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 GDC-0449 price 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 Gemcitabine nmr 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 unless 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.