that the residues identified on the S4 are the site of conversion of KCNE effects into voltage sensor movement, as alternate explanations also appear plausible. Importantly, this work presents strong evidence that the KCNE3/S1 interface observed structurally is accurate in its molecular detail, and crucial for inter-protein coupling. This study should cement the functional relevance of the KCNE3 interface with the S1 of KCNQ1 observed in a recent structure. Additionally. this work identifies key sites that perturb the modulation of KCNQ1 by KCNE proteins.

The KCNQ1 F127A-KCNE3 G73L result is compelling, it is strong conceptual evidence that the KCNE3/S1 interface forms as advertised. I think the paper would be better appreciated if it focused almost entirely on this result, and perhaps further investigate this key finding with a few more related experiments. The manuscript states that " Appropriate side-chain volumes at the interaction interface are necessary" yet could other physical/chemical concepts explain the reason for rescue?

Testing further sets of double mutations at positions 127 and 73 could test whether this apparent rescue by the swap of side chain bulk is more than just a happy coincidence, and whether bulk inversions at positions 127 and 73 could be found that produce channels even more functionally similar to WT KCNE/KCNQ

I wonder if this KCNQ1 F127A-KCNE3 G73L protein engineering could be used to knock in KCNE-resistant channels that will only respond when a mutant KCNE is expressed…possibly useful for experiments conceptually similar to DREADDs or Kevan Shokat's knob-in-hole chemical genetics.

We greatly appreciate your valuable comments. In this revised manuscript, we exclusively focused on the interaction between the S1 and KCNE3, following your suggestions. We further investigated the effect of mutations on the interaction face between the S1 and KCNE3 and successfully identified at least another pair (S57 and I145).

The idea of knock-in KCNE-resistant channels sounds cool. That might work!

line 134

"For the KCNQ1 F127A mutant, co-expression with KCNE3 F68A or V72A yielded G-V curves similar to that for KCNE3 WT, implying that F127 of the S1 segment and F68/V72 of KCNE3 are functionally coupled."

I disagree. these results just mean that all of the mutations eliminate the E3 effect. The functional coupling analysis here and in the following sentences (up to line 144) seems flawed, or maybe I am failing to understand it. What basis is there for "additive" shifting?

As you pointed out, the analysis may be flawed, and we abandoned this part in this revision. Therefore, the sentence no longer exists.

line 182

"Therefore, we concluded that M238 and V241 are the sites where the binding of KCNE3 is converted to the VSD movement's modulation."

I disagree, and suggest softening this conclusion. In combination with the structure, the possibility of this being a site of conversion does seems plausible, but these results do not make this certain. This result merely means that perturbation of these sites perturbs the coupling. This is consistent with being a site of physical coupling, or conversion, but based on this result alone, other physical possibilities also seem plausible. For example, this could be a site needed to align the site of conversion or There could be multiple sites of conversion.

As we focused on the S1 segment and abandoned the S4 analysis, this sentence no longer exists.

Figure Supplement 1D

The confocal images do clearly show surface expression, but provide little information about the density of surface KCNE1, nor association with KCNQ1. Quantitation across multiple oocytes could strengthen this control.

We agree with your comments. The confocal images only show the surface expression and do not provide any information on the association with KCNQ1. On the other hand, as seen in Figure 3 and Figure supplements 9-12, all but I76A mutant showed substantial changes in voltage dependence. Furthermore, as seen in Figure supplement 8, we confirmed that our experiment condition where 10 ng RNA of KCNQ1 co-injected with 1 ng RNA of KCNE3 is sufficient to fully modulate KCNQ1 currents at least in the cases of KCNE3 WT and two representative mutants (S57A and G73L). Therefore, we believe most KCNE3 mutants bind and modulate KCNQ1 channels.

Figure 3F, purple traces appear to be mislabeled…should be F130A +A69F?

This figure no longer exists.

Table 1: reporting the midpoints of the E1 mutations on their own (no KCNE), even if from another publication, would be helpful G is conductance, but in table 1 current amplitudes are given (minor semantic issue)

This revised manuscript reports the midpoints and other parameters for all KCNQ1 mutations (with or without KCNE3) and KCNE3 mutants (Tables supplement 1-3).

