Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region
Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region
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DOI：
10.1124/mol.104.008821
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发表时间：
2005-05-01
影响因子：
3.6
通讯作者：
Koepsell, H
中科院分区：
文献类型：
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作者：
Gorboulev, V;Shatskaya, N;Koepsell, H
皮质酮对有机阳离子转运蛋白（OCT）的亲和力具有亚型和种属依赖性。例如，皮质酮抑制转运蛋白rOCT 1和rOCT 2阳离子摄取的IC 50值分别为150和4 μ M。通过将rOCT 2的结构域和氨基酸引入rOCT 1，我们发现，假定的第10跨膜α螺旋中的三个氨基酸的交换足以增加rOCT 1对皮质酮的亲和力。rOCT 2中这些氨基酸的替换降低了对皮质酮的亲和力。这些氨基酸（rOCT 1中的Ala 443、Leu 447和Gln 448以及rOCT 2中的Ile 443、Tyr 447和Glu 448）可能位于底物结合区内，因为在rOCT 1突变体中，四乙铵（TEA）和1-甲基-4-苯基吡啶（MPP）吸收的K-m值与皮质酮抑制阳离子摄取的IC 50值的降低平行降低。在突变体rOCT 1（L447 Y/Q448 E）中，皮质酮（24 +/- 4 mu M）抑制[H-3]MPP（0.1 mu M）摄取的IC 50值显著高于抑制[C-14] TEA（10 mu M）摄取的IC 50值（5.3 +/- 1.7 mu M）。这一发现表明转运阳离子和皮质酮之间的变构相互作用。由于这种底物特异性的影响不能解释由MPP与TEA的皮质酮的差异替代，并观察到点突变后，在假定的底物区域内，数据表明，MPP或TEA结合到底物结合区，同时与皮质酮，并导致皮质酮结合位点的短程变构效应。
The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are similar to 150 and similar to 4 mu M, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K-m values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [H-3]MPP (0.1 mu M) uptake by corticosterone ( 24 +/- 4 mu M) was significantly higher compared with the IC50 value for inhibition of [C-14] TEA (10 mu M) uptake (5.3 +/- 1.7 mu M). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.