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. 2022 Nov 4:11:e81683.
doi: 10.7554/eLife.81683.

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

Go Kasuya  1 , Koichi Nakajo  1
Affiliations

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

Go Kasuya et al. Elife. .

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.

Keywords: KCNE; KCNQ1; human; ion channel complex; molecular biophysics; mouse; potassium channel; structural biology; voltage clamp fluorometry; voltage sensor; xenopus.

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Conflict of interest statement

GK, KN No competing interests declared

Figures

Figure 1.
Figure 1.. 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.
Figure 2.. 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—figure supplement 1.
Figure 2—figure supplement 1.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 F123 mutants.
(A–F) Representative current traces (A–D) and G-V relationships (E,F) of KCNQ1 F123 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (E,F).
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 F127 mutants.
(A–L) Representative current traces (A–H) and G-V relationships (I–L) of KCNQ1 F127 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (I–L). For clear comparisons, the current traces of KCNQ1 F127 mutants with KCNE3 WT shown in Figure 2C–F are redisplayed here.
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 F130 mutants.
(A–L) Representative current traces (A–H) and G-V relationships (I–L) of KCNQ1 F130 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (I–L).
Figure 2—figure supplement 4.
Figure 2—figure supplement 4.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 L134 mutants.
(A–L) Representative current traces (A–H) and G-V relationships (I–L) of KCNQ1 L134 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (I–L).
Figure 2—figure supplement 5.
Figure 2—figure supplement 5.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 I138 mutants.
(A–L) Representative current traces (A–H) and G-V relationships (I–L) of KCNQ1 I138 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (I–L).
Figure 2—figure supplement 6.
Figure 2—figure supplement 6.. Current traces and conductance-volltage (G-V) relationships of KCNQ1 L142 mutants.
(A–L) Representative current traces (A–H) and G-V relationships (I–L) of KCNQ1 L142 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (I–L).
Figure 2—figure supplement 7.
Figure 2—figure supplement 7.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 I145 mutants.
(A–L) Representative current traces (A–H) and G-V relationships (I–L) of KCNQ1 I145 mutants with or without KCNE3 WT. Error bars indicate ± SEM for n=10 in (I–L). For clear comparisons, the current traces of KCNQ1 I145 mutants with KCNE3 WT shown in Figure 2H–K are redisplayed here.
Figure 3.
Figure 3.. 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—figure supplement 1.
Figure 3—figure supplement 1.. Dose-dependent modulation of KCNQ1 by decreasing amounts of KCNE3 WT and mutants.
(A–F) Representative current traces (A–E) and conductance-voltage (G-V) relationships (B) of KCNQ1 WT co-expressed with each amount of KCNE3 WT. (G–L) Representative current traces (G–K) and G-V relationships (L) of KCNQ1 WT co-expressed with each amount of the KCNE3 S57A mutant. (M–R) Representative current traces (M–Q) and G-V relationships (F) of KCNQ1 WT co-expressed with each amount of the KCNE3 G73L mutant. Error bars indicate ± SEM for n=10 in 10 ng KCNQ1 with 1 ng KCNE3 conditions and for n=5 in the other conditions in (F, L, and R). For clear comparisons, the current traces of 10 ng KCNQ1 WT with 1 ng KCNE3 WT and mutants shown in Figures 2B, 3B and J are redisplayed here.
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 WT with the KCNE3 I61 mutants.
(A–F) Representative current traces (A–E) and G-V relationships (F) of KCNQ1 WT with the KCNE3 I61 mutants. Error bars indicate ± SEM for n=10 in (F).
Figure 3—figure supplement 3.
Figure 3—figure supplement 3.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 WT with the KCNE3 M65 mutants.
(A–G) Representative current traces (A–F) and G-V relationships (G) of KCNQ1 WT with the KCNE3 M65 mutants. Error bars indicate ± SEM for n=10 in (G).
Figure 3—figure supplement 4.
Figure 3—figure supplement 4.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 WT with the KCNE3 A69 mutants.
(A–F) Representative current traces (A–E) and G-V relationships (F) of KCNQ1 WT with the KCNE3 A69 mutants. Error bars indicate ± SEM for n=10 in (F).
Figure 3—figure supplement 5.
Figure 3—figure supplement 5.. Current traces and conductance-voltage (G-V) relationships of KCNQ1 WT with the KCNE3 I76 mutants.
(A–F) Representative current traces (A–E) and G-V relationships (F) of KCNQ1 WT with the KCNE3 I76 mutants. Error bars indicate ± SEM for n=10 in (F).
Figure 4.
Figure 4.. 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—figure supplement 1.
Figure 4—figure supplement 1.. Current traces, conductance-voltage (G-V) relationships, and ratios of conductance of the KCNQ1 F130 mutants with the KCNE3 A69 mutants.
(A–G) Representative current traces (A–E), G-V relationships (F), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (G) of the KCNQ1 F130A mutant with the KCNE3 A69 mutants. (H–N) Representative current traces (H–L), G-V relationships (M), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (N) of the KCNQ1 F130V mutant with the KCNE3 A69 mutants. Error bars indicate ± SEM for n=10 in (F, G, M,N).
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Current traces, conductance-voltage (G-V) relationships, and ratios of conductance of the KCNQ1 I138 mutants with the KCNE3 M65 mutants.
(A–H) Representative current traces (A–F), G-V relationships (G), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (H) of the KCNQ1 I138A mutant with the KCNE3 M65 mutants. (I–P) Representative current traces (I–N), G-V relationships (O), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (P) of the KCNQ1 I138W mutant with the KCNE3 M65 mutants. Error bars indicate ± SEM for n=10 in (G, H, O,P).
Figure 4—figure supplement 3.
Figure 4—figure supplement 3.. Current traces, conductance-voltage (G-V) relationships, and ratios of conductance of the KCNQ1 L142 mutants with the KCNE3 I61 mutants.
(A–G) Representative current traces (A–E), G-V relationships (F), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (G) of the KCNQ1 L142A mutant with the KCNE3 I61 mutants. (H–N) Representative current traces (H–L), G-V relationships (M), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (N) of the KCNQ1 L142W mutant with the KCNE3 I61 mutants. Error bars indicate ± SEM for n=10 in (F, G, M, and N).
Figure 4—figure supplement 4.
Figure 4—figure supplement 4.. Current traces, conductance-voltage (G-V) relationships, and ratios of conductance of the KCNQ1 I145W mutant with the KCNE3 S57 mutants.
(A–H) Representative current traces (A–F), G-V relationships (G), and ratios of conductance at –100 mV (G–100mV) and maximum conductance (Gmax) (H) of the KCNQ1 I145W mutant with the KCNE3 S57 mutants. Error bars indicate ± SEM for n=10 in (G and H).
Figure 5.
Figure 5.. 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—figure supplement 1.
Figure 5—figure supplement 1.. Conductance-voltage (G-V) and fluorescence-voltage (F-V) relationships for KCNQ1 mutants.
(A–C) Ionic currents (upper row) and fluorescence traces (lower row) of KCNQ1vcf WT (A), KCNQ1vcf F127A (B), and KCNQ1vcf I145F (C). (D and E) G-V (D) and F-V (E) relationships of KCNQ1vcf WT, KCNQ1vcf F127A, and KCNQ1vcf I145F. Error bars indicate ± SEM for n=5 in (D and E).
Figure 6.
Figure 6.. 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).
Author response image 1.
Author response image 1.. 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.
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