Akinesia and freezing caused by Na+ leak‐current channel (NALCN) deficiency corrected by pharmacological inhibition of K+ channels and gap junctions

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The Na+ leak‐current channel (NALCN) is a nonselective cation channel that mediates transmembrane flux of Na+, Ca++, and K+ ions (Ren, 2011; Cochet‐Bissuel et al., 2014). Along with the channel, there are two conserved auxiliary subunits, encoded by unc‐79 and unc‐80, which promote the membrane localization and stabilization of the channel and/or modulate channel gating and kinetics (Jospin et al., 2007; Yeh et al., 2008; Lu et al., 2010). The Caenorhabditis elegans homologs of mammalian NALCN, UNC‐77 (NCA‐1) and NCA‐2, function redundantly to regulate locomotion (Humphrey et al., 2007). Animals with loss‐of‐function (lf) mutations that affect the C. elegans NALCNs (NCA‐1 and NCA‐2) and associated proteins (UNC‐79 and UNC‐80) have low levels of spontaneous movement (akinesia), an abnormally extended posture, and faint or freeze in response to touch on the tail (Sedensky and Meneely, 1987; Yeh et al., 2008). Mutation of the single NALCN ortholog in Drosophila, called narrow abdomen (na), causes hesitant walking and disturbs circadian rhythms (Nash et al., 2002). These phenotypes in NALCN‐deficient animals compare closely with certain features/symptoms of Parkinson's disease (PD) or primary progressive freezing gait (Bonnett et al., 2014). Rare genetic mutations in human NALCN cause disordered movement, developmental delay, hypotonia, and intellectual impairment (Cochet‐Bissuel et al., 2014; Chong et al., 2015). Therefore, understanding how the lf mutations disrupt the function of the NALCN in C. elegans may provide broader insights into how this protein regulates locomotion in mammals and how it may contribute to certain movement and neuropsychiatric disorders.
The mechanisms through which loss‐of‐function in NALCN causes akinesia and freezing are not known. Hippocampal neurons of Nalcn knockout mice are hyperpolarized by around 10 mV (Lu et al., 2007) relative to control neurons demonstrating a role in the maintenance of the resting membrane potential. This means that neurons lacking the NALCN will be more difficult to depolarize or that neuronal output in response to a signal of fixed size will be less than normal. Consequently, akinesia may result from signal failure in key interneurons or motor neurons. In addition, the NALCN determines the balance between Na+, Ca++, and K+ following depolarization, and thus influences the duration of the depolarization–repolarization cycle (Lear et al., 2005; Bonnett et al., 2014; Gao et al., 2015). Loss of the NALCN would shift the balance toward hyperpolarization after a depolarizing stimulus (tail touch), which could explain freezing and also the low level of spontaneous movement. If relative hyperpolarization is part of the problem in NALCN‐deficient animals, it should be possible to overcome this defect by decreasing K+ flux in neurons. In addition, there may be an inadequate Ca++ response that can be corrected in animals with NALCN deficits by promoting Ca++ influx. We have tested these possibilities by pharmacological inhibition of various K+ channels and activation of voltage‐gated Ca++ channels. Here we report that these two approaches significantly improve locomotion in NALCN‐deficient strains.
Genetic analysis has revealed that concomitant knockout of NALCNs and gap junction (innexin) genes, either unc‐7 or unc‐9, corrected anesthetic and ethanol sensitivity (Sedensky and Meneely, 1987; Morgan and Sedensky, 1995), and freezing in C. elegans (Bouhours et al., 2011). According to our current model, knockout of gap junctions could be beneficial because it prevents the spread of negative hyperpolarizing signals between connected neurons or because it blocks the dissipation of positive depolarizing signals (e.g., Ca++ influx) that initiate and sustain movement. We sought to clarify the crosstalk between gap junctions and the NALCN by evaluating the effect of gap junction inhibitors on spontaneous locomotion and freezing.
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