(A) Genetic mapping of nca(gf) mutants and a schematic representation of the nca-1 genetic locus (top) and the predicted NCA-1 protein structure (bottom). The positions of the nca(gf) (hp102 and e625) and nca(lf) mutations (gk9 and tm1851) are illustrated. Numbers with yellow highlights denote the beginning and end of each ion transport motif. The amino acid residues that determine the ion selectivity in related cation channels are highlighted with orange circles.
(B) Similarity between NCA family members. The residues equivalent to hp102 and e625 are indicated. C.e.: C. elegans; D.m.: D. melanogaster; R.n.: R. norvagicus; M.m.: M. musculus; H.s.: H. sapiens.

Representative traces of spontaneous activity [(A) for nca(lf) and (E) for nca(gf)] and responses evoked in muscle by an electric stimulation of the ventral nerve cord [(B) for nca(lf) and (F) for nca(gf)] are shown.
(A–D) The nca(lf) mutant mPSCs varied between wild-type (WT) values (nca(lf), upper trace) and decreased frequency and amplitude (nca(lf), lower trace). The overall mPSC frequency (C) and evoked response amplitude (D) were decreased in nca(lf) animals as compared to wild-type at either 5 mM (nca(lf): n = 15; WT: n = 13) or 1 mM (nca(lf): n = 9; WT: n = 6) extracellular Ca²⁺. No significant change in mPSC amplitude and distribution was observed between nca(lf) and WT animals. Individual results are shown in black, mean ± SEM in gray, for recordings at 5 mM and 1 mM extracellular Ca²⁺.
(E–H) Representative traces of spontaneous (E) and evoked (F) post-synaptic currents at the neuromuscular junction at 5 mM Ca²⁺. The nca(gf) mutant mPSCs were either comparable to WT (population 1) or highly decreased in frequency and amplitude (population 2), whereas highly reduced (if any) responses could be evoked by electric stimulation of the nerve cord (F). The mPSC frequency (G) and evoked responses amplitude (H) are plotted for N2 and nca(gf) animals. Individual results are shown in black, mean ± SEM in gray, when relevant. (WT, mPSCP, n = 10, ePSP, n = 7; nca(gf), mPSP, n = 13, ePSP, n = 8) Error bars: SEM, *p ≤ 0.05, **p < 0.01. Statistic analysis was performed with Student's t-test.

(A) Upper panel: Image of HSN neuron used in calcium imaging. The HSN cell body and the synapse where Ca²⁺ imaging were performed are circled in dots. Lower panel: Sample traces of simultaneous recording of calcium spikes of both cell body and synapse, showing the synchronicity of the calcium signals.
(B) Sample traces of yellow/cyan ratio that represent the relative Ca²⁺ concentration in HSN cell bodies (left panels) or their synapses (right panels). x-axis: time in s; y-axis: yellow/cyan ratio in percent. For nca(lf) and unc-80, animals displayed traces with either silent (top) or active (bottom) Ca²⁺ transients.
(C–E) Histograms for the total spike frequency, spike interval, and size of calcium spikes detected in each strain. Arrowheads: examples of calcium spikes. The number of animals examined, n, (C) or the number of calcium spikes examined (D, E), is illustrated at the bottom of each bar. Error bar: SEM. *p < 0.05, **p < 0.005. (C) Average number of spikes/min for each genotype. There is no statistically significant difference for HSN cell bodies (cell) among all strains, or between HSN cell bodies (cell) and synapses (syn) of the same strain except for nca(lf) and unc-80. (D) Average time interval between two consecutive spikes within trains of calcium transients. There is no statistically significant difference between HSN cell bodies and corresponding synapses for all strains. (E) Average spike size. Wild-type, nca(lf), and unc-80 neurons displayed no statistically significant difference in spike size, but for the nca-1(gf) neurons, it was increased.
(F–H) Scatter plots for spike frequency, interspike time interval, and spike size. Each cross represents a data point. Clear and filled triangles represent mean numbers for cell body and synapse calcium transients, respectively. Red circles highlight populations of animals with silenced synapse calcium transients. Red box highlights a population of nca(gf) synapse spikes that were significantly larger than those seen in other genotypes. All statistic analysis was performed by Kolmogorov-Smirnov rank test.