As you pointed out, G should have been I. We have corrected them in the Table supplements.

Exclusion criteria for oocyte leak should be in the methods section

We only used oocytes with a holding current less than -0.2 uA at -90 mV in TEVC and -0.3 µA at -100 mV in VCF. We have added the criteria in the methods section.

Reviewer #2 (Recommendations for the authors):

In this manuscript the authors inspected the cryo-EM structure of KCNQ1 and KCNE1 and made mutations to KCNQ1 and KCNE3 residues that appear to interact in the structure. These mutated residues are located in the transmembrane segment S1 and S4 of KCNQ1 and of KCNE3. The authors found that the KCNE4 mutations F68A, V72A and I 76 A abolished the ability of KCNE3 to shift GV relation to voltages more negative than -160 mV, which is similar to the findings of their previous study that the S1 mutations F127A and F130A also abolished the ability of KCNE3 to shift GV to negative voltages. A double mutation Q1 F127A + E3 G73L rescued a large part of the GV shift, supporting the observation from the structure that KCNQ1 S1 and KCNE3 may interact among these residues for the modulatory effects of KCNE3 on channel gating. The authors also measured VSD movements in the WT and these mutant channels using VCF, and concluded that the S1-E3 interactions at the S1 site are important for the KCNE3 induced shift of VSD activation to the negative voltages. The authors then found that mutations of S4, M238A and M241A, also reduced the ability of KCNE3 to shift the GV relation, and reduce VSD activation at negative voltages. Based on these results, the authors conclude that the interactions among S1, S4 and KCNE3 at the location of these residues modulate VSD activation and stabilize the open state of the channel. The manuscript presents some interesting data, including the Q1 F127A + E3 G73L results as evidence for S1-KCNE3 interaction. However, some of the conclusions made in the manuscript seem to be based on incomplete experiments or analyses of the data.

Some questions and comments are as follows.

1) Although mutant KCNE3 was shown to express in the surface membrane, it is not known if E3 association with Q1 is reduced by the mutations. It is known that Q1:E1 at different ratio can have different stoichiometry (as shown by the author of this manuscript) and the channels with different stoichiometry show different function including G-V and kinetics. Does KCNE3 share similar properties? Additional experiments may be needed to address this concern.

Thank you for raising this critical point. We also think the stoichiometry could be various, especially when E3 expression is low, as in the case of Q1:E1. To verify the E3 expression level was high enough, we conducted KCNQ1-KCNE3 current measurements with various amounts of KCNE3 RNAs (Figure supplement 8). According to the experiments, 1 ng of KCNE3 RNA is sufficient to fully modulate 10 ng of KCNQ1 channels. We added these descriptions to explain our rational experiment design (Lines 146-148).

2) The authors used DF-160/DF60 to measure VSD activation. In VCF measurements it is not clear how VCF amplitude is related to VSD movements. This method seems to be flawed.

Thank you for pointing the problem out. We admit the DF-160/DF60 index may not be appropriate and is no longer used for evaluation.

3) It is not clear why the authors did not analyze the VCF data to show FV. The relationship between FV and GV is not analyzed either. The authors made conclusions in reference of intermediate and activated states of the VSD, but these conclusions were not backed up by data of fluorescence or current measurements.

We have added G-Vs and F-Vs for all VCF data in Figure 5.

4) KCNE3 may also contact with S3 and S5 of KCNQ1 in the cryo-EM structure. Why did not the authors inspect the residues in these motifs? In addition, the cryo-EM structure shows the conformation of only one state, but the KCNE3-KCNQ1 interactions in other states are not known. These need to be considered in the structure motivated mutagenesis study.

As you pointed out, other segments are in contact with KCNE3 in the cryo-EM structure. In this paper, however, we focused on the S1 segment in the VSD in this study. Of course, our results do not exclude any significance of the contact with the pore domain (S5 and S6).

