Fig. 4: UNC80 and UNC79 are auxiliary subunits of the NALCN channel complex.

a Protein fractionation demonstrating that UNC80 and UNC79 co-segregate with NALCN to the membrane fraction. Total proteins (T) from adult brains were centrifuged at 200,000 × g and separated into the cytosolic (supernatant, S) and microsomal membrane (pellet, P) fractions, as illustrated in the schematic diagram (upper). Each fraction was blotted with anti-NALCN, anti-UNC80, anti-UNC79, or anti-actin antibody. b A knock-in mouse line with NALCN tagged with GFP, HA, and His tags (NALCN-GFP-HA-His mice). Upper, schematic design. Lower, total brain proteins (100 μg) prepared from the triple-tagged mice and wild-type (non-tagged) mice were immunoblotted with anti-His antibody. c TTX-resistant Na⁺ leak currents recorded from neurons cultured from the KI pups (n = 5). Left, representative current traces. Right, averaged current amplitudes. 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). Numbers of neurons recorded are in parentheses. d Protein depletion demonstrating that all UNC80 and UNC79 proteins are associated with NALCN. Total brain protein lysates were prepared from the NALCN-GFP-HA-His mice. NALCN was depleted by incubating with Ni column (binding to 6-His) followed by further immune depletion with anti-GFP antibody. Lysates before (lane 1) and after (lane 2: with Ni-beads, lane 3: with α-GFP agarose) depletion were blotted with anti-NALCN, anti-UNC79, or anti-UNC80 antibody. Anti-actin was used as a control. For (a, b, d), three or more independent repeats were performed with similar results. Data are presented as mean values ± SEM. Source data are provided as a Source Data file.