Datensatz

Zusammenfassung

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na⁺ symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na⁺-dependent. The transport activity of R262 mutants increased by 30-40% in the presence of a membrane potential, indicating substrate/Na⁺ cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1' helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na⁺-coupled substrate symporters. We propose that a similar mechanism is operative in other Na⁺/H⁺-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.