A robust expression system reveals distinct gating mechanisms and calmodulin regulation of NaV1.9 channels

NaV1.9 is a voltage-gated Na+ channel subtype with unique gating properties that are poorly understood, partly due to the lack of reliable heterologous expression systems. Here, we present a transient expression protocol that produces robust mouse NaV1.9 currents, enabling direct electrophysiological comparisons with native dorsal root ganglion neurons. To further understand the low current density observed in human NaV1.9, we created chimeras with NaV1.5 and identified a role for the C-tail-specifically the IQ motif and EF-hand-in regulating current densities, likely due to a weak affinity for calmodulin. Isothermal titration calorimetry experiments indicated that, unlike other NaV channel subtypes, calmodulin binding to the C-tail is likely too weak to occur under physiological conditions. Markedly, the pre-IQ region did not influence channel expression but was responsible for conferring the characteristic depolarized voltage dependency of inactivation of NaV1.9. Our findings provide insights into the unique gating mechanisms of NaV1.9 and demonstrate the robustness of this platform for structure-function studies.