We understand that the cryo-EM structure represents one of many states. Still, our mutagenesis studies clearly show that most of the interactions between S1 and KCNE3 in the structure are required for the constitutive activity of the KCNQ1-KCNE3 channel.

5) Related to comment 4, how does this study reconcile with the studies by the McDonald lab (Melman et al. 2004 Neuron), which concluded that KCNE1 (and KCNE3) interacted with the pore domain of KCNQ1 to modulate channel activation?

As answered for comment 4, our results do not exclude any significance of the contact with the pore domain (S5 and S6). However, according to Barro-Soria et al. (PNAS, 2017), KCNE1 affects both the S4 movement and the gate, while KCNE3 affects the S4 movement and only affects the gate indirectly. Therefore, the interaction between the pore domain and KCNE3 could be less important than in the case of KCNE1.

6) Figure 2. What does "functionally coupled" mean? How is this defined? The interpretation of Figure 2 data is hard to follow and seems to be arbitrary. For example, for F127A the E3 mutations are "functionally coupled" when the GV is the same to E3 WT (Figure 2A & B), but for F130A, the E3 mutations are "functionally coupled" when the GV differs from E3 WT (Figure 2C & D).

As we answered to reviewer #1, the analysis may be flawed, and we abandoned this part in this revision. Therefore, the sentence no longer exists.

7) Figure 4. The FV of the mutant channels + E3 WT shifts to negative voltages and shows two components. The shifts need to be characterized and compared to WT KCNQ1+E3. The ratio of DF-160/DF60 in Figure 4F does not reflect the shift and is misleading (see comment 2).

Thank you for the comment. We updated the FV analysis to clearly show the two components in the Q1WT + E3 WT. Mutations on KCNQ1 diminished the first component, suggesting that the intermediate state of the VSD became harder to maintain by the mutations. Then, E3 mutants partly restored the first component (Figure 5).

8) E1 differs from E3 in sequence and function but the S4 mutations are shown to affect E1 and E3 similarly, please explain. In addition, previous studies (Melman et al. 2001 JBC) showed that the triplet amino acids in the middle of the transmembrane segment of KCNE1 and 3 can switch the function between KCNE1 and KCNE3. How does this study reconcile with the previous results?

In this manuscript, we no longer examined KCNE1 and solely focused on KCNE3. However, the functional difference between KCNE1 and KCNE3 is still an open and vital question that remains to be seen. One possible explanation is the intermediate state of KCNQ1-KCNE1 is non-conductive while the intermediate state of KCNQ1-KCNE3 is conductive. Amino acid sequence difference may contribute to the conductive state of the intermediate state.

Reviewer #3 (Recommendations for the authors):

This is an interesting manuscript dealing with the functional effect of KCNE3 on KCNQ1 function. Using the latest KCNQ1/KCNE3 CryoEM structures, they test different residues for functional effects on S4 movement or gate opening. They identify some interactions between S1 and KCNE3 and that these interactions affect the voltage sensor S4 movement via two residues in S4. This is a timely study on a physiologically important K channel involved in many diseases. My main concern is how the analysis of the data was done. But if it stands up to more rigorous analysis, I think the findings are very important and would be interesting to a general audience.

1. Pg. 7. Line 108. Is ΔF-160mV/ΔF60mV a convincing measure for determining the voltage dependence? Can authors explain more about the parameter and why they choose this one? To me, it is not clear that this parameter will give unambiguous conclusions. For example, If F1 is shifted to more depolarized potentials, then the ratio will be bigger. But if F2 is shifted to the right instead, then the ratio will also be bigger. So how distinguish between these two cases with this parameter? Why didn't the authors use even more negative voltages (-180 mV) or positive voltages (+80 mV) to get complete FVs?

As we answered reviewer #2’s comment, we admitΔF-160mV/ΔF60mV may not be appropriate. We abandoned ΔF-160mV/ΔF60mV. Instead, we used the complete F‑V curves as suggested (Figure 5).

2. Also, normalizing the FVs at +60mV if they are not saturated at +60mV will distort the FVs. Not sure how to get around this problem though?

