A substrate binding hinge domain is critical for transport-related structural changes of organic cation transporter 1.
Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane α-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl]methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH 8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.
Institute of Anatomy and Cell Biology, University of Würzburg, 97070 Würzburg, Germany.
Brigitte
B
Egenberger
Valentin
V
Gorboulev
Thorsten
T
Keller
Dmitry
D
Gorbunov
Neha
N
Gottlieb
Dietmar
D
Geiger
Thomas D
TD
Mueller
Hermann
H
Koepsell
D000818:Animals; D001665:Binding Sites; D050482:Catecholamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013379:Substrate Specificity / Q000502:physiology; D014982:Xenopus laevis
D050482:Catecholamine Plasma Membrane Transport Proteins; C493212:Slc22a1 protein, rat