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NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN's most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd ( 3+) -sensitive, NMDG (+) -impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (ILP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Nax or Cav 1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels. Show less

Pacemaker cells play a fundamental role in generating or regulating many essential biological rhythms. Spontaneous pacemaker activity is dependent on the function of an array of ion channels expressed in these cells. Recent characterization of a Na(+) leak channel (NALCN) has linked to its role in conducting the background Na(+) current that depolarizes resting membrane properties of pacemaker neurons. NALCN, along with Unc79 and Unc80, forms a protein complex that is involved in regulating intrinsic membrane and synaptic activities. In this review, we will discuss the current understanding of NALCN channel physiology and its role in regulating cell excitability and pacemaker activity. Show less

The abused volatile solvent toluene shares many behavioral effects with classic central nervous system depressants such as ethanol. Similarities between toluene and ethanol have also been demonstrated using in vitro electrophysiology. Together, these studies suggest that toluene and ethanol may be acting, at least in part, via common mechanisms. We used the genetic model, Caenorhabditis elegans, to examine the behavioral effects of toluene in a simple system, and used mutant strains known to have altered responses to other CNS depressants to examine the involvement of those genes in the motor effects induced by toluene. Toluene vapor brings about an altered pattern of locomotion in wild-type worms that is visibly distinct from that generated by ethanol. Mutants of the slo-1, rab-3 and unc-64 genes that are resistant to ethanol or the volatile anesthetic halothane show no resistance to toluene. A mutation in the unc-79 gene results in hypersensitivity to ethanol, halothane and toluene indicating a possible convergence of mechanisms of the three compounds. We screened for, and isolated, two mutations that generate resistance to the locomotor depressing effects of toluene and do not alter sensitivity to ethanol. In C. elegans, ethanol and toluene have distinct behavioral effects and minimal overlap in terms of the genes responsible for these effects. These findings demonstrate that the C. elegans model system provides a unique and sensitive means of delineating both the commonalities as well as the differences in the neurochemical effects of classical CNS depressants and abused volatile inhalants. Show less

Extracellular K⁺, Na⁺, and Ca²⁺ ions all influence the resting membrane potential of the neuron. However, the mechanisms by which extracellular Na⁺ and Ca²⁺ regulate basal neuronal excitability are not well understood. Recent findings suggest that NALCN, in association with UNC79 and UNC80, contributes a basal Na⁺ leak conductance in neurons. Mutations in Nalcn, Unc79, or Unc80 lead to severe phenotypes that include neonatal lethality and disruption in rhythmic behaviors. This review discusses the properties of the NALCN complex, its regulation, and its contribution to neuronal function and animal behavior. Show less

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans. Show less

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)'s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCN's intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion. Show less

Alternative patterns of neural activity drive different rhythmic locomotory patterns in both invertebrates and mammals. The neuro-molecular mechanisms responsible for the expression of rhythmic behavioral patterns are poorly understood. Here we show that Caenorhabditis elegans switches between distinct forms of locomotion, or crawling versus swimming, when transitioning between solid and liquid environments. These forms of locomotion are distinguished by distinct kinematics and different underlying patterns of neuromuscular activity, as determined by in vivo calcium imaging. The expression of swimming versus crawling rhythms is regulated by sensory input. In a screen for mutants that are defective in transitioning between crawl and swim behavior, we identified unc-79 and unc-80, two mutants known to be defective in NCA ion channel stabilization. Genetic and behavioral analyses suggest that the NCA channels enable the transition to rapid rhythmic behaviors in C. elegans. unc-79, unc-80, and the NCA channels represent a conserved set of genes critical for behavioral pattern generation. Show less

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the alpha1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons. Show less

Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1, 2]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action. Show less

