Three autism-associated substitutions, namely V176I, L236S and S438P (Figures 1A, 2A), reside within the membrane domain of NHE9, in positions that are evolutionarily conserved among all eukaryotic transporters. A fourth mutation, P117T, is localized to a highly variable extracellular loop and could not be modeled with certainty in the yeast ortholog (Figure 1A). Other substitutions resided outside the homology region, including the C-terminal hydrophilic tail. As seen in Figure 2A, L236 and S438 are highly conserved in all eukaryotic and prokaryotic homologues and buried in the protein core, whereas V176 is only moderately conserved and faces the lipid bilayer. Furthermore, the autism-associated variants L236S and S438P change the physicochemical nature and stereochemistry of the amino acid side chains significantly whereas the V176I change is moderate.
