NALCN Knockout Cell Lines

To investigate the functional impact of NALCN gene knockout on neuronal excitability and extracellular calcium (\(Ca^{2+}\))-mediated channel regulation, using genetically modified cell lines as a model for neurological disorders associated with NALCN mutations.

Scientific Context: The NALCN channel is essential for maintaining neuronal resting membrane potential (RMP) and excitability. Mutations in NALCN are linked to severe neurodevelopmental disorders (e.g., epileptic encephalopathies) and neonatal lethality in knockout models, underscoring its critical role in neuronal function .

Technical Challenge: Traditional methods for generating and validating NALCN knockout cell lines have been hindered by low expression efficiency and difficulty in functional characterization. Heterologous expression of NALCN requires auxiliary proteins (UNC79, UNC80, FAM155A) for robust channel activity, complicating knockout validation .

Market Need: Robust NALCN knockout cell lines are needed to model disease mechanisms and screen therapeutic candidates for NALCN-related disorders.

Cell Line Generation:Human embryonic kidney (HEK-293T) cells were engineered using CRISPR-Cas9 to disrupt the NALCN gene, targeting exons critical for channel function. Single-cell clones were isolated via limiting dilution, and genomic DNA was verified for biallelic mutations using Sanger sequencing 14.Co-expression of UNC79, UNC80, and FAM155A was omitted in knockout lines to abrogate NALCN complex formation, while wild-type (WT) controls retained these auxiliary proteins for functional comparison 4.Functional Validation:Electrophysiology: Patch-clamp recordings showed that NALCN knockout cells lacked voltage-dependent sodium currents observed in WT cells. In WT cells, the NALCN complex exhibited constitutive activity with inward currents sensitive to hyperpolarization, while knockout cells displayed no such currents, confirming functional gene disruption 56.Extracellular \(Ca^{2+}\) Sensitivity: WT cells showed reduced current amplitude in the presence of physiological \(Ca^{2+}\) (1 mM), with an \(IC_{50}\) of 319 µM for instantaneous current (I\(_{inst}\)), whereas knockout cells were insensitive to \(Ca^{2+}\) changes, validating NALCN’s role in \(Ca^{2+}\)-mediated channel block 910.Ion Selectivity: WT cells exhibited permeability to monovalent cations (\(Na^{+}, K^{+}, Cs^{+}\)) but not divalent cations (\(Ca^{2+}, Mg^{2+}\)), while knockout cells showed no cation-specific currents, confirming NALCN’s role in monovalent cation conductance 78.Phenotypic Characterization:RT-qPCR and western blotting confirmed abolished NALCN mRNA and protein expression in knockout lines. Functional assays using TLR ligands (e.g., Pam3CSK4) showed no change in neuronal signaling responses, consistent with disrupted excitability 11.

NALCN knockout cell lines provide a validated model to study the molecular mechanisms of neuronal excitability and \(Ca^{2+}\)-dependent channel regulation. The data confirm that NALCN, in complex with UNC79, UNC80, and FAM155A, mediates voltage-sensitive sodium leakage and is directly blocked by extracellular \(Ca^{2+}\), aligning with its role in maintaining RMP 19. These cell lines enable high-throughput screening for drugs targeting NALCN-related disorders, such as epileptic encephalopathies, and offer insights into how NALCN mutations disrupt neuronal function. The model’s utility is further enhanced by its ability to recapitulate physiological \(Ca^{2+}\) sensing, making it a valuable tool for translational research in neurology 1213.