The most obvious function of the tonically active background Na⁺ conductance is perhaps to balance the K⁺ leak to set the RMP, which would be at ~ −90 mV (E_K) in all the neurons if there were only basal K⁺ conductance. A tonic leak of other ions such as Ca²⁺, Mg²⁺ and H⁺ can hypothetically achieve the same goal, but excessive leak of these ions into neurons can be damaging to the cells because of the cellular metabolism’s high sensitivity to the intracellular concentrations of the ions. By varying the basal P_Na/P_K, the nervous system can have a wide range of RMPs among different neurons, a heterogeneity in neuronal intrinsic properties known to exist in the brain (Kandel et al., 2000; Llinas, 1988).

Another function of the Na⁺ conductance is to provide a regulation of the membrane potential by environmental stimuli. One such example was demonstrated in the excitation of sympathetic ganglion neurons in the frog (Jan and Jan, 1982; Kuffler and Sejnowski, 1983). In addition to small-molecule neurotransmitters such as acetylcholine, the spinal nerves also release peptide neurotransmitters such as LHRH- and substance P (SP)-like peptides to excite the sympathetic ganglion neurons. These peptides elicit “late slow” depolarization that lasts minutes (Jan and Jan, 1982; Kuffler and Sejnowski, 1983). In addition to suppressing K⁺ currents such as the M-current, the neurotransmitters can also excite the neurons through activation of a Na⁺-dependent basal cation current that is apparently carried by the basal Na⁺ leak conductance (Brown and Adams, 1980; Jones, 1985; Kuba and Koketsu, 1978). Such a mechanism of excitation through the activation of Na⁺-leak-like basal conductances has also been found in the excitation of serotonin neurons in the dorsal raphe nucleus by orexin (Liu et al., 2002), VTA dopaminergic neurons by SP and neurotensin (Farkas et al., 1996), locus coeruleus neurons by SP and muscarine (Shen and North, 1992a; Shen and North, 1992b), and pre-Bötzinger complex neurons by serotonin and SP (Pena and Ramirez, 2004; Ptak et al., 2009). Similarly, suppression of a Na⁺ leak-like current can lead to hyperpolarization by driving the RMP toward E_K, as suggested in the gastrin-releasing peptide containing retinorecipient neurons in the suprachiasmatic nucleus (SCN). In these neurons, a one-hour light exposure causes a large reduction (> 100 pA) of what appears to be a Na⁺-leak current and a hyperpolarization of membrane potential by 15 mV (LeSauter et al., 2011).

Several other channel families having a 6-TM structure also share high sequence similarity with the NALCN/Na_V/Ca_V family (Fig. 1). These include the bacterial voltage-gated Na⁺ channels (Na_VBac) (Ren et al., 2001b), the bacterial putative voltage-gated Ca²⁺ channels (Ca_VBac), and the pH-gated Ca²⁺ channels (CatSpers1-4) that are found in both vertebrates and invertebrates and, in mammals, are localized to sperm and required for male fertility (Ren and Xia, 2010). CavBac has not been electrophysiologically characterized but, because of its TFEDWTD sequence in the pore region (Fig. 2), is predicted to be Ca²⁺-selective based on mutagenesis studies done to convert the NaChBac Na⁺ channel to a Ca²⁺ channel CaChBac (Yue et al., 2002). NALCN, Ca_V, Na_V, Na_VBac, Ca_VBac, and CatSper channels contain highly homologous sequences in the S1–S4 voltage-sensing domains (VSDs) and, in particular, the channel pore regions; thus, the 24-TM channels likely evolved through duplication from 6-TM bacteria channels. Consistent with this idea, a transitional state can be observed in the two-pore segment channels (TPC), which have two six-transmembrane domain segments (12-TM) (Ishibashi et al., 2000). Mammals have two TPC channels, TPC1 and TPC2. Their overall sequences have similarity with those of the 24-TM channels, although not as high as that found among the other families. TPCs are intracellular cation channels localized on lysosomes and endosomes (Calcraft et al., 2009).

