Synaptic function is modulated by targeting and recycling of transporters and receptors to and from the astrocyte cell surface45. In the yeast model, Nhx1-mediated regulation of endosomal pH is critical for cell surface expression and turnover of membrane proteins. Therefore, we investigated if NHE9 knockdown or overexpression similarly altered the function and expression of cell surface receptors and transporters in astrocytes. Steady state levels of fluorescence-tagged transferrin were significantly elevated (by 1.75-fold) in astrocytes overexpressing NHE9 (Figure 8A, C), with corresponding stabilization of the transferrin receptor, observed upon blocking protein synthesis by cycloheximide addition (Figure 8B). Although internalized transferrin levels were not decreased upon knockdown of endogenous NHE9, treatment with shRNA effectively reversed the elevation seen in NHE9 overexpressing cells (Figure 8C). We used this gain of function phenotype to assess the three autism-associated variants in NHE9. GFP-tagged variants, L236S, S438P and V176I, were expressed at levels equivalent to wild type NHE9 in primary astrocytes (Figure 7A) and individually colocalized with Alexa Fluor-labeled transferrin (Figure 7B). After incubation with Alexa-633-transferrin for 1hr at 37°C, none of the three variants displayed
