Functional expression of CLIFAHDD and IHPRF pathogenic variants of the NALCN channel in neuronal cells reveals both gain- and loss-of-function properties

Abstract

The excitability of neurons is tightly dependent on their ion channel repertoire. Among these channels, the leak sodium channel NALCN plays a crucial role in the maintenance of the resting membrane potential. Importantly, NALCN mutations lead to complex neurodevelopmental syndromes, including infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF) and congenital contractures of limbs and face, hypotonia and developmental delay (CLIFAHDD), which are recessively and dominantly inherited, respectively. Unfortunately, the biophysical properties of NALCN are still largely unknown to date, as well as the functional consequences of both IHPRF and CLIFAHDD mutations on NALCN current. Here we have set-up the heterologous expression of NALCN in the neuronal cell line NG108-15 to investigate the electrophysiological properties of NALCN carrying representative IHPRF and CLIFAHDD mutations. Several original properties of the wild-type (wt) NALCN current were retrieved: mainly carried by external Na + , blocked by Gd 3+ , insensitive to TTX and potentiated by low external Ca 2+ concentration. However, we found that this current displays a time-dependent inactivation in the −80/−40 mV range of membrane potential, and a non linear current-voltage relationship indicative of voltage sensitivity. Importantly, no detectable current was recorded with the IHPRF missense mutation p.Trp1287Leu (W1287L), while the CLIFAHDD mutants, p.Leu509Ser (L509S) and p.Tyr578Ser (Y578S), showed higher current densities and slower inactivation, compared to wt NALCN current. This study reveals that heterologous expression of NALCN channel can be achieved in the neuronal cell line NG108-15 to study the electrophysiological properties of wt and mutants. From our results, we conclude that IHPRF and CLIFAHDD missense mutations are loss- and gain-of-function variants, respectively.

Structure of human sodium leak channel NALCN in complex with FAM155A

“…These observations indicate that the auxiliary subunits are essential for a functional NALCN ( Supplementary Fig. 1a), consistent with previous studies 30,31 . The electrophysiological properties of NALCN suggested that it tends to maintain the RMP in a manner reminiscent the Le Chatelier's principle in chemical equilibria.…”

“…Despite its physiological and pathological importance, the biophysical properties of the voltage sensitivity and ion selectivity of NALCN are still under debate. Electrophysiological studies on NALCN have been performed in heterologous expression systems such as HEK293 cells, X. laevis oocytes and the neuronal cell line NG108-15 5,30,31 . NALCN expressed in HEK293 cells was reported to exhibit a linear current-voltage relationship, suggesting that NALCN is a voltage-independent ion channel.…”

“…Other studies suggested that the VSDs of NALCN can exhibit a broader range of gating behaviors. Co-expression of NALCN, UNC79, UNC80, and FAM155A resulted in voltage dependent NALCN current 30,31 . Finally, NALCN was also reported to be only selective for monovalent cations and to be blocked by extracellular divalent cations 30 .…”

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

“…UNC79 and UNC80 are widely expressed in the brain, where they form a complex with NALCN and regulate its localization and function (5,11,34). While UNC79 is not absolutely required for NALCN activity in mouse hippocampal neurons [NALCN-like currents were observed in the absence of UNC79 (5)], both proteins are needed for NALCN function in Drosophila (34), the neuronal cell line NG108-15 (30), HEK-293T cells, and X. laevis oocytes ( Fig. 1 and fig.…”

The sodium leak channel complex is modulated by voltage and extracellular calcium

“…UNC79 and UNC80 are widely expressed in the brain, where they form a complex with NALCN and regulate its localisation and function (Yeh et al, 2008, Lu et al, 2010, Lear et al, 2013. While UNC79 is not absolutely required for NALCN activity in mouse hippocampal neurons (NALCN-like currents were observed in the absence of UNC79 (Lu et al, 2010)), both proteins are needed for NALCN function in Drosophila (Lear et al, 2013), the neuronal cell line NG108-15 (Bouasse et al, 2019), HEK293 cells and Xenopus laevis oocytes (Figures 1 and S1), highlighting potential interspecies differences in the regulatory roles of these proteins. FAM155A is pivotal for the axonal localisation of NALCN in C. elegans (Xie et al, 2013).…”