Another possibility is that a small fraction of the NCA channels could remain localized and functional in the absence of NLF-1.

We favor the second possibility because expressing either buy Forskolin NLF-1 or NCA-1 in the GABAergic motor neurons did not result in any noticeable improvement in the locomotion deficit in nlf-1 or nca(lf) mutants (data not shown). Moreover, this small behavioral difference coincided with a subtle, yet also consistent, reduction in Na+-dependent change in background leak current (ΔI; Figure 4C) and RMP (ΔV; Figure 4F) in the AVA premotor interneurons in nlf-1 when compared to that in nca(lf) mutants. We provide the first direct, physiological evidence for the NCA channel’s role in maintaining neuronal RMP. Thus, in both the nonspiking Wnt inhibitors clinical trials C. elegans and spiking vertebrate neurons, this Na+ leak channel constitutes a conserved mechanism that modulates neuronal excitability. Despite a modest sequence homology between NLF-1 and mNLF-1, and the differences in neuronal properties between vertebrate and C. elegans interneurons, mNLF-1 fully substitutes

for NLF-1 when expressed in C. elegans. mNLF-1 exhibits enriched, broad expression in the mouse brain (http://mouse.brain-map.org). shRNA-mediated knockdown of mNLF-1 effectively, albeit partially reduces the Na+ leak currents in primary cortical neuron cultures. Thus, while mNLF-1’s physiological function awaits further investigation, our current studies imply its role in the folding/trafficking of ADAMTS5 the NALCN Na+ leak channel. The ability of mNLF-1 to substitute NLF-1 in the C. elegans nervous system further highlights the conservation of machineries that modulate membrane physiology. Removing extracellular Na+ leads to a further hyperpolarization of RMP in both nca(lf) and nlf-1 mutants, indicating the presence of additional Na+ channels in modulating neuronal excitability. As the C. elegans genome does not encode voltage-gated Na+

channels, machineries that carry out the remaining Na+ conductance remain to be identified. How the Na+ leak channel affects intact neural circuit activity remains to be explored. Intriguingly, nalcn−/− mice cannot generate respiratory rhythm ( Lu et al., 2007); na flies fail to exhibit motor patterns associated with circadian cycles ( Lear et al., 2005; Nash et al., 2002; Zhang et al., 2010); the knockdown of a snail NALCN homolog compromises respiratory function ( Lu and Feng, 2011); the loss of NCA leads to disrupted rhythmicity in C. elegans locomotion ( Pierce-Shimomura et al., 2008; this study). Collectively, these phenotypes imply a requirement of this channel in neural networks generating rhythmic behaviors. Despite a broad expression in the C. elegans motor circuit, NLF-1 activity in motor neurons was neither necessary, nor sufficient to restore the continuity of locomotion.