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.
BACKGROUND. Liver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear. METHODS. We estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the Asl Neo/Neo mouse model of ASLD. RESULTS. We demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The Asl Neo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liverspecific (ApoE) promoter. CONCLUSION. Our results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction. TRIAL REGISTRATION. This study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).
Recent investigations of rodent Tmem163 suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two speciesconserved 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 micromolars. To further characterize the efflux function of
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