These interactions required its S5/P loop/S6 segment (Figure 7B, compare constructs 2 and 3). Replacing this segment with an analogous region of a P/Q/N-type VGCC UNC-2,

or a L-type VGCC EGL-19 also abolished the interaction (Figures S7B and S7C). Other NALCN channel AZD9291 components (mUNC-79 and mUNC-80), and an innexin channel (UNC-7), did not exhibit interactions with NLFs (not shown). Our molecular genetic, biochemical and physiological analyses uncover NLF-1/mNLF-1, a conserved ER regulator of a Na+ leak channel NCA/NALCN, which maintains the RMP and activity of a small premotor interneuron network responsible for the maintenance of C. elegans’ rhythmic locomotion ( Figure 7D). Our current data suggest a remarkable functional specificity of NLF-1 with a Na+ leak channel NCA. nlf-1 mutants exhibit behavioral phenotypes unique and characteristic of the loss-of-function mutants for the NCA channel components, with no additional phenotypes from nca(lf). nlf-1 null alleles do not enhance nca(lf) defects in locomotion or in AVA membrane properties. Other C. elegans

cation channel mutants, while uncoordinated in locomotion, do not faint. nlf-1 suppresses the nca(gf) movement pattern but does not suppress that of VGCC(gf) mutants. Genetically, these results place nlf-1 fairly specifically in the biological CHIR-99021 in vitro pathway as the nca genes. Consistently, all NCA channel component reporters, despite being overexpressed, exhibit drastic reduction of axonal localization in the absence of NLF-1. On the other hand,

sequence-related VGCC reporters are unaffected in nlf-1 mutants, although a subtle difference of endogenous level could be masked by reporter overexpression. NLF-1 may achieve its functional CYTH4 specificity as an auxiliary subunit unique for the Na+ leak channel. Multiple lines of evidence, however, suggest NLF-1’s role at the ER. NLF-1, as well as ectopically expressed mNLF-1, are restricted at the ER of C. elegans neurons. mNLF-1 also localizes to the ER in yeast and mammalian cells. Importantly, disrupting NLF-1’s ER localization diminishes, or severely reduces its rescuing ability of nlf-1 mutants. Although many ER proteins are promiscuous facilitators for the folding and delivery of membrane proteins, ER resident proteins with remarkable substrate and functional specificity, such as RIC-3 that facilitates the surface expression of subtype nicotinic acetylcholine receptors (Halevi et al., 2002; Lansdell et al., 2005), CALF-1 that affects axon localization of the C. elegans P/Q/N-type VGCC UNC-2 ( Saheki and Bargmann, 2009), and SARAF that interacts with STIM1 to regulate store-operated calcium entry ( Palty et al., 2012), are present. NLF-1 may represent another example of an emerging class of ER proteins with substrate specificity.