Exploring the selective inhibitory effects of Flavoxate and its analogues on two chloride channels and neuronal action potentials

Volume-regulated anion channels (VRACs) and TMEM16A calciumactivated chloride channels (CaCCs) are distinct but share some features and can co-immunoprecipitate. Currently, there are no specific inhibitors for these channels exist. Our prior research showed Flavoxate inhibits VRAC currents in HEK293 cells under hypotonic conditions. MFCA (3-methylflavone-8-carboxylic acid, a primary active metabolite of Flavoxate) and 3-methylflavone are two structural analogues of Flavoxate. To further elucidate the specific inhibitory effects of the three flavonoids on VRACs and TMEM16A/CaCCs, the current study used patch-clamp techniques to explore their effects on the two chloride channels. Additionally, we investigated the effects of the three flavonoids on action potential (AP) firing in small dorsal root ganglion (DRG) neurons utilizing the current-clamp technique. Our findings indicate that the inhibition rates of 30 μM Flavoxate, MFCA, and 3-methylflavone on VRAC currents approximately were 78%, 46%, and 35%, with corresponding half-maximal concentration (IC₅₀) values of 1.8 μM, 18.5 μM, and 37.2 μM, respectively. In contrast, the inhibition rates of these compounds on TMEM16A/CaCC currents approximately were 14%, 15%, and 24%, with IC₅₀ values of 32.8 μM, 28.3 μM, and 26.5 μM, respectively. These results suggest that Flavoxate is highly efficient and selective for VRAC inhibition, with the 8-substituent on its flavonoid core being crucial for this selectivity. All three flavonoids strongly inhibit AP firing, indicating that their core structure contributes to analgesic effects. In summary, Flavoxate is a selective VRAC inhibitor and a potential neuropathic pain inhibitor by suppressing AP firing in small DRG neurons.