List of features annotated for the collected IC sequences.

The labels are colored by their annotation category. Labels for Aquaporin 1 are shown as examples of each annotation label.

Distribution of human ICs across different families.

Each circle represents a human IC family, with the symbol at the center indicating its Group. The size of the circles is proportional to the number of sequences in that family, and the colored pies indicate the proportion of their TDL status as designated by IDG. The placement of the bubbles is based on the average co-ordinates of all the members within that family in a distribution of UMAP embeddings generated using protein embedding-based pairwise sequence alignments. An embedding-based sequence alignment approach was used to overcome the vast divergence of IC sequences with minimal sequence similarity. A family level abstraction was done to provide an intuitive view undeterred by the relationships of individual ICs across families. A detailed view of the full UMAP plot showing the placement of individual ICs is provided in Supplementary Figure 3 for reference. Auxiliary IC families at the bottom were not part of the embedding-based analysis due to the lack of a pore-containing domain, and thus placed arbitrarily at the bottom of the figure.

Orthology profiling of human ICs.

(A) Heatmap showing the percent of orthologs detected for each IC family within a given taxonomic lineage. The taxonomic groups are shown in the vertical axis with a tree on the left. Darker color represents a higher percentage of orthologs detected. Percentages were calculated as (total number of orthologs found for all ICs in a family)/(total number of organisms queried in the taxonomic lineage * number of sequences in the family) (B) Clustergram depicting the presence/absence of orthologous sequences of ICs across eukaryotic taxonomic lineages. ICs are clustered along the horizontal axis into 9 distinct clusters. Taxonomic groups are shown on the vertical axis. Each square in the heatmap is colored based on the orthology relationship found for a specific IC in a specific organism (black: one-to-one ortholog present, red: co-ortholog detected, brown: no orthology detected). (C) Results from the enrichment analysis performed on human ICs of each cluster. The x-axis shows the number of ICs in the cluster enriched for the GO term shown on the y-axis. The bars are colored based on their FDR values for the enriched term. For a full list of enriched terms, please refer to Supplementary Table 4.

Evolutionary analysis of CALHM reveals conserved pattern positions.

(A) A phylogenetic tree depicting the evolutionary relationships across all 6 CALHM members with orthologs across different taxa. (B) The conserved pattern positions conserved across all 6 CALHM members are shown as a weblogo with the red bars indicating the significance (longer bars indicate higher significance) of conservation and are mapped into a representative structure of human CALHM2. The four transmembrane helices are labeled S1-S4. Conserved residues that are targeted for mutation are highlighted using a asterisk symbol in the labels. (C) Schematic representing the location of transmembrane regions and identified conserved pattern positions in a representative human CALHM2 sequence. The residues targeted for mutations are labelled with their corresponding positions for CALHM2, 1 and 6 shown in the labels. (D) Cartoon representation of CALHM2 structure (PDB ID: 6uiv and 6uiw) in open and closed conformation, respectively. (E) List of disease variants and mutations performed in the conserved pattern positions for functional studies.

Electrophysiological studies of human CALHM1 and CALHM6.

Whole-cell voltage-clamp recordings were performed in tsA cells overexpressing wild-type CALHM1 (A–D) and wild-type CALHM6 (E–H), as well as in non-transfected cells (I–L). Currents were measured under three conditions sequentially from the same cell: 5 mM Ca2+ at 22 °C (a, e, i; black), 0 mM Ca2+ at 22 °C (B,F,J; blue), and 0 mM Ca2+ at 37 °C (C,G,K; red). Voltage steps ranged from −100 mV to +140 mV, followed by a final tail pulse at −100 mV, with a holding potential of 0 mV (protocol illustrated in the box on the right). Current-voltage (I–V) relationships were plotted in D, H, and L using mean current amplitudes (averaged across independent cells) measured at the end of the voltage steps. The arrow indicates the time point at which current amplitudes were measured. Error bars represent SEM (D, n=5; H, n=5; L, n=5)

Functional characterization of CALHM1 and CALHM6 mutants at conserved residues at 37 °C.