We presented full F-V curves and fitted them with the Boltzmann equation (Figure 5). F-Vs are normalized accordingly.

3. Pg. 9. Line 167. M238 and V241 are important for KCNE3 binding and modulation of KCNQ1, but no evidence shows that M238 and V241 interact with S1 of KCNQ1, even though they seem face to each other. In other words, the authors have not identified the functional coupling between S1 and S4. Therefore, the subtitle here is not appropriate.

We focused on the S1 segment in this manuscript and abandoned the S4 data.

4. Admittedly, KCNE3 interacts with S1 and S4, but the authors have not shown any evidence that S4 and S1 interact, and that these interactions are important for VSD modulation of KCNE1 or KCNE3. The title stating the triad interaction need to be revised. Any statement talking about triad interaction, such as line 73 and 278, should be avoided.

Because we no longer examined the triad interaction, we have changed the title accordingly.

5. Pg. 8. Line 134-138. More explanation is needed for the functional couple between KCNQ1 and KCNE3. Why similar GV curves suggested they are functionally coupled? Why the additive GV curves suggested no interaction? Is this mutant cycle analysis?

As we answered reviewers #1 and #2 comments, the “functional coupling” analysis may be flawed, and we abandoned this part in this revision. Therefore, the sentence no longer exists.

6. Pg. 9. Line 151. I understand why the authors used mutation G73L, but wouldn't it be better to switch the two residues at F127-KNCQ1 and G73-KCNE3 (F127A-G73F) and see the role of Phe in the KCNQ1-KCNE3 interaction as they did with KCNQ1-F130A-A69F-KCNE3?

Thank you for the comment. In this version, we examined five residues, including G73L, and found that G73L was the best option to restore the F127A mutant (Figure 4A-G). Therefore, Phe or its ring structure may not be required here.

7. Pg. 11. Line 199. I wonder if the mutations affect the kinetics of the S4 movement? This would be helpful understanding how mutations in S4 affect the VSD of KCNQ1 by KCNE1 and KCNE3 modulation.

Because we abandoned the S4 experiments, this part no longer exists.

8. Figure 1D and 1F. Any evidence showing that KCNE3 is indeed co-expressed with KCNQ1? For me, some mutations, such as Q1-WT-E3-I76A, show very similar current and fluorescence traces with the WT Q1-E3. This concern is also raised for Q1-F127A-E3-G73L in Figure 3B and 3D. Some conclusions in the manuscript couldn't be made without showing KCNE3 or its mutations is present in the KCNQ1 channels.

Thank you for raising this critical point. It is a little difficult to prove the KCNE3 indeed co-expressed with KCNQ1. As we answered to reviewer #2’s comment, to verify the E3 expression level was high enough, we conducted KCNQ1-KCNE3 current measurements with various amounts of KCNE3 RNAs (Figure supplement 8). According to the experiments, 1 ng of KCNE3 RNA is high enough to fully modulate KCNQ1 channels. The G73L and S57A showed similar tendencies. Most KCNE3 mutants show different z and V1/2 values, as shown in Table supplement 2, indicating they successfully modulated KCNE3. Only one exception is I76A. KCNQ1+KCNE3 I76A showed similar z and V1/2 values to KCNQ1 WT alone (Table supplements 1-2, Figure supplement 12). It may fail to bind KCNQ1.

9. Figure 1D and 1F, for the trace of -160 mV in Q1-WT-E3-WT, the fluorescence signal is lower at the tail voltage of -30 mV than at the resting voltage of -90 mV, which seems incorrect. I would expect S4 would move up higher during -30 mV than -90 mV, which means the fluorescence signal would be bigger at -30 mV than -90 mV. Please show better recordings or explain why. Please also check with Q1-F127A-E3-G73L in Figure 3D, Q1-M238A+E3 WT in Figure 4D.

10. Figure 5B. More positive voltages, at least +80 mV, should be used as in Figure 2D. The three GV curves are not even close to getting saturated at positive voltages. Please also provide GV at + 80 mV and +100 mV for Figure supplement 4B.

