Channel character of uncoupling protein-mediated transport

Abstract

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function.

Fig. 1. Classic and Eyring barrier models for typical energy barrier profiles and their limiting cases

Energy profiles are illustrated corresponding to (A) Goldman equation, (B) trapezoid barrier, (C) single-peak, and (D) double-peak energy barrier with a well (binding site). The single Eyring barrier model (C) and its modification containing an energy well (D) are described in the Sections 3.1 and 3.2, respectively. A Goldman equation, J = P· u ·(C₁·e^u - C₂ / (e^u2 - 1), is a special case of equation {8} with N approaching to infinity (Fig.1A) [42]. Alternatively a trapezoid barrier (Fig.1B) is introduced [42], for which flux J is given, J = P ·W·u·(C₁·e^u - C₂·e^−u/22) / (e^Wu/2 - e^−Wu/2), where W is a fractional width of the trapezoid (Fig.1B). At the limit W = 1, one gets the Goldman equation, whereas at the limit W = 0, equation changes to the expression for a single sharp peak (β= 0.5) (Fig.1C).

Fig. 2. Nonohmic Cl⁻ and Br⁻ uniport mediated by reconstituted UCP1 reflects a double-peak energy barrier with a well (binding site) in the middle

Rates of ion uptake in UCP1-proteoliposomes (J) for Cl⁻ (●), Br⁻ (◆), and Br⁻ in vesicles loaded with 2 mM GDP (▲) are plotted as a function of K⁺ diffusion potential ΔΨ in direct plots, where fits of the data were done using equation {11} (A). Data were linearized while plotting ln J vs. zFΔΨ/RT (B). Fits yielded (R² of 0.98) the following parameters β: 0.22 ± 0.01 for Cl⁻ flux and 0.25 ± 0.01 for Br⁻ flux into UCP1-proteoliposomes; and 0.26 ± 0.02 for Br⁻ flux into GDP-loaded UCP1-proteoliposomes. ΔΨ was calculated from the Nernst distribution of K⁺ in the presence of 1 μM valinomycin. Reconstitution of hamster brown adipose tissue UCP1 and 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorometric quantification of ion fluxes has been performed as described in [17]. UCP1-proteoliposomes contained 50 mM tetraethyl ammonium (TEA)-sulfate, 100 mM TEA- N-tris(hydroxymethyl)-methylamino-ethanesulfonic acid (TES) pH 7.2, containing 0.14 mM KCl (KBr), while the external assay medium was composed of 150 mM KCl (KBr, respectively), 25 mM TEA-TES, pH 7.2, for maximum ΔΨ, which was further decreased by proportional mixing with 150 mM TEA-Cl (TEA-Br, respectively), 25 mM TEA-TES, pH 7.2.

Fig. 3. Nonohmic SCN⁻ uniport in protein-free liposomes reflects a single-peak energy barrier

Rates of ion uptake in lecithin liposomes (J_SCN⁻) for SCN⁻ are plotted as a function of K⁺ diffusion potential ΔΨ in direct plots, where data were fitted by equation {11} (A). Data were linearized while plotting ln J vs. zFΔΨ/RT (B). The fit yielded (R² of 0.98) the parameters β of 0.49 ± 0.02. ΔΨ was calculated from the Nernst distribution of K⁺ in the presence of 10 μM valinomycin. Liposomes were prepared and SPQ fluorometric quantification of ion fluxes has been performed as described in [17]. Liposome lumen contained 50 mM TEA-sulfate, 75 mM TEA-TES pH7.2, 25 mM Li-TES, and 0.14 mM KSCN, while the external assay medium was composed of 25 mM KSCN,125 mM KTES, 25 mM Li-TES, pH 7.2, for maximum ΔΨ, which was further decreased by proportional mixing with 25 mM LiSCN, 150 mM TEA-TES, pH 7.2.

Fig. 4. Simultaneous lauric acid cycling and C₁₁-sulfonate uniport in UCP1-proteoliposomes

H⁺ efflux and K⁺ influx are plotted vs. C₁₁-sulfonate concentration. Total substrate anion concentration [C₁₁-sulfonate] plus [lauric acid] was held constant at 50 μM as [C₁₁-sulfonate] was increased. H⁺ efflux and K⁺ influx were indicated by fluorescent probes SPQ and PBFI loaded in two parallel proteoliposomal preparations, respectively. Ion fluxes were initiated by 0.1 μM valinomycin in the presence of a [K⁺ ] gradient. Reconstitution of hamster brown adipose tissue UCP1 was performed as described in [22]. UCP1-proteoliposomes contained 30 mM TEA-TES, pH 7.2, 80 mM TEA-sulfate, 0.6 mM TEA-EGTA. Reprinted from Jabůrek et al. [22].