Unlike some channels, such as Na_V and CatSper, NALCN is found in all animals studied, from humans to D. melanogaster, C. elegans, snails, sea urchins, and the placozoan Trichoplax adhaerens. Most species appear to have only one Nalcn gene; however, C. elegans, which does not have Na_V channel, has two Nalcn genes. Similar to the other 24-TM channels, NALCN is not found in plants such as Arabidopsis. Intriguingly, Saccharomyces cerevisiae (budding yeast) has only one 24-TM channel (CCH1), whose nearest homolog is NALCN, suggesting an early appearance of 24-TM channels in eukaryotes during evolution (Hong et al., 2010).

The existence of NALCN as the third branch of ion channels in the 24-TM channel family was first evident when large amount of genomic and cDNA sequences became available in the late 1990s. By searching databases with template sequences from Na_vs and high-voltage-activated Ca_Vs (the only known 24-TM channels at that time), partial sequences encoding novel channels with sequences similar to those of Na_vs and Ca_vs, especially in the pore region, were found. These include the T-type Ca_Vs (Perez-Reyes et al., 1998), TPCs (Ishibashi et al., 2000), CatSpers (Ren et al., 2001a), and NALCN from rats (named as Rb21)(Lee et al., 1999), humans (VGCNL1), C. elegans (NCA) and Drosophila (Dma1U for unique α1 subunit) (Littleton and Ganetzky, 2000). The in vivo importance of NALCN was first revealed by the findings that several alleles of an existing Drosophila mutant (na for narrow abdomen, and har for halothane resistance) have a 9 nt deletion (in the na allele) predicted to lead to a deletion/alteration of four amino acids (na) or a point mutation predicted to alter RNA splicing (har) in the Dma1U gene (Nash et al., 2002). These hypomorphic mutant flies have severely reduced expression of the NALCN ortholog. They are viable but have disruptions in circadian rhythm and sensitivity to halothane. Nalcn mutations leading to significant phenotypes were later reported C. elegans (Humphrey et al., 2007; Jospin et al., 2007; Pierce-Shimomura et al., 2008; Yeh et al., 2008) and the mouse (Lu et al., 2007) (see more discussions in later sections.)

NALCN has several unique biophysical properties (Lu et al., 2007). For example, it is voltage-independent, with a linear current (I) −voltage (V) relationship over the tested range of −100 mV to +100 mV (Fig. 2). In addition, NALCN does not inactivate. Interestingly, NALCN is the only nonselective channel found in the 24-TM channel family and is equally permeable to Na⁺, K⁺, and Cs⁺. At RMPs, which are normally close to E_K, the major charge-carrying ion for NALCN is Na⁺.

