Research ArticleNEUROSCIENCE

The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium

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Science Advances  24 Apr 2020:
Vol. 6, no. 17, eaaz3154
DOI: 10.1126/sciadv.aaz3154
  • Fig. 1 Functional expression of NALCN requires UNC79, UNC80, and FAM155A.

    (A and B) Whole-cell patch-clamp recordings from HEK-293T cells expressing NALCN-eGFP-2×FLAG (NALCN*) alone or in different combinations with UNC79 (79), UNC80 (80), and FAM155A (155) under (A) symmetrical Na+ and (B) more physiological conditions using voltage-step protocols shown on the left. Normalized I-V plots highlighting the different current components of NALCN* + 79 + 80 + 155 are shown on the right. The instantaneous current (Iinst; red) was measured in the beginning of a voltage-step change, immediately after the transient current settled. The steady-state current (Iss; blue) was measured at the end of a voltage step. Insets show tail currents (Itail; orange) immediately after repolarization to −100 mV. (C) Current responses (left) and Iinst-V plots (right, normalized to the control current) in the absence and presence of TTX, Gd3+, or verapamil under symmetrical Na+ conditions. Data in (A) to (C) are shown as mean ± SD; gray dashed lines indicate 0 nA; numbers in parentheses indicate number of individual cells used for recordings. (D) Western blot of total lysate and surface fraction proteins extracted from HEK-293T cells expressing the indicated constructs (see also fig. S2A).

  • Fig. 2 The NALCN channel complex is selective for small monovalent cations.

    (A) Representative current traces of HEK-293T cells expressing NALCN* + 79 + 80 + 155 under various bi-ionic conditions in response to the voltage protocol shown. EC, extracellular; IC, intracellular. (B) Representative current traces obtained with intracellular NMDG+ and different extracellular mono- or divalent cations using voltage-step protocols shown on the left. (C) The top panel shows currents obtained with a voltage ramp protocol (−80 to 80 mV) with different test ions for both wild-type (WT) [EEKE selectivity filter (SF), top] and the DEKA SF mutant (bottom) using intracellular NMDG+ solution; insets show only −20- to +80-mV range. (D) Reversal potentials of different test ions and their permeability relative to Na+; data are shown as mean ± SD; gray dashed lines indicate 0 nA; numbers in parentheses indicate number of individual cells used for recordings.

  • Fig. 3 Divalent cation block of the NALCN channel complex is attenuated by mutations of side chains in the putative SF region.

    (A) Current responses of HEK-293T cells expressing NALCN* + 79 + 80 + 155 in the absence and presence of 1 mM Ca2+, Mg2+, or Ba2+. Normalized I-V plots illustrate the inhibitory effects of each divalent cation on Iinst and Iss. (B) Sample traces of a HEK-293T cell expressing NALCN* + 79 + 80 + 155 exposed to 0, 0.1, and 1 mM Ca2+ under symmetrical Na+ conditions when stepping from 0 to −80 mV. Inset shows Iinst on an expanded time scale. IC50 graph shows the potency of Ca2+ inhibition on both Iinst and Iss of NALCN-mediated current. (C) Top: Alignment of the selective filter (SF) region from the four homologous domains in NALCN. The putative SF motif EEKE is highlighted in orange and other negatively charged residues selected for charge neutralization are highlighted in dark pink. Bottom: Homology model of the putative SF region of NALCN based on the structure of CaV1.1 (Protein Data Bank ID: 5GJV). (D) Representative currents from X. laevis oocytes expressing WT NALCN or alanine mutants in response to step protocols from +80 to −100 mV (HP = 0 mV) in the presence (ND96; 1.8 mM Ca2+ and 1 mM Mg2+) and absence of divalent cations (X2+-free). (E) Fold increase in inward current elicited at −100 mV for WT NALCN and SF alanine mutants in response to removal of divalent cations. Data are shown as mean ± SD; *P < 0.05; ****P < 0.0001; one-way analysis of variance (ANOVA), Dunnett’s test (against WT); gray dashed lines indicate 0 nA; numbers in parentheses indicate number of individual cells used for recordings. See fig. S3.

  • Fig. 4 NALCN voltage sensitivity primarily arises from S4 charges in domains I and II.

    (A) Left: Schematic illustration of the structural topology of NALCN, which consists of four domains (DI to DIV) connected via intracellular linkers. Each domain contains six transmembrane segments (S1 to S6), with S1 to S4 forming the VSDs and S5 to S6 forming the pore domains. The S4 segments typically carry several positively charged residues important in voltage sensing. The conserved and missing (compared to canonical VSDs) positively charged residues in each S4 segment of NALCN are highlighted. Right: Alignment of the S4 segments of the four homologous domains in hNALCN, hNaV1.1, and hCaV2.1. Positively charged side chains are highlighted in red. (B) Current traces and Iss-V plots from X. laevis oocytes expressing WT and charge-neutralized mutants in S4 of DI to DIV using the indicated protocol. (C and D) Representative traces of single and double charge-neutralized mutants in S4 of VSDI (C) and VSDII (D). (E and F) Slow and fast time constants of depolarization-elicited currents (0 to +80 mV) in ND96 (E) and hyperpolarization-elicited currents (0 to −100 mV) in X2+-free buffer (F) for WT and VSD mutants. Superimposed traces of WT (black) and selected VSD mutants (red) are shown above the bar graphs. Data are shown as mean ± SD; *P < 0.05; **P < 0.01; ****P < 0.0001; one-way ANOVA, Dunnett’s test (against WT); n.e., no effect; n.d., not determined; gray dashed lines indicate 0 nA; numbers in parentheses indicate number of individual cells used for recordings.

Supplementary Materials

  • Supplementary Materials

    The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium

    H. C. Chua, M. Wulf, C. Weidling, L. P. Rasmussen, S. A. Pless

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