We apologize for the errors and the confusion in the previous version. We do not know why this happened. It could be overcompensation of fluorescence bleaching. We abandoned the data in this version and carefully remeasured the F-V relationships in Figure 5.

As large endogenous currents appear over +80 mV, we only applied membrane potential up to +60 mV.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #1 (Recommendations for the authors):

This manuscript is a transformed version of the one submitted prior. The new data and improved presentation provide very strong evidence that the KCNQ1 S1 – KCNE3 interface observed in a cryo-EM structure is critical for KCNE3 to exert its proper functional effect on KCNQ1. The volume scanning of each residue on both sides of a double mutant cycle analysis that resulted in rescue by multiple sets of interacting pairs makes it difficult to imagine that the binding interface observed in the structure is not an interface that enables KCNE3 function.

General suggestion:

The language in the paper refers to mutants "impairing" the modulation or being "tolerated". It seemed to me that the mutations may not impair the interaction so much as change the functional output of the interaction.

Can any conclusions be reached or compelling speculations made about whether the mutations prevent E3 from binding to all or a proportion of channels vs E3 binding well to all channels, but in a distorted conformation that induces a different GV?

It looked to me in Figures2L, 3G, 4N that bulkier residues consistently positively shifted the GV, suggesting the latter in those cases. I might speculate that the exact conformation of KCNE3, which is determined by the S1-E3 interface, determines the functional impact of E3 and that could at least partially explain the functional differences between KCNEs. Such speculation is not necessary but is my conceptual takeaway from this study.

We agree the reviewer #1’s insightful speculation. As shown in Figure supplement 8G-R, no matter how much the KCNE3 RNA ratio increased, the modulation was not fully recovered in KCNE3 mutants. This result indicates the latter is likely the case. Accordingly, as reviewer #1 suggested, we stopped using the word “impairing” and “tolerated” and changed them like “changed the functional output” or “distorted” (lines 90-91, 114-115, 133-134, 138, 141, 163-164, 167-168, 172-173, 181-182, 184, 190-191, 195, 201, 207, and 232, highlighted in yellow).

Specific suggestions:

Line 112

" It seemed that the modulation depended on the side-chain volume: the more different the size was, the more significant was the impairment of modulation (Figure 2N). "

It could be helpful to quantitate what the side chain volumes are for G, A, V, L, F, W.

According to this comment, we added the volumes of amino acid residues in Figure 1D.

line 220

"which further strengthens the importance of the tight interaction between the S1 segment and KCNE3. "

I think it would be more appropriate to state that this is the strong evidence that the specific interaction between the S1 segment and KCNE3 is important. None of the single mutations really show that this precise interface is functionally critical.

According to this comment, we changed the corresponding sentence in lines 225-226.

line 265

"Overall, the results indicate that KCNE3 residues in the middle of the transmembrane segment are important for channel modulation, probably by preventing the S4 segment from going to the down position. "

Similarly I think it would be more appropriate to state that this is the strong evidence that it is an interaction between the S1 segment and KCNE3 that is important for modulating for VSD movement.

According to this comment, we changed the corresponding sentence in lines 270-272.

Reviewer #2 (Recommendations for the authors):

The authors have responded well to my critique and made several improvements of the manuscript. I now have only one comment that should be addressed:

The fits of the FVs are in some cases not constrained enough by the data, e.g. when the data do not tend to saturate at either the negative or positive range of voltages. In those cases, it is much better to just report an upward bound for the V0.5. For example, one can state that the V0.5>+60 mV for FVs that do tend to saturate at positive voltages and V0.5<-160 mV for FVs that do tend to saturate at negative voltages. The conclusions will still be the same and valid even without an exact V0.5 number, since for the single mutants you have a V0.5 that is different from +60mV or -160 mV.

We are pleased that we were able to respond to your previous comments appropriately. According to the additional comment, we revised the table (Figure 5-source data 2) as well as lines 259 and 268.

