title: The large extracellular loop of organic cation transporter 1 influences substrate affinity and is pivotal for oligomerization.
abstract: Polyspecific organic anion transporters (OATs) and organic cation transporters (OCTs) of the SLC22 transporter family play a pivotal role in absorption, distribution, and excretion of drugs. Polymorphisms in these transporters influence therapeutic effects. On the basis of functional characterizations, homology modeling, and mutagenesis, hypotheses for how OCTs bind and translocate structurally different cations were raised, assuming functionally competent monomers. However, homo-oligomerization has been described for OATs and OCTs. In the present study, evidence is provided that the large extracellular loops (EL) of rat Oct1 (rOct1) and rat Oat1 (rOat1) mediate homo- but not hetero-oligomerization. Replacement of the cysteine residues in the EL of rOct1 by serine residues (rOct1(6ΔC-l)) or breaking disulfide bonds with dithiothreitol prevented oligomerization. rOct1 chimera containing the EL of rOat1 (rOct1(rOat1-l)) showed oligomerization but reduced transporter amount in the plasma membrane. For rOct1(6ΔC-l) and rOct1(rOat1-l), similar K(m) values for 1-methyl-4-phenylpyridinium(+) (MPP(+)) and tetraethylammonium(+) (TEA(+)) were obtained that were higher compared with rOct1 wild type. The increased K(m) of rOct1(rOat1-l) indicates an allosteric effect of EL on the cation binding region. The similar substrate affinity of the oligomerizing and non-oligomerizing loop mutants suggests that oligomerization does not influence transport function. Independent transport function of rOct1 monomers was also demonstrated by showing that K(m) values for MPP(+) and TEA(+) were not changed after treatment with dithiothreitol and that a tandem protein with two rOct1 monomers showed about 50% activity with unchanged K(m) values for MPP(+) and TEA(+) when one monomer was blocked. The data help to understand how OCTs work and how mutations in patients may affect their functions.
authors: Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany.
authors: Thorsten
authors: T
authors: Keller
authors: Brigitte
authors: B
authors: Egenberger
authors: Valentin
authors: V
authors: Gorboulev
authors: Frank
authors: F
authors: Bernhard
authors: Zeljko
authors: Z
authors: Uzelac
authors: Dmitry
authors: D
authors: Gorbunov
authors: Christophe
authors: C
authors: Wirth
authors: Stefan
authors: S
authors: Koppatz
authors: Volker
authors: V
authors: Dötsch
authors: Carola
authors: C
authors: Hunte
authors: Harald H
authors: HH
authors: Sitte
authors: Hermann
authors: H
authors: Koepsell
mesh_terms: D000818:Animals; D050482:Catecholamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D009154:Mutation; D027362:Organic Anion Transport Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D050482:Catecholamine Plasma Membrane Transport Proteins; D027362:Organic Anion Transport Protein 1; D011993:Recombinant Fusion Proteins; C493212:Slc22a1 protein, rat; C421043:Slc22a6 protein, rat; D004229:Dithiothreitol