Structural Biology and Molecular Biophysics

Characterization of an early-diverging KCNE potassium-channel auxiliary subunit in the jawless vertebrate lamprey

Go Kasuya author has email address
Kaei Ryu
Buntaro Zempo
Emi Kawano-Yamashita
Koichi Nakajo author has email address

Division of Integrative Physiology, Department of Physiology, Jichi Medical University, Shimotsuke, Japan
Department of Chemistry, Biology, and Environmental Science, Faculty of Science, Nara Women’s University, Nara, Japan

https://doi.org/10.7554/eLife.111781.1

Open access
Copyright information

Figures and data

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 Omega⁶¹ and displayed using ESPript3⁶². Residues corresponding to the “triplet”^42,43 motif are highlighted with an orange square. (D) Genomic region containing the kcne locus in sea lamprey, shown from the Ensembl genome browser⁶³. 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 Supplementary Fig. 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)¹⁰ 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.

Biophysical properties of lamprey KCNQ1 and modulation by KCNE0.
(A–G) Representative current traces (A–F) and 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 F–V relationship (J) of PmKCNQ1_vcf WT expressed alone or co-expressed with PmKCNE0 WT. Error bars denote mean ± s.e.m. for n = 5 in (G,J).

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 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).

Species dependence of KCNQ1–KCNE compatibility.
(A–C) Representative current traces (A,B) and 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).

Intracellular leucine substitutions shift KCNE0 modulation toward to 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 server⁶⁴. 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. One KCNQ1, KCNE0 and CaM subunit are shown in blue, red, and green. 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 motif⁴¹. (C–F) Representative current traces (C,D), 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. Error bars denote mean ± s.e.m. for n = 5 in (E,H,I). 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).

ORF nucleotide and corresponding amino-acid sequence of KCNQ1 in Arctic lamprey.
The open reading frame (ORF) nucleotide sequence and the translated amino-acid sequence of Arctic lamprey KCNQ1 used for cloning and electrophysiological recordings.

ORF nucleotide and corresponding amino-acid sequence of KCNE0 in Arctic lamprey.
The open reading frame (ORF) nucleotide sequence and the translated amino-acid sequence of Arctic lamprey KCNE0 used for cloning and electrophysiological recordings.

Uncropped agarose gel corresponding to the gel image shown in Figure 1F.

Amino-acid sequence alignment of KCNQ1 from various species.
Amino acid sequence alignment of KCNQ1 from various human, zebrafish, sea lamprey, and vase tunicate generated with Clustal Omega⁶¹ and displayed with ESPript3⁶². The residue corresponding to the labeling site in human KCNQ1 (G219)^29,30 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), and vase tunicate (CiKCNQ1; NP_001153537.1) KCNQ1 were used.

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 Fig. 5A (B) Sequence alignment around the KCNE transmembrane segments highlighting the “triplet” region^42,43. (C,D) A representative current trace (C) and G-V relationship (D) of PmKCNQ1 WT co-expressed with the HsKCNE1-like triplet mutant of PmKCNE0 (PmKCNE0 L54T).

Sign up for email alerts