Functional interactions between nicotinic and P2X channels in short-term cultures of guinea-pig submucosal neurons

1. Functional interactions between nicotinic and P2X receptors in submucosal neurons were investigated. Whole-cell currents induced by ACh (I(ACh)) and ATP (I(ATP)) were blocked by hexamethonium and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), respectively. Currents induced by simultaneous application of the two transmitters (I(ACh+ATP)) were only as large as the current induced by the most effective of these substances. This current occlusion indicates that activation of nicotinic and P2X channels is not independent. 2. Kinetic parameters of I(ACh+ATP) indicate that they are carried through channels activated by either substance. In agreement with this interpretation, both I(ACh) and I(ATP) amplitudes were decreased when ATP and ACh were applied simultaneously, whereas no cross-desensitization was observed when nicotinic and P2X receptors were desensitized individually. 3. Current occlusion was observed at membrane potentials of -60 and +10 mV, when I(ACh) and I(ATP) were inward. However, when these currents were outward (at +40 mV), current occlusion was not observed. Current occlusion was still observed at +40 mV in experiments in which the reversal potential of these currents had been adjusted to more positive values. 4. Current occlusion occurred as soon as currents were detected (< 5 ms), was still present in the absence of Ca²⁺, Na⁺ or Mg²⁺, and after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution. Similar observations were noted after substituting α,β-methylene ATP for ATP, or GTP for GTP-γ-S in the pipette and in experiments carried out at 36, 23 and 9°C. 5. We propose that nicotinic and P2X channels are in functional clusters of at least two, and that the influx of ions through one activates (through allosteric interactions) a mechanism that inhibits the other channel.