Characterization of an early-diverging KCNE potassium-channel auxiliary subunit in the jawless vertebrate lamprey
- Division of Integrative Physiology, Department of Physiology, Jichi Medical University, Japan
- Department of Chemistry, Biology, and Environmental Science, Faculty of Science, Nara Women’s University, Japan
Figures
Figure 1 with 2 supplements
Sequence, genomic context, and transcript distribution of lamprey KCNE0.
(A–C) Amino acid sequence alignment (A), phylogenetic tree (B), and percent identity (C) of lamprey and human KCNE subunits. Alignments were generated with Clustal Omega (Madeira et al., 2024) and displayed using ESPript3 (Robert and Gouet, 2014). Residues corresponding to the ‘triplet’ (Melman et al., 2001; Melman et al., 2002) motif are highlighted with an orange square. (D) Genomic region containing the kcne locus in sea lamprey, shown from the Ensembl genome browser (Martin et al., 2023). For sequence comparison, KCNE0 from lamprey species (sea lamprey, PmKCNE0; NCBI Accession Number XP_032831116.1; Far Eastern brook lamprey, LrKCNE0; XP_061431545.1 and Arctic lamprey, LcKCNE0; see Figure 1—figure supplement 2), the five human KCNE subunits (HsKCNE1, NP_000210.2; HsKCNE2, NP_751951.1; HsKCNE3, NP_005463.1; HsKCNE4, NP_542402.4; and HsKCNE5, NP_036414.1) and zebrafish KCNE6 (DrKCNE6) (Kasuya et al., 2024) were used. (E) RNA-seq-based transcript levels of KCNQ1 (XM_061564454.1) and KCNE0 (XM_061575561.1) from 10 organs of Far Eastern brook lamprey quantified using a decoy-aware Salmon index. Values are shown as counts per million of the library (CPM). (F) RT-PCR detection of KCNQ1 and KCNE0 transcripts using cDNA synthesized from total RNA isolated from six organs of Arctic lamprey.
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Figure 1—source data 1
PDF file containing original uncropped RT-PCR gel for Figure 1F, indicating the bands and lanes used in the figure.
- https://cdn.elifesciences.org/articles/111781/elife-111781-fig1-data1-v1.zip
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Figure 1—source data 2
Original raw image file for the RT-PCR gel shown in Figure 1F.
- https://cdn.elifesciences.org/articles/111781/elife-111781-fig1-data2-v1.zip
Figure 1—figure supplement 1
Open reading frame (ORF) nucleotide and corresponding amino acid sequence of KCNQ1 in Arctic lamprey.
The ORF nucleotide sequence and the translated amino acid sequence of Arctic lamprey KCNQ1 used for cloning and electrophysiological recordings are shown. The primer-binding sites used for RT-PCR analysis in Figure 1F are indicated with orange arrows.
Figure 1—figure supplement 2
Open reading frame (ORF) nucleotide and corresponding amino acid sequence of KCNE0 in Arctic lamprey.
The ORF nucleotide sequence and the translated amino acid sequence of Arctic lamprey KCNE0 used for cloning and electrophysiological recordings are shown. The primer-binding sites used for RT-PCR analysis in Figure 1F are indicated with orange arrows.
Figure 2 with 1 supplement
Biophysical properties of lamprey KCNQ1 and modulation by KCNE0.
(A–G) Representative current traces (A–F) and conductance–voltage (G–V) relationships of lamprey KCNQ1 WT expressed alone or co-expressed with the corresponding KCNE0 WT. (H–J) Representative ionic-current and fluorescence traces (H, I) and fluorescence–voltage (F–V) relationship (J) of PmKCNQ1vcf WT expressed alone or co-expressed with PmKCNE0 WT. Error bars denote mean ± s.e.m. for n=5 in (G, J). Statistical comparisons of G–V V1/2 values for lamprey KCNQ1 WT expressed alone are provided in Supplementary file 3. V1/2 values were not determined for KCNQ1–KCNE0 complexes because the G–V relationships were nearly saturated over the tested voltage range. Source data are provided in Source data 1.
Figure 2—figure supplement 1
Amino acid sequence alignment of KCNQ1 from various species.
