Figure 3. Mutations on the Ca²⁺ binding site.

(a) Ca²⁺ influx by WT (green), D39A (blue), and the triple mutant (Q99A_N100A_E219A, purple) of the Ca²⁺ binding site in proteoliposomes. (b) Comparison of initial rates of Ca²⁺ influx in (a). (c) Ca²⁺ uptake in HEK cells expressing WT (green) or the triple mutant (purple) Fpn. (d) Fpn-specific Ca²⁺ transport activities of WT and mutants. Initial rates are subtracted from the empty control. For statistical significances in (b) and (d), Dunnett’s test was used as a post hoc test following one-way ANOVA with the WT as the control. 500 µM Ca²⁺ was used in (a–d). Binding of Ca²⁺ to WT (e), D39A (f), and H43A (g) HsFpn measured by ITC. Upper plot: raw thermogram showing the heat during binding and baseline (red line). Lower plot: integrated heat of each injection (black square) and the fit of data (red line).

Figure 3—figure supplement 1. Expression of WT and mutant Fpns in HEK cells assessed by western blot.

Figure 3—figure supplement 2. Binding of Ca²⁺, Co²⁺, or Zn²⁺ to WT and mutant Fpns measured by ITC.

Binding of Ca²⁺ to HsFpn-11F9 complex (a), TsFpn (b), and S2 mutant (c). (d) Binding of Co²⁺ to the alanine mutant of Q99 in the Ca²⁺ binding site. Titration of Ca²⁺ into the alanine mutants of Q99 (e), N100 (f), N212 (g), and E219 (h) in the Ca²⁺ binding site. Binding of Zn²⁺ to WT (i) and the alanine mutant of N212 (j) in the Ca²⁺ binding site.

Figure 3—figure supplement 3. Effect of D39A on Co²⁺ transport by Fpn.

(a) Co²⁺ influx in proteoliposomes with symmetrical NaCl buffer. 500 µM Co²⁺ was added at time zero. (b) Comparison of initial rates of Co²⁺ transport. Statistical significances were analyzed with unpaired t-test with Welch correction.