Wittern Carla Isabel, Schröder Sophie, Jensen Ole, Brockmöller Jürgen, Gebauer Lukas
Institute of Clinical Pharmacology, University Medical Center Göttingen, Göttingen, Germany.
Department for Epigenetics and Systems Medicine in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
J Biol Chem. 2024 Nov;300(11):107835. doi: 10.1016/j.jbc.2024.107835. Epub 2024 Sep 27.
Organic cation transporters (OCTs) can transport structurally highly diverse substrates. The molecular basis of this extensive polyspecificity has been further elucidated by cryo-EM. Apparently, in addition to negatively charged amino acids, aromatic residues may contribute to substrate binding and substrate selectivity. In this study, we provide a comprehensive characterization of phenylalanine 244 in OCT1 function. We analyzed the uptake of 144 OCT1 substrates for the phenylalanine 244 to alanine substitution compared to WTOCT1. This substitution had highly substrate-specific effects ranging from transport reduced to 10% of WT activity up to 8-fold increased transport rates. Four percent of substrates showed strongly increased uptake (>200% of WT) whereas 39% showed strongly reduced transport (<50% of WT). Particularly with larger, more hydrophobic, and more aromatic substrates, the Phe244Ala substitution resulted in higher transport rates and lower inhibition of the transporter. In contrast, substrates with a lower molecular weight and less aromatic rings showed generally decreased uptake rates. A comparison of our data to available transport kinetic data demonstrates that generally, high-affinity low-capacity substrates show increased uptake by the Phe244Ala substitution, whereas low-affinity high-capacity substrates are characterized by reduced transport rates. Altogether, our study provides the first comprehensive characterization of the functional role of an aromatic amino acid within the substrate translocation pathway of OCT1. The pleiotropic function further highlights that phenylalanine 244 interacts in a highly specific manner with OCT1 substrates and inhibitors.
有机阳离子转运体（OCTs）能够转运结构高度多样的底物。低温电子显微镜进一步阐明了这种广泛的多特异性的分子基础。显然，除了带负电荷的氨基酸外，芳香族残基可能有助于底物结合和底物选择性。在本研究中，我们全面表征了OCT1功能中苯丙氨酸244的作用。我们分析了与野生型OCT1相比，苯丙氨酸244突变为丙氨酸后144种OCT1底物的摄取情况。这种取代具有高度的底物特异性效应，转运活性从降低到野生型活性的10%到增加8倍不等。4%的底物摄取显著增加（>野生型的200%），而39%的底物转运显著降低（<野生型的50%）。特别是对于更大、更疏水和更具芳香性的底物，Phe244Ala取代导致转运速率更高，对转运体的抑制更低。相比之下，分子量较低且芳香环较少的底物通常摄取速率降低。将我们的数据与现有的转运动力学数据进行比较表明，一般来说，高亲和力低容量底物通过Phe244Ala取代摄取增加，而低亲和力高容量底物的特征是转运速率降低。总之，我们的研究首次全面表征了OCT1底物转运途径中芳香族氨基酸的功能作用。这种多效性功能进一步突出了苯丙氨酸244与OCT1底物和抑制剂以高度特异性方式相互作用。
