Fig. 1: Letm1 regulates mitochondrial Ca²⁺ export in neuronal mitochondria.

a, Letm1 is a protein located on the inner mitochondrial membrane where it transports one Ca²⁺ in exchange for two H⁺. It contains a Ca²⁺-binding EF-hand domain facing the mitochondrial matrix. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; MCU, mitochondrial Ca²⁺ uniporter. b, Axonal mitochondrial Ca²⁺ responses to stimulation (20 action potentials (APs), 20 Hz) in control and Letm1 KD neurons. The figure shows traces corresponding to the mean ± s.e.m. of Mito^4x-GCaMP6f in axons. c, Rate of mitochondrial Ca²⁺ decay measured as half-time decay (t_1/2) in axonal mitochondria following stimulation in control and Letm1 KD neurons. Statistical significance was assessed using a two-tailed Mann–Whitney U-test (****P < 0.0001; n = 27 for control neurons, n = 21 for Letm1 KD neurons; U = 72). d, Peak Mito^4x-GCaMP6f responses (ΔF/F₀) after stimulation. Non-significance (NS) was determined using a two-tailed Mann–Whitney U-test (P = 0.578; n = 27 for control neurons, n = 21 for Letm1 KD neurons; U = 256). e, Baseline mitochondrial Ca²⁺ concentration (nM) in control and Letm1 KD neurons. Non-significance was determined using a two-tailed unpaired t-test (NS, P = 0.6023, n = 16 control neurons, n = 11 Letm1 KD neurons, t₂₅ = 0.5279). f, Half-time decay in axonal mitochondria following stimulation in control, Letm1 KD neurons, Letm1 KD neurons expressing rat Letm1 wild-type protein and Letm1 KD neurons expressing rat Letm1 protein with the EF-hand domain mutated. P values were determined by a Kruskal–Wallis test followed by Dunn’s multiple-comparisons test against the control group (****P < 0.0001; n = 19 for control, n = 13 for Letm1 KD, n = 7 for wild-type (WT) Letm1 rescue and n = 8 for EF-hand mutant Letm1. Post hoc P values; control versus Letm1 KD, P = 0.0001; control versus Letm1 KD + Letm1 rescue, P = 0.3126; Letm1 KD + Letm1ΔEF-hand rescue, P = 0.0022). g, Dendritic mitochondrial Ca²⁺ responses to a single glutamate uncaging pulse in a single spine in control neurons and Letm1 KD neurons. Traces correspond to the mean ± s.e.m. of Mito^4x-GCaMP6f. h, Half-time decay in dendritic mitochondrial Ca²⁺ following stimulation in control and Letm1 KD neurons. P value was determined using a two-tailed Mann–Whitney U-test (***P < 0.0001, n = 63 dendrites for control, n = 47 dendrites for Letm1 KD, U = 809). i, Fold change in half-time decay in Letm1 KD neurons in axonal and dendritic compartments. P value was determined using a two-tailed unpaired t-test (****P < 0.0001, with n = 33 for axons and n = 47 for dendrites, t₇₈ = 4.959). Data are represented as the mean ± s.e.m. See Extended Data Figs. 1 and 2.

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