Whole-cell voltage-clamp recordings were performed in tsA cells overexpressing wild-type CALHM1 (A-C), CALHM1 mutant (I109W) (D-F), wild-type CALHM6 (G-I), and CALHM6 mutants (Y51A (J-L)and W113A(M-O)). Currents were measured under two conditions: 0 mM Ca²LJ at 37 °C (A,D,G,J,M; red) and 0 mM Ca²LJ at 37°C plus 100 µM Gd³LJ (B,E,H,K,N; blue), with both conditions recorded sequentially from the same cell. Voltage steps ranged from −80 mV to +120 mV, followed by a final tail pulse at −80 mV, with a holding potential of 0 mV (protocol shown in the box on the right). Current-voltage (I–V) relationships were plotted in C,F,I,L, and O using mean current amplitudes (averaged across independent cells) measured at the end of the voltage steps. The arrow indicates the time point at which the current was measured. The number of independent measurements (cells) were: n = 5 (C); n = 5 (F); n = 5 (I); n = 5 (L); n=5 (O). Error bars represent SEM. (P,Q) Current amplitudes obtained using a two-step voltage protocol (from +120 mV to −80 mV; protocol shown in the box on the right) are compared between wild-type CALHM1 and its mutants (P) and between wild-type CALHM6 and its mutants (Q). Each dot represents an independent measurement (cell), and bar represents the mean current amplitude across cells. The number of independent measurements (cells) for each bar in P and Q are shown from left to right: 5, 8, 5, 8, 6, 7, 5, 5, 5, 6, 7, 7 (P); 5, 6, 5, 7, 7, 5, 6, 6 (Q). Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test, comparing each mutant to wild type (*p < 0.05; **p < 0.01; ***p < 0.001).

RAG annotation pipeline used for validating the annotation of ion specificity and gating mechanism

Upset plot showing the overlap of IC sequences based on their UniProt IDs across the current study, the KEGG database, GtoP and Pharos.

UMAP embeddings plot of all human IC sequences.

The UMAP embeddings were generated based on the pairwise similarity scores of the protein embedding alignment generated for all pairs of human ICs. Shapes of the markers indicate the different IC groups (circle: VGIC, square: LGIC, diamond: Chloride channel, triangle: Other, plus: Unclassified) and the colors indicate different IC families within each group as shown in the legend above.

Expression analysis of wild-type CALHM1, wild-type CALHM6, and their mutants.

(A,B) Representative gels showing CALHM1 and its mutants (A), and CALHM6 and its mutants (B). CALHM1 and CALHM6 signals were detected using in-gel fluorescence of the C-terminal GFP tag, while β-actin was detected by Western blotting as a loading control. (C,D) Quantification of total protein expression levels of wild-type CALHM1 and its mutants (C), and wild-type CALHM6 and its mutants (D). Each dot represents an independent measurement (transfection) and error bars represent SEM (c, n = 3; d, n = 3). Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test, comparing each mutant to wild type (*p < 0.05; **p < 0.01; ***p < 0.001). (E,F) Surface biotinylation assays of wild-type CALHM1 and its mutants (E), and wild-type CALHM6 and its mutants (F), detected using the C-terminal GFP tag by in-gel fluorescence.

Functional characterization of CALHM1 and CALHM6 mutants at conserved residues at 22 °C.

Current amplitudes obtained using a two-step voltage protocol (from +120 mV to −80 mV; protocol shown in the box on the right) are compared between wild-type CALHM1 and its mutants (A, B) and between wild-type CALHM6 and its mutants (C, D). The cells analyzed here are the same as those in Figure 5P, Q. Briefly, for each cell, currents were measured sequentially under three conditions: 5 mM Ca2+ at 22 °C, 0 mM Ca2+ at 22 °C, and 0 mM Ca2+ at 37 °C. The currents from the first two conditions are plotted here, while currents at 0 mM Ca2+ at 37 °C are shown in Figure 5R, S. Each dot represents an independent measurement (cell), and bar represents the mean current amplitude across cells. The number of independent measurements (cells) for each bar in a–d are shown from left to right: 5, 8, 5, 8, 6, 7, 5, 5, 5, 6, 7, 7 (A, B); 5, 6, 5, 7, 7, 6, 6, 5 (C, D). Statistical analysis was performed using one-way ANOVA with Bonferroni’s post hoc test, comparing each mutant to wild type (*p < 0.05; **p < 0.01; ***p < 0.001).

Conserved pattern positions identified within the clade for CALHM2,4,5 and 6.

A) Phylogenetic tree of CALHM sequences where the orange star indicates the clade for CALHM2,4,5 and 6. B) The identified pattern positions are mapped into a representative structure of human CALHM2 (PDB: 6uiw). C) Weblogo showing the conserved pattern positions. The red bar indicates the significance of conservation where a taller bar indicates higher significance.