Transmembrane 163 (TMEM163) protein effluxes zinc

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 proposed to bind zinc, perturbs protein 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. Multiple sequence alignment and phylogenetic analyses showed that TMEM163 is related to distinct members of the cation diffusion facilitator (CDF) protein family. To further characterize the efflux function of TMEM163, we substituted alanine in two homologous aspartate residues (D124A/D128A) and 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 that TMEM163 effluxes zinc, and it should now be designated ZNT11 as a new member of the mammalian CDF family of zinc efflux transporters.

Transmembrane 163 (TMEM163) Protein: A New Member of the Zinc Efflux Transporter Family

“…Nevertheless, the tissue expression of TMEM163 is consistent with tissues that also express other ZNT proteins (Cuajungco & Kiselyov, 2017). It is worth noting that the relative mRNA tissue expression of mouse Tmem163 parallels that of its human counterpart (Sanchez et al, 2019), despite the initial claims that the gene is only exclusively expressed in the mouse brain (Barth et al, 2011;Burré et al, 2007). At the cellular level, TMEM163 localizes to the plasma membrane and membrane-bound compartments such as lysosomes, endosomes, and synaptic vesicles (Burré et al, 2007;Cuajungco et al, 2014).…”

“…In a recent paper, it was revealed that TMEM163 modulates the activity P2X3 receptor (Salm et al, 2020) and while this function helps to show an increasing importance of TMEM163, a second finding in this study relates to the SLC30 family. It was found that TMEM163 was able to alter the expression of the P2X3R protein (Salm et al, 2020), which could further solidify the phylogenetic relationship between ZNT9 and TMEM163 proteins (Sanchez et al, 2019), since ZNT9 has been demonstrated to influence and regulate specific transcriptional targets (Chen, Kim, & Stallcup, 2005). It would be interesting to further prove the hypothesis that ZNT9 and TMEM163 share a similar function in cells that impact normal and pathological states in human health.…”

“…Transmembrane 163 protein (TMEM163), recently characterized as a zinc efflux protein (Sanchez, Ali, Escobar, & Cuajungco, 2019), is also known as synaptic vesicle 31 (SV31) (Burré, Zimmermann, & Volknandt, 2007). TMEM163 is a 31.5 kDa protein that binds divalent cations such as zinc, nickel, and copper, although the protein does not bind as strongly for the latter two metals (Barth, Zimmermann, & Volknandt, 2011).…”

“…At the cellular level, TMEM163 localizes to the plasma membrane and membrane-bound compartments such as lysosomes, endosomes, and synaptic vesicles (Burré et al, 2007;Cuajungco et al, 2014). As a new member of the CDF superfamily, and specifically the SLC30 family, phylogenetic analysis showed that TMEM163 is evolutionary related to ZNT9 (Sanchez et al, 2019). In terms of amino acid sequence identity and similarity, however, TMEM163 appears to be closely related to ZNT4 (Table 1).…”

“…Interestingly, ZNT8 isoforms hare phylogenetically similar, but their amino acid sequence percent identity and similarity scores are also low upon our analysis. Nevertheless, TMEM163 is a zinc efflux transporter that should be reclassified as a ZNT11 protein that is encoded by the SLC30A11 gene (Sanchez et al, 2019).…”