Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Molecules. 2018 Mar 22;23(4):732. doi: 10.3390/molecules23040732.
Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases-to a lesser extent-and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, a detailed understanding of ENT function at the molecular level has remained elusive. ScENT1 (formerly known as Function Unknown Now 26 or FUN26) is the only known ENT ortholog endogenously expressed in , and a proteoliposome assay system was used to study homogenously overexpressed and purified ScENT1 (wildtype relative to L390A and F249I mutants). L390 and F249 are highly conserved residues and were found to alter transporter function. L390A produced a reduction of mean transport activity while F249I increased mean substrate translocation relative to wildtype protein. However, both mutations resulted in transport of UTP-a novel gain of function for any ENT. These residues were then mapped onto an ab initio model of FUN26 which suggests they function in substrate translocation (L390) or cytoplasmic gating (F249). Furthermore, wildtype, L390A, and F249I were found to be sensitive to the presence of alcohols. Ethanol attenuated ScENT1-mediated transport of uridine by ~50%. These findings further demonstrate functional similarities between ScENT1 and human ENT isoforms and support identification of FUN26 as ScENT1, the first ENT isoform in .
平衡核苷转运蛋白（ENTs）是多跨膜转运蛋白，负责将核苷、核苷碱基（在较小程度上）和核苷类似物治疗药物穿过细胞膜进行转运。ENTs 以扩散控制的双向方式发挥作用，据认为它们利用交替访问转运机制。然而，ENT 功能的详细分子水平理解仍然难以捉摸。ScENT1（以前称为功能未知现在 26 或 FUN26）是唯一在 中内源性表达的 ENT 直系同源物，并且使用蛋白脂质体测定系统研究了均匀过表达和纯化的 ScENT1（相对于 L390A 和 F249I 突变体的野生型）。L390 和 F249 是高度保守的残基，被发现改变了转运蛋白的功能。L390A 导致平均转运活性降低，而 F249I 相对于野生型蛋白增加了底物的平均转运。然而，这两种突变都导致 UTP 的转运 - 这是任何 ENT 的新获得的功能。然后将这些残基映射到 FUN26 的从头模型上，这表明它们在底物转运（L390）或细胞质门控（F249）中发挥作用。此外，发现野生型、L390A 和 F249I 对醇的存在敏感。乙醇使 ScENT1 介导的尿苷转运减少了约 50%。这些发现进一步证明了 ScENT1 和人 ENT 同工型之间的功能相似性，并支持将 FUN26 鉴定为 ScENT1，这是 中的第一个 ENT 同工型。
