Action potential afterdepolarization mediated by a Ca 2+-activated cation conductance in myenteric AH neurons

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 ( I ADP) 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 I ADP 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 I ADP. The I ADP was also inhibited when CsCl-filled patch pipettes were used. The I ADP was blocked or substantially decreased in amplitude in the presence of N-type Ca 2+ channel antagonists, ω-conotoxin GVIA and ω-conotoxin MVIIC, respectively, and was eliminated by external Cd 2+, indicating that it was dependent on Ca 2+ entry. The I ADP was also inhibited by ryanodine (10–20 μM), indicating that Ca 2+-induced Ca 2+ release was involved in its activation. Niflumic acid consistently inhibited the I ADP with an IC 50 of 63 μM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca 2+ channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca 2+ channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca 2+-channel blocker, is generated by the opening of Ca 2+-activated non-selective cation channels following action potential-mediated Ca 2+ entry mainly through N-type Ca 2+ channels. Ca 2+ release from ryanodine-sensitive stores triggered by Ca 2+ entry contributes significantly to the activation of this current.