, 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 Selleck PD98059 et al., 2005). This rescue experiment with pore mutants also provided the in vivo evidence confirming that NALCN is indeed an ion channel. Currently, the only available high-resolution structure in the NALCN/CaV/NaV/ CatSper/NaVBac superfamily is that of a bacterial voltage-gated Na+ channel isolated from Arcobacter butzleri (NaVAb) ( Payandeh et al., 2011). Given the overall sequence similarity, especially in the pore regions, between NaVAb and other channels in these families, the structure of the NaVAb homotetramer likely has many of the key signatures of NaVs, CaVs, and NALCN. The overall structure of NaVAb is similar to that of the KVs and is composed of an S1-S4

VSD and a channel pore formed by S5-S6. Unique to NaVAb is a large fenestration on the side of the pore. NaVAb also has an additional pore helix (P2) in addition to the helix (P) also found in KV UMI-77 concentration channels. This P2 helix is C-terminal to the P helix and contains the tryptophan residue (W) of the T/SxE/DxW signature found in all the 24-TM channels ( Payandeh et al., 2011; Figure 3B). In addition, C-terminal to the tryptophan residue in the P2 helix are several amino acids that have been shown to influence channel selectivity, as demonstrated for the bacterial voltage-gated Na+ channel NaChBac Evodiamine ( Ren et al., 2001b and Yue et al., 2002). In the homotetrameric NaVAb channel, the four glutamate (E) residues in the T/SxE/DxW

pore signature form the narrowest ring in the pore filter. In the NALCN protein, one of the glutamate residues in repeat III is replaced by a lysine. Many of the Ca2+/Na+ channels consist of multiple subunits. For example, the NaV complex is composed of a pore-forming α subunit and two transmembrane auxiliary subunits, β1 and β2 (Catterall et al., 2002). Similarly, high voltage-gated CaVs contain the pore-forming α1 subunit, an intracellular β subunit, an α2/δ subunit, and, in some cells such as skeletal muscle cells, a transmembrane γ subunit (Catterall, 2011). Likewise, CatSper channels contain four pore-forming subunits (CatSper1–4) and at least three membrane-spanning auxiliary subunits (β, γ, and δ) (Chung et al., 2011 and Ren and Xia, 2010). The subunit composition of the low-voltage gated CaVs (T-type) is not known. Many of the non-pore-forming, auxiliary subunits are essential for various aspects of basic channel function (Arikkath and Campbell, 2003). The elucidation of the NALCN complex has been greatly facilitated by genetic studies in Drosophila, C. elegans, and mice.