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Targeted mutagenesis of negatively charged amino acids outlining the substrate translocation path within the human organic cation transporter 3.
Redeker, Kyra-Elisa M; Schröder, Sophie; Dücker, Christof; Brockmöller, Jürgen; Gebauer, Lukas.
Afiliación
  • Redeker KM; Institute of Clinical Pharmacology, University Medical Center Göttingen, D-37075 Göttingen, Germany. Electronic address: kyra-elisamaria.redeker@med.uni-goettingen.de.
  • Schröder S; Department for Epigenetics and Systems Medicine in Neurodegenerative Diseases, German Center for Neurodegenerative Diseases (DZNE), D-37075 Göttingen, Germany.
  • Dücker C; Institute of Clinical Pharmacology, University Medical Center Göttingen, D-37075 Göttingen, Germany.
  • Brockmöller J; Institute of Clinical Pharmacology, University Medical Center Göttingen, D-37075 Göttingen, Germany.
  • Gebauer L; Institute of Clinical Pharmacology, University Medical Center Göttingen, D-37075 Göttingen, Germany.
Biochem Pharmacol ; 223: 116188, 2024 05.
Article en En | MEDLINE | ID: mdl-38580166
ABSTRACT
Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3. E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site. In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas de Transporte de Catión Orgánico / Aminoácidos Límite: Humans Idioma: En Revista: Biochem Pharmacol Año: 2024 Tipo del documento: Article Pais de publicación: Reino Unido
Texto completo
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Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas de Transporte de Catión Orgánico / Aminoácidos Límite: Humans Idioma: En Revista: Biochem Pharmacol Año: 2024 Tipo del documento: Article Pais de publicación: Reino Unido