Functional Evaluation of Autism-Related Mutations in the Na+ (k+)/ H+ Exchanger NHE9

A number of neurological disorders including autism, have been linked to mutations in a subgroup of intracellular Na+(K+)/H+ exchangers, which include NHE6 and NHE9 that localize to recycling and late endosomes respectively. These are thought to mediate the electrically silent counter transport of cations with protons. Yeast ortholog, Nhx1, has been shown to regulate endosomal pH and control vesicular trafficking. Here, we examine the functional consequence of missense mutations in NHE9 associated with autism, using yeast as a first approach. Further, we extend our observations from yeast to a glial cell model, providing the first insight into the neurobiological basis of NHE dysfunction.Previously, a 3-dimensional model of the human NHE1 was constructed using the crystal structure of NhaA, Na+/H+ antiporter from Escherichia coli, as template. As the endosomal NHE display significant similarity to NHE1, we used the NHE1 model as a template and used a unique modeling approach to model these other human transporters. Three NHE9 mutations (S438P, L236S, V176I) could be readily extrapolated to yeast Nhx1 from sequence conservation. We used site-directed mutagenesis to replace the yeast residues with the human equivalent, as well as the disease-associated mutation. Mutants were expressed in nhx1 deletion background and evaluated for pH, salt and trafficking phenotypes. While S43P and L236S mutations led to loss of function phenotypes in yeast, unexpectedly, V167I had no discernable effect. The latter may reveal isoform specific functions that remain to be identified or may call to question the functional contribution to the disease phenotype. In a parallel approach, these NHE9 mutations were expressed in glial cells for functional evaluation in a mammalian cell model. We observed altered trafficking and surface expression of glutamate transporters, consistent with elevated glutamate levels reported in patient brains.