NHE9 variants S438P and L236S, identified in autism patients with and without co-morbid epilepsy respectively, consistently scored as loss of function mutations in both yeast and astrocyte models. This validates predictions from the structural model placing them within highly conserved transmembrane regions of a helical bundle, central to the ion transport mechanism of the bacterial orthologs NhaA and NhaP35,48–50. In contrast, the Val176Ile variant, found in a patient without seizures, lies in a more variable region peripheral to the transport domain. While it was phenotypically silent in the simpler yeast cell, functional deficits were uncovered in astrocytes suggesting additional roles in the mammalian protein, possibly via protein interactions. For example, CHP and RACK1, two non-selective binding partners of other NHE isoforms, were also shown to bind NHE9 in a heterologous system, although the functional relevance of the interaction was not established30. The bacterial and eukaryotic homologues vary significantly in their N-terminal sequences up to and including the third transmembrane helix, and the mammalian protein structure may differ from the NhaA template in this region. In this respect it is noteworthy
