A robust expression system reveals distinct gating mechanisms and calmodulin regulation of Na _V 1.9 channels

Na_V1.9 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 Na_V1.9 currents, enabling direct electrophysiological comparisons with native dorsal root ganglion neurons. To further understand the low current density observed in human Na_V1.9, we created chimeras with Na_V1.5 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 Na_V 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 Na_V1.9. Our findings provide insights into the unique gating mechanisms of Na_V1.9 and demonstrate the robustness of this platform for structure-function studies.