Fig. 3: Haploinsufficient reduction in UNC80 is associated with severe intellectual disability. | Nature Communications

Fig. 3: Haploinsufficient reduction in UNC80 is associated with severe intellectual disability.

From: Intellectual disability-associated UNC80 mutations reveal inter-subunit interaction and dendritic function of the NALCN channel complex

Fig. 3

a Sanger sequencing chromatograms confirming the whole-exome sequence findings of genetic variations in the proband leading to protein changes of Q340H, P341S (left), and E1295Q (right). Upper: from the proband; lower: control. Codons encoding Q340 (CAG), P341 (CCC) and E1295 (GAA) are underlined. b Protein sequence alignments showing conservation in the Q340, P341 (left) and E1295 (right) regions. Accession numbers of the sequences used are NM_032504 (human), NP_780719 (mouse), XP_015144792 (chicken), XP_009300567 (zebrafish), and XP_011676014 (sea urchin). c Western blots with proteins prepared from non-transfected HEK293T cells and those transfected with mouse UNC80 wild type, Q341H;P342S (corresponding to human Q340H;P341S) or E1296Q (corresponding to human E1295Q) mutants, blotted with anti-UNC80 antibody (upper) or anti-actin (lower) for loading control. More than three independent repeats were performed with similar results. d TTX-resistant Na+ leak current recorded from cultured UNC80 KO hippocampal neurons transfected with wild-type UNC80, E1296Q, Q341H;P342Q, or mixture of the two mutants, as indicated. Representative currents are in the left four subpanels and averaged current amplitudes are summarized in the right. Recordings were from −70 mV and were done with bath solutions with varying [Na+] (140 mM or 14 mM) and [Ca2+] (2 mM (2 Ca) or 0.1 mM (0.1 Ca)) (see Fig. 2 legend for details). Data are presented as mean values ± SEM. Numbers of neurons recorded are in parentheses. In the right bar graph, two sample t test of each group against the “−/−; WT UNC80” group (n = 17) was performed and the p values are as follows. ΔILNa (2 Ca): “−/−; E1296Q” (n = 17, p ≤ 0.001), “−/−; E1296Q + Q341H;P342S” (n = 10, p ≤ 0.001), “−/−; Q341H;P342S” (n = 9, p ≤ 0.001)). ΔILNa (0.1 Ca): “−/−; E1296Q” (p ≤ 0.001), “−/−; E1296Q + Q341H;P342S” (p ≤ 0.001), “−/−; Q341H;P342S” (p ≤ 0.001). ΔILCa (0.1 Ca–2 Ca): “−/−; E1296Q” (p ≤ 0.001), “−/−; E1296Q + Q341H;P342S” (p ≤ 0.001), “−/−; Q341H;P342S” (p ≤ 0.001). Asterisk “*” indicates p < 0.05. Source data are provided as a Source Data file.

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