, 1996). In addition, the charged residues in the S4s, especially of domains III and IV, are also important for Nav 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 Ku-0059436 cell line that of the EAG (KCNH2) Kv 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 MK-8776 in vivo with S4 form the voltage-sensor paddle as shown in the crystal structure of Kv 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 Casein kinase 1 of them into homotetrameric Kv channels (Bosmans et al., 2008 and Xu et al., 2010). The second unique feature of NALCN is its pore filter (Figure 3B). The selectivity filter in CaV, NaV, and KV 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 and Payandeh et al., 2011). In CaVs, 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 and Yang et al., 1993). NaVs have a DEKA motif in the analogous position (Figure 3B). NALCN has an EEKE motif, a combination of the EEEE (CaV) and DEKA (NaV) 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 Nav, 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 CaVs into EEKE enables the otherwise highly Ca2+-selective channels permeable to monovalent ions ( Parent and Gopalakrishnan, 1995, Tang et al., 1993 and Yang et al., 1993).