Experimental conditions can obscure the second high-affinity site in LeuT

Figure 4: Intact S1 and S2 sites are required for Na⁺-coupled transport by LeuT. (a) Dissociation of 1 μM ³H-Leu from LeuT-WT by dilution into 50 mM Na⁺ (+Na) and into Na⁺ -free (−Na) media. Samples were preincubated in the presence of 0.1% DDM (▪) or 0.3% DDM (□) for ~500 h. Release of ³H-Leu trapped in the S1 site of LeuT assayed in 0.1% DDM was achieved by the addition of 2.5 μM leucine (L). (b) Binding of 500 nM ³H-Leu to 0.4 pmol LeuT-WT (black), LeuT-L400S (red) or LeuT-F253A (blue) in 0.1% DDM measured by SPA (lower panel) for 16 h using nonconcentrated (solid bars) or previously concentrated (open bars) material. Binding of 500 nM ³H-Leu to 0.4 pmol of protein was also assayed after reconstitution of previously concentrated or nonconcentrated LeuT-WT, LeuT-L400S or LeuT-F253A into proteoliposomes (upper panel). Equilibrium binding in proteoliposomes was carried out for 4 h in the presence of 25 μg gramicidin ml⁻¹ (5-min pretreatment) to dissipate the Na⁺ electrochemical gradient, followed by capture of LeuT-containing proteoliposomes onto 0.22-μm nitrocellulose filters and subsequent scintillation counting. (c) Time course of Na⁺ -coupled uptake of 1 μM ³H-Ala in proteoliposomes reconstituted with LeuT-WT, LeuT- L400S or LeuT-F253A from nonconcentrated (solid black square, red inverted triangle and blue triangle, respectively) or concentrated (open black square, red inverted triangle and blue triangle, respectively) material, or in control liposomes (open black circle). Error bars in all panels are the s.e.m. of triplicate determinations from representative experiments that were repeated two or more times.