Consistent with its unique functional properties, the NALCN protein also has two quite unusual structural features that separate itself from the other 20 members of the 24-TM family: these are the S4 segments and the pore region sequences. In K_Vs, Ca_Vs, and Na_Vs, charged residues (lysine, arginine: K/R) are present in every third position along the S4 segments. In NALCN, the S4 segments have fewer charged residues (13 vs. the 21 found in Na_V1.1 or Ca_V1.1). The S4 of domain IV (IVS4) of NALCN has only two charged residues, while Ca_Vs and Na_Vs have more than four, and these two residues are not evenly spaced in an every third position manner (Fig. 3A). In Na_vs, all the S4s contribute to channel activation, although unequally (Bezanilla, 2000; Kontis et al., 1997). MTSET accessibility studies also show that, in Na_v1.1, two of the charged residues in IVS4 (missing at NALCN’s corresponding positions) move from internally accessible positions to extracellularly accessible ones (Yang et al., 1996). In addition, the charged residues in the S4s, especially of domains III and IV, are also important for Na_v inactivation (Cha et al., 1999). However, difference in the S4s alone may not explain why NALCN is voltage insensitive and doesn’t have inactivation. Indeed, a mutant tetrameric K⁺ channel can still be voltage-gated even when artificially engineered to have only one 6-TM subunit (equivalent to one of the four domains in the 24-TM channels) with an intact S4 but the other three without any charged residues in their S4s (Gagnon and Bezanilla, 2009). On the other hand, cyclic-nucleotide-gated (CNG) channels made of tetramers of 6-TM proteins are only weakly voltage-sensitive despite having charged residues in their S4s. Indeed, when the S4 of CNGA2 is used to replace that of the EAG (KCNH2) K_v channel, it is fully functional in sensing voltage changes and in supporting a voltage-gated K⁺ channel (Tang and Papazian, 1997). It therefore remains possible that NALCN’s voltage insensitivity lies in regions besides the S4s, such as the C-terminal part of S3 and the S3–S4 linker that together with S4 form the voltage-sensor paddle as shown in the crystal structure of K_v channels (Jiang et al., 2003). Alternatively, NALCN’s VSDs may be functional but there is “defect” in the coupling between voltage-sensing and channel gating. The functionality of NALCN’s four VSDs can be tested by transferring each of them into homotetrameric K_v channels (Bosmans et al., 2008; Xu et al., 2010).

The second unique feature of NALCN is its pore filter (Fig. 3B). The selectivity filter in Ca_V, Na_V, and K_V is surrounded by the VSDs and is formed by the S5–S6 pore (P) loops that are contributed by each 6-TM domain (Doyle et al., 1998; Jiang et al., 2003; MacKinnon, 1995; Miller, 1995; Payandeh et al., 2011). In Ca_Vs, the Ca²⁺ selectivity requires one glutamate (E) or aspartate (D) residue contributed from each of the four homologous repeats (EEEE motif) in the pore filter (Heinemann et al., 1992; Yang et al., 1993). Na_Vs have a DEKA motif in the analogous position (Fig. 3B). NALCN has an EEKE motif, a combination of the EEEE (Ca_V) and DEKA (Na_V) motifs. The EEKE motif is conserved in NALCN homologs in mammals, D. melanogaster and C. elegans. NALCN from the fresh water snail Lymnaea stagnalis has an EKEE motif (Lu and Feng, 2011). In Na_v, mutating the DEKA motif into DEKE converts the Na⁺ selective channel into a channel conducting primarily Na⁺ but also some K⁺ and Ca²⁺ (Schlief et al., 1996). Likewise, mutating the EEEE motif of Ca_Vs into EEKE enables the otherwise highly Ca²⁺-selective channels permeable to monovalent ions (Parent and Gopalakrishnan, 1995; Tang et al., 1993; Yang et al., 1993). Therefore, NALCN’s EEKE motif, and perhaps together with sequences surrounding it, is likely responsible for the channel’s relative lack of selectivity among monovalent cations (Williamson and Sather, 1999; Yue et al., 2002). The in vivo significance of NALCN’s EEKE motif has been demonstrated by the finding that a mutant cDNA encoding an EEEE motif, when transgenically expressed in the Drosophila na mutant, is much less capable of rescuing the phenotypes than the wild-type cDNA (Lear et al., 2005). This rescue experiment with pore mutants also provided the in vivo evidence confirming that NALCN is indeed an ion channel.

The elucidation of the NALCN complex has been greatly facilitated by genetic studies in Drosophila, C. elegans, and mice. Several lines of evidence suggest that the NALCN complex contains the pore-forming subunit, NALCN itself, and two additional proteins, UNC79 and UNC80 (ortholog of C. elegans Unc-79 and Unc-80, respectively). First, mutations in Unc79 and Unc80 in Drosophila, C. elegans, and the mouse have essentially identical phenotypes to those of Nalcn mutants. Second, mutation in one of these genes also affects the protein levels and/or localization of the others (Humphrey et al., 2007; Jospin et al., 2007; Nakayama et al., 2006; Pierce-Shimomura et al., 2008; Yeh et al., 2008). Defining evidence that UNC79 and UNC80 form a physical complex with NALCN came from co-immunoprecipitation experiments in mouse brain showing that antibody against any one of the three could bring down the others (Lu et al., 2009; Lu et al., 2010). Whether there are other core subunits in this complex awaits the purification of the protein complex and a determination of the subunit composition.

