Fig. 6: UNC80’s C-terminus truncated in human patients is not required for whole-cell I_NALCN but is essential for survival.

a, b I_NALCN (a done as in Fig. 5b, averaged amplitudes at −100 mV given in the bar graph) or Na⁺-leak current (ΔI_LNa) (b) from HEK293T cells (a) or cultured UNC80 KO neurons (b recorded with 2 mM Ca²⁺ (2 Ca) or 0.1 mM Ca²⁺ (0.1 Ca) in the bath) transfected with full-length or mutant UNC80 truncated at L2654 (aa1–2653). c Surface biotinylation assays. Left, representative western blots. Surface proteins in cells transfected with cDNA combinations as indicated were biotinylated, isolated using streptavidin-conjugated beads, and probed with α-NALCN. Total protein (whole-cell lysate, WCL) was probed with α-NALCN or α-GFP. In the lower panel, GFP alone in the control lane migrated at ~20 kDa (outside the molecular range shown) and is not visible in the blot. Right, quantification. Signal intensity of each band from cells co-transfected with NALCN and full-length or truncated UNC80 was normalized to that from cells co-transfected with NALCN and GFP. d Sanger sequencing of the knock-in mice (L2654*, lower) and WT (upper). In the L2654* mice, the leucine-encoding codon (CTA, L2654) is mutated to a stop codon (TAA,*). e Representative appearances of WT and homozygous L2654* mutant P0 pups. For apnea phenotypes, see Supplementary Movie 2. f ΔI_LNa recorded from neurons cultured from WT and L2654* pups. Data are presented as mean values ± SEM. In the bar graphs, numbers of recordings or repeats are in parentheses. Two sample t tests were performed. Asterisk “*” indicates p < 0.05. p values are as follows. a (against the “full-length” group): “Mock” (p ≤ 0.001), “1–2653” (p ≤ 0.001), b (between the group of “−/−; UNC80 full-length” (full-length UNC80-transfected) and the group of “−/−; (1–2653)” (truncation mutant-transfected)): ΔI_LNa (2 Ca) (p ≤ 0.001), ΔI_LNa (0.1 Ca) (p ≤ 0.001), ΔI_LCa (0.1 Ca–2 Ca) (p ≤ 0.001), c (against the group of “GFP” (GFP-transfected)): “full-length” (p = 0.025), “1–2653” (p = 0.049). Source data are provided as a Source Data file.