Evolutionary Analysis of Vertebrate KCNH Voltage-Gated Potassium Channels and Spatial Expression of kcnh Genes in Zebrafish Embryos

Figures

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  Figure 1. Alignment of zebrafish Kcnh channel P-loop and S4 domains. Zebrafish Kcnh channels possess one P-loop and six transmembrane domains. Only sequences around the P-loop and S4 are shown. The P-loop and the potassium pore domains (T-X-G-Y/F-G) are boxed in a black line. The S6 transmembrane domain next to the filter domain is also boxed in a gray line.

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  Figure 2. The zebrafish KCNH channels have protein structures similar to those of human KCNH1. (A) Human KCNH1 subunit structure from an experiment on RCSB PDB. (B) Human KCNH1 structure predicted by AlphaFold. (C–P) Predicted zebrafish Kcnh subunit protein structures. Models are color-coded by pLDDT confidence scores (dark blue: pLDDT > 90; light blue: 90 > pLDDT > 70; yellow: 70 > pLDDT > 50; and orange: pLDDT < 50). There is no PAS domain in zebrafish Kcnh6b. (Q) Illustration of the KCNH tertiary structure, modified from the reference [38]. The KCNH channel is a tetramer, but only two subunits are shown here. cNBHD (cyclic nucleotide-binding homology domain) is illustrated as a blue oval. The light blue circle indicates the intrinsic ligand-binding site. PAS, Per-Arnt-Sim domain. S1–S6, transmembrane domains.

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  Figure 3. Maximum likelihood phylogeny of vertebrate KCNH channels. Representative species (human, rat, mouse, chicken, turkey, frog, snake, turtle, coelacanth, zebrafish, tilapia, platy fish, stickleback, medaka, spotted gar, shark, lancelet, tunicate, roundworm, and fruit fly) were analyzed. Numbers at each node indicate the supporting values based on 1000 bootstrap replicates. Branch lengths are proportional to the expected number of replacements per site. The phylogeny was inferred with the JTT model plus gamma distribution using RaxML (8.2.12). The three subtypes of Kcnh channels are grouped together and indicated with different colored shades. The zebrafish Kcnh channels are highlighted in blue. The channel protein name is labeled on the edge of the phylogenetic tree. The protein sequence reference number is included in the phylogenetic tree to avoid confusion when genes are duplicated. The duplicated channels are indicated by black lines next to their names, ending with a and b.

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  Figure 4. The syntenic analyses of the duplicated kcnh genes in representative vertebrate species. (A) Vertebrate kcnh2 synteny: traf5-hhat-kcnh1-rcor3-hhat. The zebrafish kcnh1a gene is linked to hhat, as in sharks and humans. (B) The vertebrate kcnh5 synteny is relatively stable in shark, spotted gar, human, and mouse species. In zebrafish, this conserved synteny is split into two short ones with kcnh5a and kcnh5b. (C) The kcnh2 synteny, map4-sspo-aoc1-kcnh2, is kept well in zebrafish and electric eel compared to the spotted gar. Only map4l is linked with kcnh2b in both zebrafish and electric eel. The human KCNH2 is linked only to AOC1, whereas the mouse Kcnh2 is not linked to any other genes in the spotted gar synteny. (D) Vertebrate kcnh6 synteny, kcnh6-dcaf7-taco1. In zebrafish, kcnh6a is linked to dcaf7, and kcnh6b is linked to taco1. (E) The vertebrate kcnh7 synteny is relatively long and has 11 genes in the elephant shark and spotted gar genomes. The kcnh7a gene is linked to the majority member of the synteny, while the kcnh7b gene is linked to two or three genes in both zebrafish and tilapia. (F) The vertebrate kcnh4 synteny is also relatively conserved. The zebrafish kcnh4a gene…

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  Figure 5. The kcnh1a, kcnh1b, kcnh5a, and kcnh5b gene (EAG channel) expression during zebrafish embryogenesis. Whole-mount in situ hybridization of zebrafish embryos at 15-somite stage, 15S (A,E,I,M,Q,U,Y,CC), 24 hpf (B,F,J,N,R,V,Z,DD), 48 hpf (C,G,K,O,S,W,AA,EE), and 72 hpf (D,H,L,P,T,X,BB,FF). The anterior is to the left in all the whole-mount images, and the dorsal is to the top in all transverse sections (B’,F’,J’,N’,R’,V’,Z’,DD’). The white dashed lines indicate the positions of sections left to the panel or transverse sections in the inserts of corresponding panels. The letters below or around the dashed lines correspond to the section panels. (A–D). Lateral view of gene expression of kcnh1a. (E–H). Dorsal view of gene expression of kcnh1a. (I–L). Lateral view of gene expression of kcnh1b. (M–P). Dorsal view of gene expression of kcnh1b. (Q–T). Lateral view of gene expression of kcnh5a. (U–X). Dorsal view of gene expression of kcnh5a. (Y–BB). Lateral view of gene expression of kcnh5b. (CC–FF). Dorsal view of gene expression of kcnh5b. Higher magnification (100×) of Rohon–Beard neurons is shown in the inserts of corresponding panels (B,J,R). Scale bars (250 μm) are at the bottom row…

