ABSTRACT
Recent investigations of rodent Tmem163 suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two species-conserved aspartate (D123A/D127A) residues perturbs its function. Direct corroboration, however, is lacking whether it is an influx or efflux transporter in cells. We hypothesized that human TMEM163 is a zinc effluxer based on its predicted protein characteristics. We used cultured human cell lines that either stably or transiently expressed TMEM163 and pre-loaded the cells with zinc to determine transport activity. We found that TMEM163-expressing cells exhibited significant reduction of intracellular zinc levels as evidenced by two zinc-specific fluorescent dyes and radionuclide zinc-65. The specificity of the fluorescence signal was confirmed upon treatment with TPEN, a high-affinity zinc chelator. Radionuclide zinc-65 assay revealed that the efflux activity of TMEM163 has an apparent Km value of 8 micro-molars. To further characterize the efflux function of TMEM163, we substituted alanine into two conserved aspartate residues (D124A/D128A) previously proposed as zinc binding sites. We also performed site-directed mutagenesis of several conserved amino acid residues identified as non-synonymous single nucleotide polymorphism (S61R, S95C, S193P, and E286K). We found a significant reduction of zinc efflux upon cellular expression of D124A/D128A or E286K protein variant when compared with wild-type, suggesting that these particular amino acids are important for normal protein function. Taken together, our findings demonstrate for the first time that TMEM163 effluxes zinc and is a new member of the cation diffusion facilitator (CDF) family that contributes to zinc homeostasis in specific cell types.
Footnotes
New data were added to support the findings that TMEM163 is a zinc efflux transporter such as the use of radionuclide zinc-65 assay. Michaelis-Menten kinetics of zinc efflux transport by TMEM163 was calculated to be 8 micromolars. Data to support the association of TMEM163 with other cation diffusion facilitator (CDF) family of zinc transporters were also included such as multiple sequence alignment and phylogenetic tree diagram. Cell surface biotinylation and Western blot revealed that some protein variants of TMEM163 (non-synonymous single nucleotide polymorphism) have altered plasma membrane localization and post-translational modification.