Division of Pharmacology & Toxicology, College of Pharmacy, Institute for Cellular & Molecular Biology; and Institute for Neuroscience, The University of Texas at Austin, 3.510E BME, 107 W. Dean Keeton, Austin, TX 78712, USA.
Metallomics. 2018 Aug 15;10(8):1053-1064. doi: 10.1039/c8mt00115d.
SLC30 proteins belong to the cation diffusion facilitator (CDF) superfamily of metal transporters. SLC30A10 mediates manganese efflux, while other SLC30 members transport zinc. Metal specificity of CDFs may be conferred by amino acids that form a transmembrane metal binding site (Site A). Site A of zinc-transporting CDFs, such as SLC30A1/ZnT1, have a HXXXD motif, but manganese transporters, such as SLC30A10, harbor a NXXXD motif. This critical histidine-to-asparagine substitution, at residue 43, was proposed to underlie manganese transport specificity of SLC30A10. However, we recently discovered that asparagine-43 was dispensable for manganese efflux in HeLa cells; instead, glutamate-25, aspartate-40, asparagine-127, and aspartate-248 were required. In contrast, another group reported that asparagine-43 was required in a chicken cell line. The goal of this study was to resolve the divergent results about the requirement of the crucial asparagine-43 residue. For this, we compared the manganese efflux activity of four cell types that stably over-expressed SLC30A10wild-type (WT), SLC30A10N43A or SLC30A10E25A: physiologically-relevant hepatic HepG2 and neuronal AF5 cells, HEK cells, and embryonic fibroblasts from Slc30a10-/- mice. In all cell types, manganese efflux activity of SLC30A10N43A was comparable to WT, while SLC30A10E25A lacked activity. Importantly, unlike SLC30A10, the histidine residue of the HXXXD motif of SLC30A1/ZnT1 was required for zinc transport. These results imply that the mechanisms of ion coordination within the transmembrane domain of SLC30A10 substantially differ from previously-studied CDFs, suggest that factors beyond Site A residues may confer metal specificity to CDFs, and improve understanding of the pathobiology of manganese toxicity due to mutations in SLC30A10.
SLC30 蛋白属于阳离子扩散促进剂（CDF）金属转运蛋白超家族。SLC30A10 介导锰外流，而其他 SLC30 成员则转运锌。CDF 的金属特异性可能由形成跨膜金属结合位点（Site A）的氨基酸赋予。锌转运 CDF 的 Site A，如 SLC30A1/ZnT1，具有 HXXXD 基序，但锰转运蛋白，如 SLC30A10，则具有 NXXXD 基序。这种关键的组氨酸到天冬酰胺取代，残基 43，被认为是 SLC30A10 锰转运特异性的基础。然而，我们最近发现天冬酰胺 43 在 HeLa 细胞中对锰外排是可有可无的；相反，谷氨酸 25、天冬氨酸 40、天冬酰胺 127 和天冬氨酸 248 是必需的。相比之下，另一组报道天冬酰胺 43 在鸡细胞系中是必需的。本研究的目的是解决关于关键天冬酰胺 43 残基需求的分歧结果。为此，我们比较了四种稳定过表达 SLC30A10野生型（WT）、SLC30A10N43A 或 SLC30A10E25A 的细胞类型的锰外排活性：生理相关的肝 HepG2 和神经元 AF5 细胞、HEK 细胞和 Slc30a10-/- 小鼠的胚胎成纤维细胞。在所有细胞类型中，SLC30A10N43A 的锰外排活性与 WT 相当，而 SLC30A10E25A 则没有活性。重要的是，与 SLC30A10 不同，HXXXD 基序的组氨酸残基对 SLC30A1/ZnT1 的锌转运是必需的。这些结果表明，SLC30A10 跨膜结构域内离子配位的机制与之前研究的 CDF 有很大不同，表明除 Site A 残基以外的因素可能赋予 CDF 金属特异性，并提高对由于 SLC30A10 突变引起的锰毒性的病理生物学的理解。
