Block of sensory neuronal Na⁺ channels by the secreolytic ambroxol is associated with an interaction with local anesthetic binding sites

Voltage-gated Na⁺ channels (Na_v) regulate the excitability of sensory neurons and are potential targets for novel analgesics. The secreolytic ambroxol reduces pain-related behavior in rodents and alleviates pain in humans. With properties resembling those of local anesthetics, ambroxol has been reported to block Na⁺ currents in sensory neurons with a preference for tetrodotoxin-resistant (TTXr) Na⁺ currents encoded by Na_v1.8. However, the molecular determinants for ambroxol-induced block of Na⁺ channels and a preferential block of Na_v1.8 opposed to tetrodotoxin-sensitive (TTXs) Na_v α-subunits have not been studied in detail. By means of whole-cell voltage clamp recordings, we studied the effects of ambroxol and local anesthetics on the recombinant TTXr subunit Na_v1.8, on TTXs Na_v α-subunits and on mutants of Na_v1.4 that are insensitive to local anesthetics. Tonic and use-dependent block by ambroxol was strongly alleviated in local anesthetic-insensitive Na_v1.4 mutants. Use-dependent block, but not tonic block was significantly stronger on Na_v1.8 than on TTXs channels. The TTXs subunit Na_v1.3 displayed the least degree of use-dependent block by ambroxol. The local anesthetics mepivacaine and S(-)-bupivacaine also blocked Na_v1.8 and TTXs channels differentially. While mepivacaine displayed a preferential use-dependent block of Na_v1.8, S(-)-bupivacaine displayed a preference for TTXs Na⁺ channels. Our data show that ambroxol acts as a typical local anesthetic on Na⁺ channels interacting with specific residues in the S6 segments. This property probably meditates the analgesic effect of ambroxol. Ambroxol preferentially blocks Na_v1.8, however shares this property with established local anesthetics like mepivacaine.