UNC79 and UNC80 are well conserved among animals but share no obvious sequence similarity with any other protein with known function (Humphrey et al., 2007; Jospin et al., 2007; Lu et al., 2009; Lu et al., 2010). Both are large proteins (2654 aa and 3326 aa, respectively, in humans), larger than any known auxiliary subunit in the 24-TM channel family. Despite their size, there are no obviously identifiable protein domains in UNC79 and UNC80. The lack of sequence similarity among the auxiliary subunits of the NALCN, Na_Vs, Ca_Vs, and CatSper suggest that, unlike the pore-forming subunits, the auxiliary subunits evolved independently.

In heterologous expression systems, UNC80 and NALCN have been shown to interact (Lu et al., 2009; Lu et al., 2010). UNC79 and UNC80 also associate with each other, and UNC79 requires the presence of UNC80 to associate with NALCN. These data suggest a NALCN complex model whereby UNC80 serves as a bridge between UNC79 and NALCN (Fig. 4).

Perhaps the most common phenotype resulting from mutations in any one of the three NALCN complex components is the disruption in rhythmic behaviors. In mammals, the rhythmic contraction of the diaphragm muscle used for breathing is directly controlled by electrical signals from the nerves. The respiratory rhythms are generated in regions such as the pre-Bötzinger complex (PBC) in the brain stem through network mechanisms and/or together with pacemaking mechanisms (Feldman et al., 2003; Ramirez et al., 2004). Nalcn mutant mouse pups have severely disrupted respiratory rhythm. Wild-type newborn pups have a rhythmic breathing at a frequency of about one breath per second. In the Nalcn mutant, the breathing is characterized by 5 seconds of apnea followed by 5 seconds of breath. This disrupted breathing rhythm represents an “electrical defect,” as the rhythmic electrical discharges recorded from wild-type C4 nerves are essentially absent in the Nalcn mutant (Lu et al., 2007). NALCN is also expressed in the spinal cord. Whether it is involved in the central pattern generators used for rhythmic locomotion such as walking and running requires studies using conditional knockouts with NALCN disrupted in the spinal cord.

Defects in rhythmic behaviors are also obvious in the Drosophila melanogaster, and C. elegans mutants. In the fly, hypomorphic alleles of the Nalcn ortholog (Na), though viable, display altered circadian locomotor rhythms (Nash et al., 2002). Under diurnal light/dark (LD) cycle, the mutant flies have more activity in the darkness but suppressed activity in the light cycles, a pattern “inverted” to what’s seen in the wild-type. When released to free-running condition in constant darkness (DD) from the entrainment of diurnal LD cycles, many mutant flies quickly become arrhythmic. In addition, the mutant flies do not seem to have the light-on response characterized by a marked increase in locomotor activities in the wild-type when light is turned on. Indeed, the mutant’s activities quickly decrease (Nash et al., 2002).

In both Unc79 and Nalcn mutants, flies also have a “fainter” locomotion phenotype characterized by “hesitant” walking and frequent disruptions of the rhythmic, smooth movements shown in wild-type flies climbing up vial walls after being tapped to the bottom (Humphrey et al., 2007; Nash et al., 2002). Similarly disrupted locomotion rhythms are found in C. elegans Nalcn (NCa), unc-79, and unc-80 mutants (Jospin et al., 2007; Pierce-Shimomura et al., 2008; Yeh et al., 2008). In the worm, at least two rhythmic locomotion patterns with quite distinct kinematics are used: the animal crawls while in solid food but switches to swimming when dropped into liquid (Pierce-Shimomura et al., 2008). A smooth switch between the two behaviors requires sensory neurons. In a forward genetic screening, unc-79 and unc-80 mutants were identified to have relatively normal crawling (though with the “fainter” phenotype) in solid but unable to switch to a normal swimming pattern. Indeed, the mutant worms become paralyzed in the liquid instead of expressing a smooth swimming locomotion pattern as seen in the wild-type. A similar swimming phenotype also exists in the C. elegans NCa mutant (Pierce-Shimomura et al., 2008). In the snail Lymnaea stagnalis, the rhythmic bursting of action potentials in the pacemaker RPeD1 neurons are abolished and the respiratory behaviors of the animal are disrupted when NALCN is knocked down with siRNA (Lu and Feng, 2011).

