Figure 4. Interplay between Fe²⁺/Co²⁺ and Ca²⁺ in HsFpn.

(a) Ca²⁺ binding in the presence of 2 mM Co²⁺. (b) Co²⁺ binding in the presence of 2 mM Ca²⁺. (c) Fe²⁺ transport into proteoliposomes in the presence or absence of Ca²⁺. The external [Fe²⁺] is 50 µM. ‘1:1’ indicates symmetrical [Ca²⁺] at 1.25 mM and “10:1” indicates 1.25 mM Ca²⁺ inside and 0.125 mM Ca²⁺ outside. 1 mM sodium ascorbate was included in all samples. All fluorescence traces are subtracted from corresponding blank controls using vesicles with no HsFpn. (d) Comparison of initial rates of Fe²⁺ transport in (c). One-way ANOVA was used for statistical analysis. (e) Ca²⁺ (1 mM) uptake by HEK cells expressing HsFpn in the presence (dark yellow) or absence (green) of 80 µM Fe²⁺ that has been pre-loaded into the cells. Uptake is monitored by jGCaMP7s which is co-expressed in cytosol. Cells transfected with an empty vector (dark and light gray) serve as negative controls. Ca²⁺ transport at different concentrations of Fe²⁺ (f) or Co²⁺ (g). Normalized initial rates of Fpn-specific Ca²⁺ uptake were used to represent relative Ca²⁺ transport activities. Data were fitted (black solid line) to an inhibitory dose-response equation to calculate IC₅₀ values.

Figure 4—figure supplement 1. Fe²⁺/Co²⁺ transport into proteoliposomes by Fpn in the presence or absence of Ca²⁺.

(a) Co²⁺ transport with or without 1.25 mM of intra-vesicular Ca²⁺. (c) Fe²⁺ transport with or without 0.5 mM of intra-vesicular Ca²⁺. (e) Co²⁺ transport with or without 0.5 mM of intra-vesicular Ca²⁺. (b), (d), and (f) Comparison of initial rates in (a), (c), and (e). Liposome samples were diluted (10×) into outside buffers containing 50 µM Co²⁺ or Fe²⁺. ‘1:1’ or ‘10:1’ denotes the ratio of [Ca²⁺] inside:outside achieved after 10×dilution. 1 mM sodium ascorbate was included in (c). All fluorescence traces were subtracted from their corresponding no protein controls. One-way ANOVA was used for statistical analysis.

Figure 4—figure supplement 2. Ca²⁺ transport in the presence of Fe²⁺ or Co²⁺ in HEK cells.

(a) Schematic of the Ca²⁺ transport assay in Fe²⁺-loaded HEK cells co-expressing Fpn and the Ca²⁺ sensor jGCaMP7s. (b) Transition metal ions do not interact with jGCaMP7s as indicated by unaltered fluorescence readings during loading of Fe²⁺ (top) or Co²⁺ (bottom) into HEK cells expressing Fpn (orange or pink traces) or transfected with empty vectors (gray traces). Black arrows indicate additions of metal ions. (c) Ca²⁺ transport at different extracellular pHs in the presence of 20 µM Co²⁺. Two-way ANOVA: among different pHs, p=0.717; among empty, WT, and S2 mutant, p<0.0001; interaction, p=0.130. (d) and (e) Fpn-specific Ca²⁺ transport in the presence of Fe²⁺ or Co²⁺ loaded inside HEK cells. The concentrations of Fe²⁺ or Co²⁺ used during the loading step are indicated to the right of each trace. The Fe²⁺ stock was prepared with a 10-fold molar excess of sodium ascorbate. The raw traces of the empty control group were subtracted from the corresponding traces of the Fpn WT group. Normalized initial rates calculated from the datasets are shown in Figure 4f–g.

Figure 4—figure supplement 3. Proposed transport mechanisms of metal ions in Fpn.

(a) Electrogenic Ca²⁺ uniport in the absence of Fe²⁺. (b) Electroneutral Fe²⁺/2H⁺ antiport in the absence of Ca²⁺. (c) Reduced Ca²⁺ flux in the presence of Fe²⁺. Higher concentration of Fe²⁺ (orange ramp) shows larger inhibition on Ca²⁺ transport. The inward-facing model is generated based on the bacterial homolog structure (PDB ID 6BTX). The four-helical bundle (pale green) in NTD, and TM7 and TM11 (light orange) in CTD are shown as cylinders and overlaid on silhouettes (grey) of the whole transporter. S1 and S2 of Fe²⁺ are indicated as black forks with two tines to represent two ligand residues in each site. The Ca²⁺ binding site is indicated as a black circle. When Fe²⁺ is bound to S1, the Ca²⁺ binding site is drawn as an incomplete circle to indicate its partial disruption. Similarly, the S1 becomes a half fork when Ca²⁺ is bound. H⁺ (blue), Fe²⁺ (orange), and Ca²⁺ (green) are shown as spheres. The black dashed line and shaded green spheres of Ca²⁺ in (c) indicate reduced Ca²⁺ release and occupancy.