Extended Data Fig. 2: Functional characterization of NfMCU.

a, Calcium uptake in E. coli expressing empty vector (black trace) or NfMCU (blue trace), demonstrating that heterologously expressed NfMCU can confer uniporter function to bacteria. Arrows indicate addition of 20 µM CaCl₂ to the reaction solution. b, Concentration-dependent calcium uptake in E. coli expressing empty vector (black trace) or NfMCU (blue trace). Each point represents the rate of calcium uptake normalized to the uptake rate at 90 µM CaCl₂. c, Concentration-dependent inhibition of NfMCU by GdCl₃ in E. coli expressing NfMCU. Each point represents the rate of calcium uptake normalized to the uptake rate without GdCl₃ in the reaction. d, Concentration-dependent inhibition of Ca²⁺ uptake in E. coli expressing NfMCU by the MCU-specific inhibitor Ru360. Each point represents the rate of calcium uptake normalized to the uptake rate without Ru360 in the reaction. The Ru360 concentration required to reduce the Ca²⁺ uptake rate by half (IC₅₀) is calculated to be 0.5 μM. e, In light of the contradiction of the oligomeric state between NfMCU and cMCU-∆NTD, we also designed an in vitro functional assay by reconstituting our purified tetrameric channel into liposomes. Unable to obtain currents of NfMCU by electrophysiology—probably owing to the channel’s small conductance—we performed a radioactive ⁴⁵Ca flux assay on proteoliposomes loaded with 150 mM KCl. Shown here is the time-dependent ⁴⁵Ca²⁺ uptake by empty liposomes or NfMCU proteoliposomes incubated with ⁴⁵Ca²⁺ in a reaction buffer containing 150 mM KCl (red points) or 150 mM NMDG (blue points). The result demonstrates that in a reaction solution containing 150 mM NMDG and the K⁺-selective ionophore valinomycin (to generate an electrical driving force), NfMCU proteoliposomes exhibited time-dependent ⁴⁵Ca²⁺ uptake while the same proteoliposomes in a reaction solution containing valinomycin and 150 mM KCl (to eliminate electrical driving force) accumulated ⁴⁵Ca²⁺ only slightly better than empty liposomes, recapitulating the voltage-dependent Ca²⁺ uptake property of the MCU. f, Concentration-dependent inhibition of NfMCU proteoliposomes by Ru360, demonstrating that Ru360 also blocks NfMCU ⁴⁵Ca²⁺ uptake in a concentration-dependent manner up to 50%, which is expected given an equal distribution of two channel orientations in the liposomes. Each point represents radioactivity measured after 30 min reaction normalized to sample without Ru360. All data in a–f are shown as mean ± s.e.m. (n = 3 independent experiments).

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