Cellular zinc levels are influenced by the interaction between the mucolipidosis IV‐associated TRPML1 protein and TMEM163 protein (996.5)

Abstract

Dysfunctional Mucolipin‐1 (TRPML1) ion channel protein causes the human lysosomal storage disorder Mucolipidosis type IV (MLIV). Cells affected by MLIV have significantly higher intracellular zinc levels compared to normal cells; however, the mechanism of zinc elevation remains unknown. Using a membrane‐based yeast two‐hybrid technique, we identified a putative zinc‐binding protein called transmembrane‐163 protein (TMEM163) as an interacting partner for TRPML1. We confirmed interaction by co‐immunoprecipitation and mass spectrometry. We hypothesized that the interaction between TMEM163 and TRPML1 plays a role in regulating TMEM163, and thus intracellular zinc. RNA interference targeting both TMEM163 and TRPML1 in SH‐SY5Y neuroblastoma cells resulted in significant increase of intracellular zinc when both proteins were knocked down. To determine how interaction of TRPML1 and TMEM163 could be responsible in regulation of intracellular zinc, we co‐expressed and analyzed expression of tagged constructs of both proteins using human embryonic kidney (HEK)‐293 cells. Evidence from cell surface biotinylation showed that co‐expressed TRPML1 resulted in reduced TMEM163 localized within the plasma membrane of HEK‐293 cells. This result possibly explains the reduction of cellular zinc flux when TRPML1 is co‐expressed with TMEM163. Overall, our data suggest that both TRPML1 and TMEM163 proteins play a role in intracellular zinc regulation. Thus, the loss of TRPML1 protein function and/or the reduction of TMEM163 protein could result in zinc dyshomeostasis in MLIV.Grant Funding Source: Supported by NIH R15‐NS070774 and CSUF Intramural Grants