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Transmembrane (TMEM)‐163 protein is a novel zinc transporter
Author(s) -
Cantrell Quinlan W,
Silva Joshua,
Nguyen Cathleen,
Hildebrand Leanne D,
Rivas Tiffany,
Shoemaker Ryan,
Rojas America,
Cuajungco Math P
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.878.10
Subject(s) - hek 293 cells , complementary dna , microbiology and biotechnology , transfection , biology , endosome , chemistry , transport protein , transporter , gene , solute carrier family , intracellular , biochemistry
A mutation or deletion of the MCOLN1 gene which codes for the Mucolipin‐1 (TRPML1) ion channel protein causes the human lysosomal storage disorder called Mucolipidosis IV (MLIV). An elevation of chelatable zinc, particularly in lysosomes, is observed in cells affected by MLIV compared to normal cells. The mechanism behind the zinc buildup in MLIV cells remains elusive; however, evidence suggests that it is due to the failure of non‐functional TRPML1 to mediate the release of zinc from the lysosomes or failure in lysosomal exocytosis. We recently identified TMEM163 as an interacting partner of TRPML1. We hypothesize that TMEM163 is a transporter that facilitates zinc flux in normal cells, but that the loss of TRPML1 function in MLIV cells leads to abnormal TRPML1‐TMEM163 protein interaction such that the cells uncontrollably accumulate zinc. To infer function as a zinc transporter, we first studied the membrane topology of heterologously expressed TMEM163 protein. Contrary to its predicted orientation, we found that both the N‐ and C‐terminus regions of TMEM163 lie at the extracellular rather than intracellular milieu. To test functional similarities between TMEM163 and several known zinc transporters (ZNTs), we cloned the cDNA of TMEM163 and specific ZNTs into the pBI‐CMV dual‐expression construct (containing a minimal CMV promoter vector). Single cDNA constructs were created as controls. The single gene and dual gene constructs were then heterologously expressed in human embryonic kidney (HEK)‐293 cells. Twenty‐four hours post‐transfection, treated and control HEK‐293 cells were then exposed to zinc chloride (ZnCl 2 , 100 mM) for 3 and 5 hours. Spectrofluorometric end‐point assay using the zinc‐specific Fluozin‐3 AM dye showed that the single TMEM163 expression in cells produces intracellular zinc accumulation upon 5 hours of ZnCl 2 exposure, while co‐expression of TMEM163 with ZNT4, but not other ZNTs, showed a significant synergistic effect on intracellular zinc levels at both time points tested. Overall, these results suggest that TMEM163 may serve a similar role as those of the ZNTs in transporting zinc, but that it is an influxer. Future studies relating to TMEM163‐TRPML1 protein interaction should provide new research opportunities to further understand the involvement of zinc in the underlying neuropathology and progressive cell degeneration observed in MLIV disease. Support or Funding Information NIH R15‐NS070774, NSF MCB‐0920127, and NIH T34‐GM008612‐20