Reviewer #3 (Recommendations for the authors):

In this interesting study the interaction between the S1 segment of KCNQ1 and the transmembrane segment of KCNE3 was shown to determine the shift of voltage dependence of activation of both the voltage sensor and the conductance. Guided by the KCNQ1-KCNE3 structure that was previously solved using cryo-EM, extensive mutations suggested that the S1-KCNE3 interaction depended on the size of the side chain of residues in these segments. Changing side chain sizes in either S1 or KCNE3 altered the conductance shifts, and at two pairs of S1-KCNE3 interaction residues, the complementary size changes rescued voltage shifts for both conductance and voltage sensor activation. The data are clearly presented with compelling conclusions. These results provide molecular mechanism for subunit modulation of a physiologically important ion channel. Some comments are as follows.

We appreciate your valuable comments. The responses to each comment are as follows.

1. It is known that KCNE1, which is an important KCNQ1 regulatory subunit in the heart, also shifts voltage sensor activation to more negative voltages similarly as KCNE3. This study only focuses on KCNE3. While the study is well done with interesting results, the results would be more significant for publishing in eLife if some of the KCNQ1 mutations can be coexpressed with KCNE1 to examine if similar interactions proposed here are important for KCNE1 function. Such experiments do not have to be extensive as for the KCNE3 study. The insights can be very valuable. It seems that the previous version of the manuscript included some KCNE1 results. The authors should not cut out these results in the revised manuscript.

Thank you for this constructive comment. Following this comment, we performed a preliminary mutational analysis of KCNQ1 F127 mutants (F127A, F127V, F127L, and F127W) to investigate the functional relationship between the S1 segment and KCNE1 (Author response image 1). We chose the F127 residue here since its modulation by KCNE3 depends on its side-chain volume size (Figure 2A-G, N), and the KCNQ1 F127A mutant-KCNE3 G73L mutant pair restored the modulation of KCNQ1 by KCNE3 (Figure 4A-G). All the F127 mutants potentiated KCNE1-dependent half-activation voltage (V1/2) shifts. Moreover, the more different side chain sizes induced the more significant V1/2 shifts. (Please note that the G-V curve for F127A+E1 is omitted in the Cover letter figure 1K because the curve is shifted far in the positive direction and is impossible to fit with the Boltzmann function.) These results suggest that, at least for the F127 residue, the modulation by KCNE1 depended on the side-chain volume, as in the case of KCNE3. However, we would like to keep the KCNE1 results for the next manuscript because we think it is necessary to show all the scanning results of S1 residue to claim possible side-chain volume dependence on the KCNE1 modulation. (We hope the next one will lead to an exciting result that can suffice to submit eLife.) Adding the partial F127 residue result to this manuscript may confuse readers. We would still be happy to add the KCNE1 results to the current manuscript if reviewer #3 thinks it is indispensable.

Author response image 1
Download asset Open asset
Functional effects of KCNQ1 F127 mutants on KCNQ1 modulation by KCNE1.

(A-N) Representative current traces (A-J) and G-V relationships (K-N) of KCNQ1 WT and F127 mutants with or without KCNE1 WT. (O) The half-activation voltage of KCNQ1 WT and F127 mutants with (filled bars) or without (open bars) KCNE1 WT. > 60 means over 60 mV. Error bars indicate ± s.e.m. for n = 5 in (I-M). ** and *** indicate **P < 0.01 and ***P < 0.001.

2. The authors addressed the comments from previous reviews on the issue of association of mutant KCNE3 with KCNQ1. 10 ng KCNQ1 was used to express the channel complex with 1 ng mutant KCNE3. To support that this method allows a full association of KCNE3 with KCNQ1, the functional dependence on different concentrations of KCNE3 RNA, a high and a low concentration, should be shown for at least a couple of KCNE3 mutations.

We have already presented the suggested experiment for KCNE3 S57A and G73L mutants in Figure 3—figure supplement 1G-R. We chose the KCNE3 mutants since they are functionally coupling with KCNQ1 I145F and F127A mutants, respectively (Figure 4). In both KCNE3 mutants, 0.3 ng KCNE3 RNA is enough to achieve maximum modulation with 10 ng KCNQ1 WT RNA. These results support that our expression condition through the experiment (10 ng KCNQ1 RNA and 1 ng KCNE3 RNA) is reasonable.

