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. 2021 Jun 2;4:666. doi: 10.1038/s42003-021-02114-0

Fig. 1. Genetic variation in human NCLX is linked to mental retardation and affects mitochondrial calcium efflux rates.

Fig. 1

a Genealogical tree of the affected family. Affected retarded siblings are marked in black. The location of the SLC8B1 gene in chromosome 12 is indicated. Both parents are carriers of the P367S variant of NCLX. b Predicted structure of NCLX. Yellow circle—location of P367 in the putative fifth transmembrane α-helix, adjacent to the catalytic site of NCLX. c P367 is phylogenetically conserved. Sequence alignment of a protein segment around P367 (red square) in various mammals and in fish illustrating its conservation across vertebrate species. d NCLX expression levels in HEK293T cells. Representative Western blot (n = 3 independent samples) of HEK cells expressing myc-WT NCLX (WT) or myc-NCLXP367S (P367S). mit – mitochondrial fraction (positive for the mitochondrial marker VDAC), cyt – cytoplasmic fraction (negative for VDAC). Anti myc immunoblotting revealed similar expression levels. Ctl – cells transfected with a control plasmid (pcDNA3.1 + ). e SH-SY5Y cells expressing NCLXP367S exhibit slower mitochondrial calcium efflux. Cepia2-mt and WT NCLX or NCLXP367S were co-expressed in SH-SY5Y cells in which the endogenous NCLX was knocked down by shRNA, and mitochondrial transients (Cepia2-mt fluorescence) were induced by bath application of ATP (n = 62 and 59 WT and P367S traces, mean ± SEM ΔF/F0). f Quantification of calcium efflux rates in e. A linear fit of a 150 s period after calcium levels started to decline served to determine the initial calcium efflux rate (0.166 ± 0.016, 0.073 ± 0.011 arbitrary units/minute in WT-NCLX and NCLXP367S expressing cells). ***p = 8e–6, Mann–Whitney u test (Z = −4.45, U = 990).