(A) A transcriptional GFP reporter driven by the nca-1 promoter (left panels) or unc-80 promoter (right panels) is active in neurons in the nerve ring (NR), and ventral nerve cord (VNC) motoneurons. Activity of the nca-1 promoter is also seen in the HSN neuron whereas the unc-80 promoter has activity in the vulval muscles (VM).
(B) An antibody against NCA-1 showed specific staining (arrowheads) in the nerve ring (NR) and along nerve cords in wild-type (top left panel) animals that was absent in nca-1(tm1851) deletion mutants (bottom left panel). Similarly, anti-RFP antibody showed specific immunoreactivity in the nerve ring (NR) of UNC-80::RFP (hpIs98) expressing animal (top middle panel) but not in wild-type animals not carrying the transgene (bottom middle panel). * ns: nonspecific staining persisted in negative controls where animals do not express NCA-1 or UNC-80::RFP protein (out of the focal plane for the nca-1 panel). An antibody against UNC-79 showed specific and similar staining at the nerve ring in wild-type animals (right top panel) that disappeared in unc-79 mutants (right bottom panel).
(C) Wild-type animals were co-stained with anti-NCA-1 antibody (red) and anti-UNC-10 (green).
(D) unc-80;hpIs98 animals co-stained with anti-RFP antibody (red) and anti-UNC-10 (green) showed poor colocalization. Scale bar: 5μm.

(A) Wild-type (WT), nca(gf)hp102, unc-80(e1272), hp102;unc-80(e1272), and unc-79(e1279) animals co-stained with anti-NCA-1 (red) and anti-UNC-10 antibodies (green, as internal staining control). NCA-1 staining was present in wild-type and hp102 animals but disappeared in unc-80, unc-79, and nca(gf)hp102;unc-80 animals.
(B) Staining with anti-RFP antibodies in wild-type (negative control), hpIs98 (UNC-80::RFP), nca(lf);hpIs98, and unc-79;hpIs98 animals (left panels). Specific nerve ring staining (arrow) of UNC-80::RFP disappeared in nca(lf) and unc-79 animals. UNC-10 staining was present in the same animals (right panels). Scale bar: 5 μm.

The results of propidium iodide cell death assays in HEK293T cells are graphically represented. Assays were done on mock-transfected (untransf) HEK293T cells, or cells transfected with various combination of DNA constructs that express UNC-80, NCA-1, truncated NCA-1 (NCA-1Δ), the rat (rNALCN) homologue of NCA-1, and the Kv4.2 potassium channel (as an additional control), either in the absence of any blockers (A), or in the presence of 100 μM verapamil (B) or of 10 μM gadolinium (C). The graph presented resulted from three independent sets of experiments. Statistical significance was analyzed by one-way ANOVA followed by post-hoc Student-Newman-Keuls tests. **p < 0.01, significantly different from the mock-transfection control.

Top: A schematic representation of a C. elegans neuron with en passant synapses. Ca²⁺ transients in the cell body are likely contributed by influx from L-type VGCCs and release from the intracellular calcium pools. At the synapse, depolarization signals (red waves) initiate calcium influx through P/Q-type VGCCs at the active zone. NCA channels (NCA-1, NCA-2, UNC-80, and UNC-79) regulate membrane excitability along the axon to allow the propagation of some depolarization signals. Bottom: In nca(lf) mutants some depolarization signals fail to propagate along the axon while others can still reach synapses. In nca(gf) mutants, the signals are amplified, resulting in increased synaptic activity, which indirectly regulates active zone distribution.