Amino acid sequence alignment of KCNQ1 from human, zebrafish, lampreys, and vase tunicate generated with Clustal Omega (Madeira et al., 2024) and displayed with ESPript3 (Robert and Gouet, 2014). The residue corresponding to the labeling site in human KCNQ1 (G219) (Osteen et al., 2010; Osteen et al., 2012) is highlighted with an orange circle. For sequence alignment, human (HsKCNQ1; NCBI Accession Number NP_000209.2), zebrafish (DrKCNQ1; NP_001116714.1), sea lamprey (PmKCNQ1; XP_075921450.1), Far Eastern brook lamprey (LrKCNQ1; XP_061420438.1), Arctic lamprey (LcKCNQ1; see Figure 1—figure supplement 1), and vase tunicate (CiKCNQ1; NP_001153537.1) KCNQ1 were used.
Figure 3
Regions of KCNE0 important for KCNQ1 modulation.
(A) Schematic diagram of N- and C-terminal truncation constructs of PmKCNE0. (B–F) Representative current traces (B–E) and conductance–voltage (G–V) relationship (F) of PmKCNQ1 WT expressed alone or co-expressed with each of the N-terminally truncated PmKCNE0 constructs. (G–N) Representative current traces (G–M) and G–V relationship (N) of PmKCNQ1 WT expressed alone or co-expressed with each of the C-terminally truncated PmKCNE0 constructs. Error bars denote mean ± s.e.m. for n=5 in (F, N). (O–T) Confocal images of oocytes expressing C-terminally eGFP-tagged PmKCNE0 WT or truncation constructs, and an uninjected oocyte (control). Source data are provided in Source data 1.
Figure 4
Species dependence of KCNQ1–KCNE compatibility.
(A–C) Representative current traces (A, B) and conductance–voltage (G–V) relationship (C) of HsKCNQ1 WT expressed alone or co-expressed with PmKCNE0 WT. (D–F) Representative current traces (D, E) and G–V relationship (F) of DrKCNQ1 WT expressed alone or co-expressed with PmKCNE0 WT. (G–I) Representative current traces (G, H) and G–V relationship (I) of CiKCNQ1 WT expressed alone or co-expressed with PmKCNE0 WT. (J–L) Representative current traces (J, K) and G–V relationship (L) of PmKCNQ1 WT co-expressed with HsKCNE1 WT or HsKCNE3 WT. Error bars denote mean ± s.e.m. for n=5 in (C, F, I, L). Source data are provided in Source data 1.
Figure 5 with 1 supplement
Intracellular leucine substitutions shift KCNE0 modulation toward a KCNE4-like inhibitory effect.
(A) Close-up view of the interface between KCNQ1 and KCNE0 within the PmKCNQ1–PmKCNE0–PmCaM complex structure generated by SwissModel server (Waterhouse et al., 2018). The model was built with amino acid sequences of PmKCNQ1 (XP_075921450.1), PmKCNE0 (XP_032831116.1), and PmCaM (XP_032811771.1), using the human KCNQ1–KCNE3–CaM structure (PDB: 6V00) as a template. Single KCNQ1, KCNE0, and CaM subunits are shown in blue, red, and green, respectively. The other regions are shown in gray. Residues used for motif-based mutagenesis are shown as sticks. Molecular graphics were prepared with CueMol (http://www.cuemol.org/). (B) Sequence alignment around the intracellular region corresponding to the KCNE4-related juxtamembrane tetra-leucine motif (Ciampa et al., 2011). (C–F) Representative current traces (C,D), conductance–voltage (G–V) relationship (E), and current amplitude at +60 mV (F) of PmKCNQ1 WT co-expressed with PmKCNE0 WT, PmKCNE0 H75L, or PmKCNE0 P73L/F74L/H75L. In (E), error bars denote mean ± s.e.m. for n=5. In (F), error bars denote mean ± s.e.m. for n=8. Statistical significance among the three constructs was assessed using one-way ANOVA followed by Tukey–Kramer multiple-comparison test. Significant differences are indicated by asterisks (***p<0.001). Source data are provided in Source data 1.
Figure 5—figure supplement 1
Attempted conversion of KCNE0 toward a KCNE1-like effect.