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  Figure 6. Gene expression of the kcnh2a, kcnh2b, kcnh6a, kcnh6b, kcnh7a, and kcnh7b genes (ERG channel) during zebrafish embryogenesis. Whole-mount in situ hybridization of zebrafish embryos at 15S stage (A,E,I,M,Q,U,Y,CC,GG,KK,OO,SS), 24 hpf (B,F,J,N,R,V,Z,DD,HH,LL,PP,TT), 48 hpf (C,G,K,O,S,W,AA,EE,II,MM,QQ,UU), and 72 hpf (D,H,L,P,T,X,BB,FF,JJ,NN,RR,VV). The anterior is to the left in all the whole-mount images, and the dorsal is to the top in all transverse sections (B’,F’,J’,N’,R’,V’,Z’,DD’,HH’,LL’,PP’,TT’). The white dashed lines indicate the positions of the sections to the left of the panel. The letters below or around the dashed lines correspond to the section panels. (A–D). Lateral view of gene expression of kcnh2a. (E–H). Dorsal view of gene expression of kcnh2a. (I–L). Lateral view of gene expression of kcnh2b. (M–P). Dorsal view of gene expression of kcnh2b. (Q–T). Lateral view of gene expression of kcnh6a. (U–X). Dorsal view of gene expression of kcnh6a. (Y–BB). Lateral view of gene expression of kcnh6b. (CC–FF). Dorsal view of gene expression of kcnh6b. (GG–JJ). Lateral view of gene expression of kcnh7a. (KK–NN). Dorsal view of gene expression of kcnh7a. (OO–RR). Lateral view…

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  Figure 7. The gene expression of the ELK channel genes kcnh3, kcnh4a, kcnh4b, and kcnh8 during zebrafish embryogenesis. Whole-mount in situ hybridization of zebrafish embryos at the 5S stage. (A,E,I,M,Q,U,Y,CC), 24 hpf (B,F,J,N,R,V,Z,DD), 48 hpf (C,G,K,O,S,W,AA,EE), and 72 hpf (D,H,L,P,T,X,BB,FF). The anterior is to the left in all the whole-mount images. The white dashed lines indicate the positions of the transverse sections in the inserts of corresponding panels (B,F). (A–D). Lateral view of gene expression of kcnh3. (E–H). Dorsal view of gene expression of kcnh3. (I–L). Lateral view of gene expression of kcnh4a. (M–P). Dorsal view of gene expression of kcnh4a. (Q–T). Lateral view of gene expression of kcnh4b. (U–X). Dorsal view of gene expression of kcnh4b. (Y–BB). Lateral view of gene expression of kcnh8. (CC–FF). Dorsal view of gene expression of kcnh8. Scale bars (250 μm) are located at the bottom row of images. dm, dermomyotome; e, eye; fb, forebrain, hb, hindbrain; hcrt, hypocretin (HCRT) neurons in the hypothalamus, mb, midbrain; n, neural tube; nt, notochord; ob, olfactory bulbs; oo, olfactory organ; op, optic vesicles; ov, otic vesicles; p, pallium; pg, pineal gland; rb,…

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  Figure 8. Model of vertebrate Kcnh gene evolution. Based on our phylogenetic and syntenic analysis, we proposed an evolutionary model for vertebrate potassium channels. (A) Whole-genome duplication events (1R–3R) in vertebrates. The horizontal blue bars indicate the WGD events. The vertebrate phylogenetic tree was illustrated based on the evolution of fibrillar collagens [26,27]. (B) The three Kv subtypes (EAG, ERG, and ELK) have already evolved in invertebrates. There is at least one gene for each subtype in most invertebrates. (C) Two consecutive WGDs (R1 and R2) happened at the dawn of vertebrates. So, all the Kcnh genes were doubled twice in the vertebrate ancestor. Some of the duplicated genes were lost after the two WGDs: 2 in the EAG subfamily, 1 in the ERT subfamily, and 1 in the ELK subfamily. (D) The teleost-specific WGD further reshaped the gene numbers in this group of animals. Most gene duplicates were maintained, except that one copy of kcnh3 and one copy of kcnh8 were lost in teleosts.

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  Figure 9. Schematic summary of the kcnh gene expression in 24–72 hpf zebrafish embryos. Only major embryonic structures are illustrated. The representative embryonic organs or tissues are color-coded. The kcnh genes expressed in the corresponding regions are listed in boxes of the same color. (A) At 24 hpf. (B) At 48 hpf. (C) At 72 hpf. The size of the illustration is not proportional to the actual zebrafish embryos.