A Modified Neuronal Cell Line for High Throughput Analysis of Voltage-Gated Sodium Channels

Abstract

Neuronal voltage-gated sodium (NaV) channels are important therapeutic targets for pain, epilepsy and other neurological conditions. Heterologous expression strategies are widely used for drug discovery and for studying naturally occurring mutations associated with genetic disorders. Certain NaV channels do not express well in typical laboratory cell lines (e.g., HEK293), but may be active in neural derivatives. However, neuronal cell lines have endogenous NaV currents that confound measurements. To overcome this limitation, we modified the genome of ND7/23 cells to have reduced endogenous sodium currents. Using RNA-Seq analysis, we determined that ND7/23 cells express rat and mouse Scn9a (NaV1.7) and to a lesser extent Scn8a (NaV1.6), which correlated with a large endogenous TTX-sensitive current. Next we used genome editing with CRISPR/Cas9 targeted to mouse and rat NaV1.7 to eliminate this endogenous channel. Clonal populations of modified cells were screened by automated patch-clamp recording and a single subline (ND7/no-Nav) was identified that exhibited ∼85% lower sodium current than the parental line (ND7/23=-38.7±2.1 pA/pF, ND7/no-Nav=-4.9±0.6 pA/pF). PCR analysis of ND7/no-Nav cell DNA revealed frameshift mutations of mouse and rat NaV1.7, and immunoblot analysis revealed a substantially lower level of NaV1.7 protein. Lastly, we demonstrated that ND7/no-Nav cells can be used as a cellular platform for heterologous expression of a human neuronal NaV channel (Nav1.1). Cells electroporated with Nav1.1 cDNA exhibited robust whole-cell currents by automated patch-clamp recording that peaked at 0 mV (−38.3±1.8 pA/pF) and had a voltage dependence of activation defined by V½ = −13.2±0.4 (n = 47). In summary, we engineered a neuronal cell line expressing very low levels of endogenous sodium currents that can be used for heterologous expression of human neuronal Nav channels that should have value for discovery of neuronal NaV modulators.