Impaired posttranslational processing and trafficking of an endosomal Na super(+)/H super(+) exchanger NHE6 mutant ( Delta super(370)WST super(372)) associated with X-linked intellectual disability and autism

Na super(+)/H super(+) exchanger NHE6/SLC9A6 is an X-linked gene that is widely expressed and especially abundant in brain, heart and skeletal muscle where it is implicated in endosomal pH homeostasis and trafficking as well as maintenance of cell polarity. Recent genetic studies have identified several mutations in the coding region of NHE6 that are linked with severe intellectual disability, autistic behavior, ataxia and other abnormalities. One such defect consists of an in-frame deletion of three amino acids ( super(370)Trp-Ser-Thr super(372), Delta WST) that adjoin the predicted ninth transmembrane helix of the exchanger. To better understand the nature of this mutation, a NHE6 Delta WST construct was generated and assessed for its effects on the biochemical and cellular properties of the transporter. In transfected fibroblastic CHO and neuroblastoma SH-SY5Y cells, immunoblot analyses showed that the mutant protein was effectively synthesized, but its subsequent oligosaccharide maturation and overall half-life were dramatically reduced compared to wild-type. These changes correlated with significant accumulation of Delta WST in the endoplasmic reticulum, with only minor sorting to the plasma membrane and negligible trafficking to recycling endosomes. The diminished accumulation in recycling endosomes was associated with a significant decrease in the rate of endocytosis of cell surface Delta WST compared to wild-type. Furthermore, while ectopic expression of wild-type NHE6 enhanced the uptake of other vesicular cargo such as transferrin along the clathrin-mediated recycling endosomal pathway, this ability was lost in the Delta WST mutant. Similarly, in transfected primary mouse hippocampal neurons, wild-type NHE6 was localized in discrete puncta throughout the soma and neurites, whereas the Delta WST mutant displayed a diffuse reticular pattern. Remarkably, the extensive dendritic arborization observed in neurons expressing wild-type NHE6 was noticeably diminished in Delta WST-transfectants. These results suggest that deletion of super(370)Trp-Ser-Thr super(372) leads to endoplasmic reticulum retention and loss of NHE6 function which potentially impacts the trafficking of other membrane-bound cargo and cell polarity.