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

Abstract

Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

Figure 1

LeuT has two high affinity Leu binding sites. Equilibrium dialysis was performed in 150 mM Tris/Mes, pH 7.5, 50 mM NaCl, 1 mM TCEP, 0.1 (w/v)% n-dodecyl-β, D-maltopyranoside (DDM) using 4 pmol of LeuT-WT (a), -L400S (b), and -F253A (c) in the presence of increasing concentrations of ³H-Leu ranging from 0.5 nM to 5 μM. Dissociation constants (K_d) and molar binding ratios were determined by fitting the data with a hyperbolic non-linear regression model and shown in the text. (d) Clomipramine inhibits Leu binding to the S2 site. Equilibrium dialysis using LeuT-WT (black), -L400S (red), or -F253A (blue) was performed at 1 μM ³H-Leu in the presence (open bars) or absence (solid bars) of 1 mM clomipramine (CMI). Data shown are from representative experiments that were repeated ≥2 times.

Figure 2

Impairment of Leu binding to the S2 site in LeuT. Equilibrium binding was performed by means of the scintillation proximity assay (SPA) with 0.4 pmol LeuT-WT (a), -L400S (b), or -F253A (c) in the presence of increasing concentrations of ³H-Leu in 0.1 (w/v)% n-dodecyl-β, D-maltopyranoside (DDM). The solid symbols represent protein that was assayed without further treatment after IMAC purification, whereas the open symbols indicate protein samples that were concentrated 10-fold 72 h prior to performing the SPA. Data are from a representative experiment and error bars indicate the SEM of triplicate determinations. To determine the kinetic constants, data of independent experiments (n≥2) were subjected to one-site binding global fitting, yielding stoichiometries of 1.95±0.06 and 1.02±0.04 for non-concentrated and concentrated LeuT-WT, respectively, with a K_d of 29.8±3.4 nM and 33.3±4.8 nM. For LeuT-L400S the stoichiometry and K_ds of the non-concentrated and concentrated sample, were 1.0±0.05 and 0.99±0.05, and 54.6±11.6 nM and 64.9±11.1 nM, respectively, whereas the stoichiometry and K_d for non-concentrated LeuT-F253A was 1.01±0.03 and 77.8±3.3 nM. Concentrating LeuT-F253A greatly impaired Leu binding, thereby precluding meaningful data fits. (d) Representative equilibrium dialysis experiment using concentrated LeuT-WT (□), -L400S (▽), or -F253A (△). Whereas LeuT-F253A exhibit only marginal Leu binding activity (n=2), LeuT-WT and -L400S revealed stoichiometries of 1.03±0.03 (n=3) and 1.05±0.04 (n=2) with a K_d of 45.8±10.7 nM and 67.8±14.3 nM, respectively. The composition of the assay buffer used for equilibrium dialysis was identical to that used for the experiments shown in Fig. 1.

Figure 3

Effect of detergent on LeuT binding activity. 500 nM ³H-Leu binding by 0.4 pmol LeuT-WT (a), -L400S (c), or -F253A (c) was assayed with the SPA after IMAC in the presence of 0.1 (w/v)% DDM (■, ▼, ▲, respectively), 0.3 (w/v)% DDM (□, ▽, △), or 1.17 (w/v)% OG (○,○,○) and plotted as a function of time. (d) Effect of DDM on Leu equilibrium binding. IMAC-purified LeuT-WT (in 0.1 (w/v)% DDM) was pre-incubated with 0.3 (w/v)% DDM for the indicated periods of time and subjected to SPA-mediated binding of 500 nM ³H-Leu in 0.1 (w/v)% DDM (■) or 0.3 (w/v)% DDM (□) for 16 h (equilibrium). Error bars in all panels are the SEM of triplicate determinations from representative experiments that were repeated ≥2 times.

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 pre-incubated in the presence of 0.1 (w/v)% DDM (■) or 0.3 (w/v)% DDM (□) for ~500 h. Release of ³H-Leu trapped in the S1 site of LeuT assayed in 0.1 (w/v)% DDM was achieved by the addition of 2.5 μM Leu (L). (b) Binding of 500 nM ³H-Leu to 0.4 pmol LeuT-WT (black), -L400S (red), or -F253A (blue) in 0.1 (w/v)% DDM measured by SPA (lower panel) for 16 h using non-concentrated (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 non-concentrated LeuT-WT, -L400S, or -F253A into proteoliposomes (upper panel). Equilibrium binding in proteoliposomes was performed for 4 h in the presence of 25 μg gramicidin/mL (5-min pre-treatment) 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, - L400S, or -F253A from non-concentrated (■,▼, ▲, respectively) or concentrated (□, ▽, △ ) material or in control liposomes (○). Error bars in all panels are the SEM of triplicate determinations from representative experiments that were repeated ≥2 times.