The authors tested whether mutant strains of Caenorhabditis elegans with altered sensitivity to volatile anesthetics have altered responses to GABA or GABA-agonists. They determined the ED50s of the wild-type strain N2 and two mutant strains of C. elegans to a GABA-mimetic ivermectin (IVM) and to GABA. unc-79, a strain with increased sensitivity to halothane, was more sensitive than N2 to IVM and GABA. unc-9, a strain that suppresses the increased sensitivity of unc-79 to halothane, was less sensitive than N2 to IVM and GABA. The authors also tested whether doses of GABA or IVM and volatile anesthetics were additive in their effects on C. elegans. Halothane (2.1%) did not shift the ED50 of IVM, but was antagonistic to GABA. Enflurane (4%) was antagonistic to both IVM and GABA. However, ED50s of halothane and enflurane were unchanged in the presence of IVM (35 nM) or GABA (150 mM). The authors conclude that GABA by itself does not appear to mediate halothane or enflurane sensitivity in C. elegans. Show less

The mechanism and site(s) of action of volatile anesthetics are unknown. In all organisms studied, volatile anesthetics adhere to the Meyer-Overton relationship--that is, a ln-ln plot of the oil-gas partition coefficients versus the potencies yields a straight line with a slope of -1. This relationship has led to two conclusions about the site of action of volatile anesthetics. (i) It has properties similar to the lipid used to determine the oil-gas partition coefficients. (ii) All volatile anesthetics cause anesthesia by affecting a single site. In Caenorhabditis elegans, we have identified two mutants with altered sensitivities to only some volatile anesthetics. These two mutants, unc-79 and unc-80, confer large increases in sensitivity to very lipid soluble agents but have little or no increases to other agents. In addition, a class of extragenic suppressor mutations exists that suppresses some altered sensitivities but specifically does not suppress the altered sensitivity to diethyl ether. There is much debate concerning the molecular nature of the site(s) of anesthetic action. One point of discussion is whether the site(s) consists of a purely lipid binding site or if protein is involved. The simplest explanation of our observations is that volatile anesthetics cause immobility in C. elegans by specifically interacting with multiple sites. This model is in turn more consistent with involvement of protein at the site(s) of action. Show less

The authors studied the wild type strain, N2, and three mutant strains of the nematode, Caenorhabditis elegans, in order to measure genetically produced changes in responses to nine volatile anesthetics. They determined the anesthetic ED50s of N2 for thiomethoxyflurane, methoxyflurane, chloroform, halothane, enflurane, isoflurane, fluroxene, flurothyl, and diethylether. The log-log relationship of the oil-gas partition coefficients (O/G) and the ED50s of these agents for N2 yields a straight line with a slope of -.997 with a R2 of .98 over a range of O/G (at 37 degrees C) from 48 to 7230. When the O/Gs are corrected to 22 degrees C, the slope is -.964 with an R2 of .98. This relationship is similar to that found in other animals. Two mutant strains, unc-79 and unc-80, show altered responses to these anesthetics. These strains are two to three times more sensitive than N2 to anesthetics with an O/G greater than that of halothane (220 at 37 degrees C), yet they differ little from N2 in response to anesthetics with lower O/Gs. unc-79 and unc-80 are about 30% more sensitive than N2 to diethylether. The double mutant unc-79; unc-80 is more sensitive to halothane, isoflurane, and fluroxene than is either mutant alone. The authors believe these data indicate an alteration at the site of action of volatile anesthetics in unc-79 and unc-80. They also postulate that the interaction of unc-79 and unc-80 indicate these genes code for enzymes in a common pathway, and that unc-79 precedes unc-80 in this pathway. Show less

The nematode Caenorhabditis elegans appears to be a useful model for studying the action of volatile anesthetics. A mutant strain that is hypersensitive to the widely used anesthetic halothane was described earlier. The mutation is now shown to be an allele of unc-79. Other alleles of unc-79 are also associated with hypersensitivity to halothane. A strain with a mutation in a second gene, unc-80, is also hypersensitive to halothane. Nematodes bearing mutations in both unc-79 and unc-80 are slightly more sensitive to halothane than those bearing only one of these mutations. Mutations in a third gene, unc-9, suppress both unc-79 and unc-80. Nematodes bearing the suppressor mutations alone have normal sensitivity to halothane. These results show that sensitivity to halothane can be altered by mutations in several different genes. Show less