Sodium plays a key role in determining the basal excitability of the nervous systems through the resting "leak" Na(+) permeabilities, but the molecular identities of the TTX- and Cs(+)-resistant Na(+) leak conductance are totally unknown. Here we show that this conductance is formed by the protein NALCN, a substantially uncharacterized member of the sodium/calcium channel family. Unlike any of the other 20 family members, NALCN forms a voltage-independent, nonselective cation channel. NALCN mutant mice have a severely disrupted respiratory rhythm and die within 24 hours of birth. Brain stem-spinal cord recordings reveal reduced neuronal firing. The TTX- and Cs(+)-resistant background Na(+) leak current is absent in the mutant hippocampal neurons. The resting membrane potentials of the mutant neurons are relatively insensitive to changes in extracellular Na(+) concentration. Thus, NALCN, a nonselective cation channel, forms the background Na(+) leak conductance and controls neuronal excitability.

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SUMMARY In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca2+ regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca2+'s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca2+]e activates a NALCN channel-dependent Na+-leak current (IL-Na). The coupling between [Ca2+]e and NALCN requires a Ca2+-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, IL-Na is insensitive to changes in [Ca2+]e, and reducing [Ca2+]e fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca2+ influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G-protein-dependent fashion.

Several neurotransmitters act through G-protein coupled receptors (GPCR) to evoke a "slow" excitation of neurons1 , 2. These include peptides, such as substance P (SP) and neurotensin (NT), as well as acetylcholine and noradrenaline. Unlike the fast (~ ms) ionotropic actions of small molecule neurotransmitters, the slow excitation is not well understood at the molecular level, but can be mainly attributed to suppressing K + currents and/or activating a non-selective cation channel3 -9 . The molecular identity of this cation channel has yet to be determined; similarly how the channel is activated and its relative contribution to neuronal excitability induced by the neuropeptides are unknown. Here, we show that, in the hippocampal and ventral tegmental area neurons, SP and NT activate a channel complex containing NALCN and a large novel protein UNC-80. The activation by SP through NK1R (a GPCR for SP) is via a unique mechanism: it does not require G-protein activation but is dependent on Src family kinases (SFKs). These findings identify NALCN as the cation channel activated by SP receptor, and suggest that UNC-80 and SFKs, rather than a G-protein, are involved in the coupling from receptor to channel.NALCN is a neuronal cation channel carrying a small background leak Na + current at the resting membrane potential 10 . When overexpressed in HEK293T fibroblast cells, it generates a Na + -permeable cation channel that is voltage-independent, non-inactivating, tetrodotoxin (TTX)-resistant and Gd 3+ -blockable 10 . It is not known whether, like background K + channels, NALCN is also regulated by neuromodulators, but the biophysical and pharmacological properties of the NALCN currents (I NALCN ) closely resemble those of the SP-activated cation channel currents (I SP ) studied in several brain regions [11][12][13][14] .To test the possibility that I SP requires NALCN, we recorded I SP in wild-type and Nalcn knockout 10 (Nalcn −/− ) neurons via patch clamp with measures taken to minimize K + channel effects and to block voltage-gated Na + channel and synaptic currents. In 16 of 34 wild-type hippocampal pyramidal neurons held at −67 mV, an inward current (> 50 pA) developed withinCorrespondence and requests for materials should be addressed to D.R. (dren@sas.upenn.edu). * these authors contributed equally. † Present address: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, P. R. China.Author Contributions. BL did recordings from neurons ( Fig. 1, Fig. 2, Fig. 3, Supplementary Fig. 2) and all the HEK293T cells (Fig. 4, Supplementary Figs. 1,(7)(8)(9)(10). YS contributed to neuronal recordings (Fig. 1, Fig. 2, Supplementary Figs. 3 and 4). SD contributed to work in Fig. 2. HW, YW and JL did the protein work (Fig. 4, Supplementary Fig. 6). DR started the project, designed experiments, and developed the cDNA constructs. BL and DR wrote the paper.Author Information. The sequence of mUNC80 is deposited in GenBank under accession number FJ...