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ABSTRACT: We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10–20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (IADP) in the presence of TEA (10–20 mM) reversed at −38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the IADP to −58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na+ and K+ to this current were estimated to be about 1:5. Substitution of external Na+ with N-methyl D-glucamine blocked the current while replacement of internal Cl− with gluconate did not block the IADP. The IADP was also inhibited when CsCl-filled patch pipettes were used. The IADP was blocked or substantially decreased in amplitude in the presence of N-type Ca2+ channel antagonists, ω-conotoxin GVIA and ω-conotoxin MVIIC, respectively, and was eliminated by external Cd2+, indicating that it was dependent on Ca2+ entry. The IADP was also inhibited by ryanodine (10–20 μM), indicating that Ca2+-induced Ca2+ release was involved in its activation. Niflumic acid consistently inhibited the IADP with an IC50 of 63 μM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca2+ channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca2+ channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca2+-channel blocker, is generated by the opening of Ca2+-activated non-selective cation channels following action potential-mediated Ca2+ entry mainly through N-type Ca2+ channels. Ca2+ release from ryanodine-sensitive stores triggered by Ca2+ entry contributes significantly to the activation of this current.