Bröer Angelika, Balkrishna Sarojini, Kottra Gabor, Davis Sarah, Oakley Aaron, Bröer Stefan
Research School of Biology, Australian National University, Canberra, ACT 0200, Australia.
Mol Membr Biol. 2009 Aug;26(5):333-46. doi: 10.1080/09687680903150027. Epub 2009 Jul 30.
The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na(+) and Cl(-)-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na(+)-binding sites and a putative Cl(-)-binding site. Mutation of all residues in the two proposed Na(+)-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na(+)-binding site 1) and mutants S20A, S20G, S20G/G405S (Na(+)-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na(+)-binding site 1 caused a decrease of the Na(+)-K(0.5), whereas mutations of site 2 increased the K(0.5). Mutation of Na(+)-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates (36)Cl(-) demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na(+)/1Cl(-)-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.
亚氨基酸转运体系统在肠道和肾脏中脯氨酸和羟脯氨酸的转运过程中发挥着至关重要的作用。其分子相关物已被鉴定并命名为SIT1或亚氨基酸转运体（SLC6A20）。对该转运体的初步表征显示它依赖于Na(+)和Cl(-)，但其化学计量关系仍未明确。通过基于细菌亮氨酸转运体LeuT的结构进行同源建模，我们确定了两个高度保守的Na(+)结合位点和一个假定的Cl(-)结合位点。对两个假定的Na(+)结合位点中的所有残基进行突变后发现，其中大多数对于摄取是必不可少的，并且会使转运体完全失活。然而，A22V突变体（Na(+)结合位点1）以及S20A、S20G、S20G/G405S突变体（Na(+)结合位点2）具有部分活性，并对其进行了进一步表征。通量研究表明，Na(+)结合位点1的突变导致Na(+)-K(0.5)降低，而位点2的突变则增加了K(0.5)。Na(+)结合位点1的突变还改变了亚氨基酸转运体的离子选择性。亚氨基酸转运体能够主动转运(36)Cl(-)，这表明所提出的氯离子结合位点在该转运体中发挥作用。在不同保持电位下进行的积累实验和通量测量表明，该转运体可作为一种2Na(+)/1Cl(-)-脯氨酸共转运体发挥作用。所提出的同源模型有助于研究与亚氨基甘氨酸尿症相关的亚氨基酸转运体中的突变。