3. Line 98 and afterward: "∆G", should it be "G"? Please explain in the text that it is conductance.

Thank you for the suggestion, and we are sorry for our misconversion. DG in line 98 and afterward is DG (δ-G). We used this parameter to show the relative conductance at -100 mV and explained it in lines 103-105. In the revised version, we corrected the misconversion.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Please include the KCNE1 results shown in the rebuttal in the manuscript, and address the mechanistic implications of these results. As reviewer #3 noted, the sequence of F127 mutations in KCNQ1 seem to shift the GV of both +KCNE1 and +KCNE3 in a similar way, even though KCNE1 and KCNE3 shift the GV of KCNQ1 in opposing fashions. This is very interesting, broadens the significance of the findings to the KCNE family generally, and deserves thoughtful mechanistic discussion. Also, please show data quantifying the reproducibility of the Cover Letter Figure 1D results show to back the claim that the F127A+E1 GV is shifted far in the positive direction.

Reviewer #3 (Recommendations for the authors):

The authors have responded to my comments. Yes, please include the KCNE1 results shown in the rebuttal in the manuscript, and a discussion of the comparison between KCNE1 and KCNE3 results will be significant since KCNE1 and KCNE3 modulate channel function with drastic differences. These results, on the other hand, do not show mechanistic differences. What would this comparison mean to the mechanism studied in this manuscript?

We appreciate the reviewer’s constructive comments. We have added the new Figure 6 of KCNQ1 F127 mutants co-expressed with KCNE1 WT. We have also added the Results section (lines 274-286) and the Discussion section (lines 350-361). As discussed in the manuscript, we hypothesize the S1 segment may have a similar role in the KCNE1 and KCNE3 modulations, which is to help stabilize the intermediate state of the voltage sensor domain. Further extensive study is required for the possible role of the S1 segment in the KCNE1 modulation.

https://doi.org/10.7554/eLife.81683.sa2

Article and author information

Author details

  1. Go Kasuya

    Division of Integrative Physiology, Department of Physiology, Jichi Medical University, Shimotsuke, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    gokasuya@jichi.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1756-5764

  2. Koichi Nakajo

    Division of Integrative Physiology, Department of Physiology, Jichi Medical University, Shimotsuke, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    knakajo@jichi.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0766-7281

Funding

Japan Society for the Promotion of Science (19K23833)

  • Go Kasuya

Japan Society for the Promotion of Science (20H03200)

  • Go Kasuya

Japan Society for the Promotion of Science (17K08552)

  • Koichi Nakajo

Japan Society for the Promotion of Science (21K06786)

  • Koichi Nakajo

Salt Science Research Foundation (2219)

  • Go Kasuya

JICHI MEDICAL UNIVERSITY YOUNG INVESTIGATOR AWARD

  • Go Kasuya

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

Acknowledgements

We thank Dr. Yuichiro Fujiwara (Kagawa University) and the Nakajo laboratory members for their valuable discussions. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Nos. 19K23833 and 20H03200 to GK and 17K08552 and 21K06786 to KN), by JICHI MEDICAL UNIVERSITY YOUNG INVESTIGATOR AWARD to GK, and by The Salt Science Research Foundation (Grant No. 2219) to GK.

Ethics

All experiments were approved by the Animal Care Committee of Jichi Medical University (Japan) under protocol no. 18027-03 and were performed according to guidelines.

Senior Editor

  1. Richard W Aldrich, The University of Texas at Austin, United States

Reviewing Editor

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

Reviewer

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

Version history

  1. Preprint posted: April 6, 2021 (view preprint)
  2. Received: July 29, 2022
  3. Accepted: November 3, 2022
  4. Accepted Manuscript published: November 4, 2022 (version 1)
  5. Version of Record published: November 17, 2022 (version 2)

Copyright

© 2022, Kasuya and Nakajo

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

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  1. Go Kasuya
  2. Koichi Nakajo
(2022)
Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex
eLife 11:e81683.
https://doi.org/10.7554/eLife.81683

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