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 ICs across different families.

Each circle represents an 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 studied status as designated by IDG. Families are placed based on their distribution of UMAP embeddings generated using protein embedding-based pairwise sequence alignments (Supplementary Figure 2).

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 3.

Evolutionary analysis of CALHM reveals conserved pattern positions.

A) A phylogenetic tree depicting the evolutionary relationships across all 6 CALHM paralogs with representative sequences from humans, mouse and rat. B) Schematic representing the location of transmembrane regions and identified conserved pattern positions in a representative human CALHM2 sequence. C) The conserved pattern positions conserved across all 6 CALHM paralogs are shown as a weblogo with the red bars indicating the significance (longer bars indicate higher significance) and are mapped into a representative structure of human CALHM2. 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.

A,C,E. Representative current traces in the presence of 5 mM Ca2+ in the bath solution at 22°C (left), 0 mM Ca2+ at 22°C (middle), and 0 mM Ca2+ at 37°C (right) from non-transfected tsA201 cells (A), or tsA201 cells overexpressing wild-type CALHM1 (C), and wild-type CALHM6 (E), recorded in the whole-cell patch-clamp configuration. Voltage clamps were applied from −100 mV to 140 mV (200 ms each step) with a final tail pulse at −100 mV (200 ms), with a holding potential of 0 mV. B,D,F. The mean current amplitudes at the end of each pulse step of the experiments in (A,C,E) were plotted as a function of clamp voltage. Error bars represent SEM (CALHM1: n=5, CALHM6: n=3).

Current amplitudes of wild-type CALHM1, wild-type CALHM6, and mutants at conserved pattern positions in human CALHM1 and CALHM6.

Currents were recorded at 120 mV in the presence of 5 mM Ca2+ in the bath solution at 22°C, 0 mM Ca2+ at 22°C, and 0 mM Ca2+ at 37°C. Bars represent the mean. One-way ANOVA with Bonferroni’s post hoc test was used for statistical analysis (*p<0.05. **p<0.01, ***p<0.001).