(A) Molecular phylogeny of the Ca²⁺ channel γ subunit family. The alignment used to infer the tree was done independently for full-length proteins and for conserved regions alone (defined as translation start to the final residue of the fourth predicted transmembrane domain), and resulted in identical topologies. The number of trees with a particular node among 10,000 bootstrap replicates is indicated at the node (values for conserved domain trees shown above the horizontal line and for full length proteins below). A member of the related Claudin protein family, Claudin 4, was included in the comparison and defined as the out-group. Branch lengths are arbitrary and do not correspond to genetic distances. This tree is unrooted. (B) Pair-wise amino acid identity among the Ca²⁺ channel γ_1–5 subunits and mouse Claudin 4. Percent identity determined after alignment by the BLAST2 program is shown above the horizontal and by the ALIGN program below the horizontal. Homology between Claudin 4 and the γ subunits was below the threshold of detection by BLAST2. (MC4) Mouse Claudin 4; HG1, 3, 4, and 5 refer to human Ca²⁺ channel γ subunits; MG2 refers to the mouse γ₂ subunit; (n.a.), not applicable.