<p>Whole-cell voltage patch clamp studies have shown that in bovine adrenal chromaffin cells held at -70 mV, a single 5-ns, 5 MV/m pulse activates a membrane conductance carried partially by Na⁺ through TRPC4/5 channels and the sodium leak channel (NALCN). Here we used fluorescence imaging with the Na⁺ indicator ING-2 to further investigate Na⁺ influx pathways. A 5-ns, 5 MV pulse elicited a tetrodotoxin-insensitive rise in ING-2 fluorescence that exhibited a similar electric field dependency and response to pulse-pair stimulation as the inward current. Increases in ING-2 fluorescence were partially inhibited by the NALCN inhibitor CP96345, the TRPC4/5 channel inhibitor M084, or the broad spectrum TRP channel inhibitor La³⁺, and fully blocked by the combination of these agents, suggesting involvement also of a La³⁺-sensitive Na⁺ pathway. Cholesterol depletion with methyl-β-cyclodextrin or PIP₂ synthesis inhibition with wortmannin also reduced the response. Full inhibition was achieved with the selective L-type voltage-gated Ca²⁺ channel (VGCC) inhibitors nitrendipine, verapamil, or diltiazem but not the broad-spectrum inhibitor Cd²⁺. Inhibitors of N- and P/Q-type VGCC had no effect. Fluorescence Ca²⁺ imaging in voltage-clamped GCaMP6f-expressing murine chromaffin cells held at -70 mV revealed a nitrendipine-sensitive, Cd²⁺-insensitive increase in intracellular Ca²⁺ that accompanied the inward current. These results provide evidence that nanoelectropulse-induced Na⁺ influx into chromaffin cells involves several Na⁺ influx pathways that are facilitated by Ca²⁺ influx via an L-type-like Ca²⁺ channel associated with cholesterol-rich membrane domains. Investigating such membrane effects is essential for developing nanosecond electric pulse technologies for stimulating and/or modulating excitable cells.</p> Graphic Abstract <p></p>