The reason for the apparent conservation of NALCN’s role in rhythmic behaviors is a matter of speculation. In Drosophila, the expression of NA (NALCN ortholog) itself doesn’t seem to have a circadian oscillation. In addition, the oscillation of key circadian proteins in the “central clock” such as PERIOD appears normal in the fly na mutant. These findings suggest that NA is not part of the core circadian oscillator but rather, the defect in circadian rhythms of locomotor activities in the mutant is a result of disruption in the coupling between the central clock and the neuronal networks controlling locomotion (Lear et al., 2005; Nash et al., 2002). Consistent with this prediction, the pacemaker neurons in the mutant have higher level of PDF (pigment-dispersing factor, a neuropeptide released by the pacemaker neurons to coordinate circadian behaviors in the flies), suggesting a potential decrease in the release of PDF (and/or an increase in production) by these neurons in the mutant (Lear et al., 2005). Transgenic expression of NA in circadian pacemaker neurons in the na mutant using the Gal4-UAS system restores the circadian phenotypes (Lear et al., 2005). Remarkably, NA expression in a small subset of the neurons (~ 20 DN1 dorsal neurons) is sufficient to rescue some of the phenotypes, including the acute light-on locomotor activity response (Zhang et al., 2010). It’s not clear whether any residual function of the NA protein in the hypomorphic mutant used for the rescue experiment, if present, plays a supporting role in the other neurons. How NA contributes the fly circadian responses remains further investigated, but it’s interesting to note that mammalian NALCN is activated by neuropeptides in hippocampal, VTA and pre-Bötzinger complex pacemaking neurons (Lu et al., 2009; Pena and Ramirez, 2004; Ptak et al., 2009), and the channel appears to be controlled by light input in the SCN (LeSauter et al., 2011).

In autonomously firing neurons and pacemaking neurons within a local circuitry, NALCN as a channel that leaks Na⁺-mediated current may provide a constant, non-inactivating, depolarizing force used to generate or modulate the rhythmic electrical activities for the control of behaviors (Atherton and Bevan, 2005; Jackson et al., 2004; Khaliq and Bean, 2010; Ptak et al., 2009; Raman et al., 2000; Russo et al., 2007). Oscillation of membrane potential is not restricted to neurons in the brain and spinal cord, but rather can be found throughout the body and is perhaps best characterized in the SA node and conduction system cells of heart. The depolarizing force during the diastole cycle in the heart is a result of interplay of several ion channels, but HCNs (I_h) are generally believed to be the major contributor (DiFrancesco, 2006; Vassalle, 1995). However, HCN knockout adult mice have roughly normal (Herrmann et al., 2007) or reduced heartbeat rates (Baruscotti et al., 2011), and the rate acceleration by sympathetic stimulation is intact, suggesting additional important player in heart rate regulation. NALCN is also highly expressed in the heart (Lee et al., 1999). The use of conditional Nalcn knockout mice should clarify whether NALCN plays a role in heartbeat control. Likewise, NALCN is expressed in pancreatic β cells, where the rhythmic oscillation of E_m is coupled to cell glucose metabolism and the secretion of insulin. Thus, NALCN channel may also play a role in the regulation of insulin secretion (Swayne et al., 2009).