(A) Close-up view of the interface between KCNQ1 and KCNE0 within the PmKCNQ1–PmKCNE0–PmCaM complex corresponding to the structure shown in Figure 5A. (B) Sequence alignment around the KCNE transmembrane segments highlighting the ‘triplet’ region (Melman et al., 2001; Melman et al., 2002). (C, D) A representative current trace (C) and conductance–voltage (G–V) relationship (D) of PmKCNQ1 WT co-expressed with the HsKCNE1-like triplet mutant of PmKCNE0 (PmKCNE0 L54T). In (D), error bars denote mean ± s.e.m. for n=5. Source data are provided in Source data 1.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Lethenteron camtschaticum) | Arctic lamprey | Commercial source | RRID:NCBITaxon_980415 | Commercially obtained Arctic lamprey originally captured in the Ishikari River (Hokkaido, Japan) |
| Strain, strain background (Xenopus laevis, female) | Xenopus laevis frogs | Hamamatsu Seibutsu Kyouzai; http://www.h-seibutsu.co.jp/ | RRID:NCBITaxon_8355 | Used for oocyte expression experiments |
| Recombinant DNA reagent | pGEMHE vector | Liman et al., 1992 | Expression vector for cRNA synthesis | |
| Other | Alexa Fluor 488 C5 maleimide | Thermo Fisher Scientific | Cat# A10254 | VCF labeling |
| Commercial assay or kit | HiScribe T7 ARCA mRNA Kit | New England Biolabs | Cat# E2065S | cRNA synthesis |
| Commercial assay or kit | NucleoSpin RNA Plus | MACHEREY-NAGEL | Cat# 740984 | RNA extraction |
| Commercial assay or kit | PrimeScript II | TaKaRa | Cat# 6210 | Reverse transcription |
| Software, algorithm | pCLAMP 10.7 | Molecular Devices | RRID:SCR_011323 | Electrophysiology acquisition and analysis |
| Software, algorithm | Igor Pro | WaveMetrics | RRID:SCR_000325 | VCF analysis |
| Software, algorithm | fastp | Chen et al., 2018; Chen, 2025 | RRID:SCR_016962 | RNA-seq preprocessing |
| Software, algorithm | FastQC | Babraham Bioinformatics | RRID:SCR_014583 | Quality control |
| Software, algorithm | MultiQC | Ewels et al., 2016 | RRID:SCR_014982 | QC summary |
| Software, algorithm | Salmon | Patro et al., 2017 | RRID:SCR_017036 | Transcript quantification |
| Software, algorithm | CueMol | http://cuemol.org/en/ | RRID:SCR_019052 | Molecular graphics |
Additional files
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Supplementary file 1
Summary of fastp quality control.
Quality-control statistics generated by fastp for all RNA-seq runs. For each run, total reads before and after trimming (million reads), percentage of reads retained after trimming (%), mean read length of R1 and R2 before and after trimming (bp), and the percentage of bases with Phred quality ≥30 (Q30) before and after trimming (%) are shown. ‘Reads’ denote paired-end fragments.
- https://cdn.elifesciences.org/articles/111781/elife-111781-supp1-v1.xlsx
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Supplementary file 2
Quantification summary using Salmon.
Summary of transcript detections for Far Eastern brook lamprey KCNQ1 (XM_061564454.1) and KCNE0 (XM_061575561.1) using Salmon. For each run, the number of detected fragments, total processed fragments, and CPM (counts per million of the library) are shown. ‘Fragments’ denote paired-end read pairs (one read pair counted as one fragment).
- https://cdn.elifesciences.org/articles/111781/elife-111781-supp2-v1.xlsx
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Supplementary file 3
Summary of electrophysiological parameters and V1/2 statistical comparisons.
Summary of electrophysiological parameters for all constructs. For each condition, maximum tail current amplitude (Imax) and Boltzmann fit parameters (V1/2 and z) are reported. For VCF experiments, parameters from single- or double-Boltzmann fits are reported as appropriate. Values are mean ± s.e.m.; n indicates the number of oocytes. In the statistical comparisons, p-values indicate pairwise comparisons of G–V V1/2 values. For datasets containing more than two groups (Figures 2G and 3F and N), one-way ANOVA was first performed, followed by Tukey–Kramer multiple-comparison tests. For two-group comparisons (Figure 4C, F, I, and L), an unpaired two-tailed Welch’s t-test was used. V1/2 values were not statistically compared when reliable Boltzmann fits could not be obtained because the G–V relationships were nearly saturated over the tested voltage range.
- https://cdn.elifesciences.org/articles/111781/elife-111781-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/111781/elife-111781-mdarchecklist1-v1.pdf
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Source data 1
Excel file with numerical electrophysiology data acquired in this study.
- https://cdn.elifesciences.org/articles/111781/elife-111781-data1-v1.xlsx
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Characterization of an early-diverging KCNE potassium-channel auxiliary subunit in the jawless vertebrate lamprey
eLife 15:RP111781.
https://doi.org/10.7554/eLife.111781.3
