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. 2019 Aug 13;9:11791. doi: 10.1038/s41598-019-48071-x

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The NALCN current for wt, Y578S and L509S variants is potentiated in low extracellular Ca²⁺ with no change in reversal potential. (A) Representative traces obtained on cells held at −40 mV (HP) during application/wash of an extracellular Na⁺-free solution (replaced with NMDG) with high (2 mM) or low (0.1 mM) extracellular Ca²⁺ for a mock (NLF1)-transfected cell, a NALCN/NLF1 (wt)-transfected cell, a NALCN CLIFAHDD mutant (L509S)/NLF1-transfected cell, and a NALCN CLIFAHDD mutant (Y578S)/NLF1-transfected cell. (B) Density of the external Na⁺-dependent background current (Na⁺-free) with high (2 mM, filled bars) or low (0.1 mM, hatched bars) extracellular Ca²⁺ for mock (NLF1)-transfected cells (−0.008 ± 0.029 pA/pF in [Ca²⁺]_e = 2 mM and −0.073 ± 0.028 pA/pF in [Ca²⁺]_e = 0.1 mM, n = 9, P = 0.136), NALCN/NLF1 (wt)-transfected cells (−0.303 ± 0.046 pA/pF in [Ca²⁺]_e = 2 mM and −0.712 ± 0.115 pA/pF in [Ca²⁺]_e = 0.1 mM, n = 9, p = 0.004). NALCN CLIFAHDD mutant (L509S)/NLF1-transfected cells (−0.529 ± 0.148 pA/pF in [Ca²⁺]_e = 2 mM and −1.347 ± 0.188 pA/pF in [Ca²⁺]_e = 0.1 mM, n = 16, P = 0.001), and NALCN CLIFAHDD mutant (Y578S)/NLF1-transfected cells (−0.792 ± 0.126 pA/pF in [Ca²⁺]_e = 2 mM and −2.466 ± 0.415 pA/pF in [Ca²⁺]_e = 0.1 mM, n = 16, P = 0.001). Statistical significance was calculated with an unpaired student’s t test (C) I/V relationship in the −30 mV to + 30 mV range of the Na⁺-dependent component recorded in low concentration of extracellular Ca²⁺ (0.1 mM) of wt NALCN (n = 8), NALCN-Y578S (n = 12) and NALCN-L509S (n = 18) currents. (D) Mean reversal potential (E_Rev) of the Na⁺-dependent component of wt NALCN (E_rev = 36.55 ± 12.91 mV, n = 8), NALCN-Y578S (E_rev = 40.22 ± 2.99 mV, n = 12) and NALCN-L509S (E_rev = 46.22 ± 4.70 mV, n = 18) currents. Each dot represents the E_Rev of 1 cell. For each cell, the E_Rev was calculated from the linear part of its I/V relationship as illustrated in (C). Statistical significance was calculated with a Tukey’s multiple comparisons test.