Evolution and functional divergence of the anoctamin family of membrane proteins

Abstract

Background: The anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family.

Results: Our results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins.

Conclusions: Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family.

Figure 1

Maximum likelihood tree of the anoctamin protein family. The phylogenetic tree constructed with the program PhyML shows the evolutionary relationship of the anoctamin protein family. Several possible duplication time points are indicated with black arrows. Non-vertebrate anoctamins are depicted with red color. The unit of branch length is the expected fraction of amino acids substitution.

Figure 2

Average hydropathy plot of 166 homologues of vertebrate bestrophins. Hydropathy plot was generated from 166 vertebrate sequences as given in Additional file 1 using TMAP server which predicts transmembrane segments from an aligned set of proteins. Amino acid numbering corresponds to the numbers from the multiple sequence alignment. Black boxes depict predicted TMD's. RL = re-entry loop

Figure 3

Evolution of the protein domains in the anoctamin protein family. A, Schematic representation of protein domains of anoctamin proteins in vertebrates. TMD, transmembrane domain; RL, re-entry loop; PDZ, PDZ domain, N-Gly, N-glycosilation site; PKA, protein kinase A phosphorylation site; cNMP, cyclic nucleotide-monophosphate binding domain. B, Amino acid sequence alignment of the representative anoctamin protein members containing putative cNMP binding site.

Figure 4

Site specific profiles for evolutionary rate changes in the vertebrate anoctamin protein family. A, The posterior probabilities of functional divergence for vertebrate anoctamins ano1, ano2 and ano4 were obtained with Diverge. Individual cut-off values for each comparison are marked with red horizontal lines. B, Residues with predicted functional divergence between anoctamin subfamilies are mapped onto the membrane topology model of ano 1.

Figure 5

Ka/Ks ratios and anoctamin 1 protein structure. The results of Ka/Ks analysis on multiple alignment of ano1 proteins. Above the alignment, amino acids divergent between ano1, ano2 and ano4, are depicted with asterisk. Below the alignment is a histogram of the Ka/Ks ratios for each ungapped column of the alignment. IN/OUT indicates orientation with respect to the plasma membrane. Alignment shading indicates alignment quality.