Analysis of whole‐cell currents by patch clamp of guinea‐pig myenteric neurones in intact ganglia
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abstract
Whole‐cell patch‐clamp recordings taken from guinea‐pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time‐independent, caesium‐sensitive, inward‐rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half‐activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e‐fold change. Ih has relatively fast, voltage‐dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium‐dependent current that was poorly sensitive to TTX. In AH neurones, the post‐action‐potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω‐conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω‐agatoxin IVA (0.2 μm). There was no additivity between the effects of the three conotoxins, which indicates the presence of N‐ but not of P/Q‐type Ca2+ channels. A residual Ca2+ current, resistant to all toxins, but blocked by 0.5 mm Cd2+, could not generate IAHP. This patch‐clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea‐pig duodenum are under the control of four major currents, IAHP, Ih, an N‐type Ca2+ current and a slowly inactivating Na+ current.
