Figure 6. Mrs2 gain-of-function mutant augments mMg²⁺ uptake and prevents Ca²⁺-overload mediated PTP opening.

(a) Schematic of full-length Mrs2 with its functional domains (Mrs2^WT). Mutations in Mrs2^WT are highlighted in red (Mrs2^ΔGMN and Mrs2^ΔMBS). (b) Representative Western blot probed with antibodies specific for Flag shows the expression of Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS in HEK293 WT cells. (c-g) Simultaneous measurement of Δψm and mMg²⁺ in HEK 293 cells expressing Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS. Mean traces of Δψm (c) and [Mg²⁺]_out (d). (e) Quantification of the basal Δψm before the addition of 1 mM Mg²⁺ pulse. (f) Quantifying the rate of mMg²⁺ uptake as a function of the decrease in [Mg²⁺]_out after 1 mM Mg²⁺ pulse. (g) Quantification of the [Mg²⁺]_out after CCCP addition. Data represents Mean ± SEM; *P<0.05, **P <0.01; n = 4. (h-l) Simultaneous measurement of Δψm and mCa²⁺ in HEK 293 cells expressing Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS. Mean traces of Δψm (h) and [Ca²⁺]_out (i). (j) Quantification of the basal Δψm before adding 20 μM Ca²⁺ pulse. (k) Quantification of the rate of mCa²⁺ uptake as a function of the decrease in [Ca²⁺]_out after 20 μM Ca²⁺ pulse. (l) Quantification of the [Ca²⁺]_out after CCCP addition. Data represent Mean ± SEM; **P<0.01, ***P <0.001; n = 4. (m) Mean traces of oxygen consumption rate (OCR) in HEK 293 cells expressing Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS using pyruvate as a substrate and after sequential exposure to Oligomycin (a), FCCP (b), and rotenone/antimycin (c). (n) Quantification of basal, maximal OCR, proton leak, and ATP-coupled respiration. Data indicate Mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001; n = 10. (o) Quantification of total cellular ATP levels in HEK 293 cells expressing Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS. (p-s) Simultaneous measurement of Δψm and mCa²⁺ in HEK 293 cells expressing Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS. Mean traces of Δψm (p) and [Ca²⁺]_out (q) after a series of 10 μM Ca²⁺ pulses. (r) Quantification of the basal Δψm before adding the first 10 μM Ca²⁺ pulse. (s) Quantification of the number of Ca²⁺ pulses handled before the loss of Δψm. Bar represents Mean ± SEM; **P < 0.01, ***P < 0.001; n = 4–7. (t) Mean traces of mitochondrial calcein fluorescence in HEK 293 cells expressing Mrs2^WT, Mrs2^ΔGMN, and Mrs2^ΔMBS before and after adding ionomycin (5 μM).