Figure 2. Inhibition of EcoDMT by Br-BIT.

(A) EcoDMT mediated Mn²⁺ transport into proteoliposomes in presence of Br-BIT assayed by the quenching of the fluorophore calcein trapped inside the vesicle. Br-BIT was added at different concentration (5–200 µM) to the outside of the liposomes. The transport is started by the addition of the potassium ionophore Valinomycin (Val) and 5 µM MnCl₂. To equilibrate the Mn²⁺ ions, the ionophore Calcimycin (Cal) was added at the end of the run. Schematic figure (left) depicts the sidedness of inhibition which is responsible for the remaining activity at large inhibitor concentrations. (B) Transport kinetics of EcoDMT at the indicated Br-BIT concentrations. The solid lines are fits to the Michaelis-Menten equation assuming similar kinetic properties of transport for both orientations of the transporter. The observed kinetic parameters thus describe apparent values obtained from an average of transport properties in inside-out and outside-out orientations. Values show averages of six technical replicates obtained from three independent proteoliposome preparations, errors are s.e.m.

Figure 2—figure supplement 1. Characterization of EcoDMT inhibition.

(A) Scheme of proteoliposome-based assay. The metal sensitive fluorophore Calcein (yellow stars) was incorporated into proteoliposomes. Valinomycin (Val) was added to dissipate the membrane potential established upon Mn²⁺ transport into the vesicles. The time-dependent quenching of the Calcein fluorescence (blue stars) was recorded for several minutes. As a control, the metal ion ionophore Calcimycin (Cal) was added at the end of the assay to equilibrate the Mn²⁺ ions. (B) To determine the orientation of the transporters in the proteoliposomes the accessibility of the C-terminus of EcoDMT was probed using a C-terminally His-tagged version of EcoDMT in which the tag was preceded by a 3C protease cleavage site (EcoDMT-3C-His). The Coomassie blue stained SDS-PAGE gel shows the cleavage of the tag upon addition of the 3C protease to the outside of unilammellar vesicles (3C to Liposomes) and incubation for 2 hr. The 3C protease was subsequently removed from the outside solution and the vesicles were solubilized in detergent and digested with 3C protease (3C after solubilization). As a control, the cleavage of the tag was also assessed with purified EcoDMT-3C-His before (3C at start) and after addition of detergent (3C after excess det. addition). Upon addition of the 3C protease to the outside of proteoliposomes (third sample from the left), the His-tag is cleaved in about 50% of the transporters, which suggests an about equal ratio of outside-out and outside-in orientations. (C) Transport properties of EcoDMT WT assayed at the indicated Mn²⁺ concentrations. D-E, EcoDMT mediated Mn²⁺ transport into proteoliposomes in presence of compound 2, TMBIT (D) or compound 5, oBr-BIT (E). Inhibitor concentrations are indicated.