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

Abstract

1. Functional interactions between nicotinic and P2X receptors in submucosal neurons were investigated. Whole-cell currents induced by ACh (IACh) and ATP (IATP) were blocked by hexamethonium and PPADS), respectively. Currents induced by simultaneous application of the two transmitters (IACh+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 IACh+ATP indicate that they are carried through channels activated by either substance. In agreement with this interpretation, both IACh and IATP 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 IACh and IATP 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 alpha, beta-methylene ATP for ATP, or GTP for GTP-gamma-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.

Figure 1. Whole-cell outward and inward currents induced by either ATP or ACh show similar desensitization kinetics

Outward and inward currents induced by ATP (A) and ACh (B), and recorded at approximately equidistant membrane potentials from the reversal potential for these currents. Both agonists were applied as indicated by bars. Currents of panels A and B are from two different submucosal neurons. Vertical calibration bar labels indicate outward (upper label) and inward currents (lower label).

Figure 2. Whole-cell inward currents induced by ACh (I_ACh) and ATP (I_ATP) in submucosal neurons are not additive, revealing a current occlusion

A shows recordings from one neuron of a typical experiment and B shows the mean values of seventeen experiments. Currents were induced by application of either ACh (1 mm) or ATP (1 mm) and by the simultaneous application of both agonists (I_ACh+ATP). I_ACh and I_ATP were recorded 5 min before and 5 min after I_ACh+ATP. For the outer two bars, the downward error bar indicates s.e.m. for each individual current and the upward error bar s.e.m. for the expected currents (I_expected =I_ACh+I_ATP). The mean I_ACh+ATP was significantly lower (***P < 0.001) than I_expected. C, onset of the currents recorded from another submucosal neuron showing that I_ACh+ATP is smaller than I_expected from the onset of these currents. The expected current shown in C is a graph representing the addition of I_ACh and I_ATP. Whole-cell currents were measured at a holding potential of −60 mV.

Figure 3. Desensitization kinetics of currents induced by simultaneous application of ACh and ATP (I_ACh+ATP) cannot be explained by the desensitization kinetics of the currents induced by application of ACh (I_ACh) or ATP (I_ATP) alone

A, representative recordings from a submucosal neuron of I_ACh, I_ATP and I_ACh+ATP (grey current traces). The desensitization of I_ACh+ATP and I_ACh were better fitted by the sum of three exponential functions (continuous black lines), whereas I_ATP desensitization was better fitted by the sum of two exponential functions. Note that I_ACh+ATP desensitizes faster than I_ATP but slower than I_ACh. B, bars represent the mean ±s.e.m. values of the τ values of these exponential functions. The first exponential (τ₁) of I_ACh was significantly smaller (*P < 0.05) than τ₁ of I_ACh+ATP. τ values of the second and third exponentials of these currents were not different, however. The τ values of the second and third exponentials of I_ACh+ATP desensitization were also not different from those of the first and second exponentials of I_ATP. Exponential fits were performed using the data from the current peak to the ‘steady-state’ component. In these experiments agonists were applied for approximately 3 min and the holding potential was −60 mV.

Figure 4. Inhibitory interactions between nicotinic and P2X receptors required the presence of functional channels

A, recordings from a neuron in which ATP induced only a small initial current (I_ATP), indicating few functional P2X channels in this cell, but with a prominent response to ACh. Note that ATP did not modify either the amplitude or the kinetics of the current induced by ACh (I_ACh). Similar results were obtained when I_ATP was blocked with a P2X receptor antagonist (60 μm PPADS; not shown). B, currents induced by ACh, ATP and ACh + ATP in the presence of hexamethonium (1 mm). Note that ACh did not modify the kinetics of I_ATP. Similar results were obtained when I_ACh was blocked with a nicotinic receptor antagonist (not shown). I_ACh (C) and I_ATP (D) were recorded 5 min before (left recordings) and during continuous application of the other agonist. Note that currents induced by this second application have similar kinetics and amplitude to control currents. These experiments were repeated in four neurons with similar results. The four sets of currents were recorded in four different submucosal neurons and were taken at a holding potential of −60 mV.

Figure 5. Currents induced by ATP (I_ATP) and ACh (I_ACh) were additive when they were outward

Data shown are from experiments carried out at two holding potentials of +10 mV (A and B; n = 5) and +40 mV (C and D; n = 10). Two sets of recordings from two typical experiments are shown in A and C and the mean values of similar experiments are shown in B and D, respectively. Currents were induced by application of either ACh (1 mm) or ATP (1 mm) and by simultaneous application of both agonists (I_ACh+ATP). I_ACh and I_ATP were recorded 5 min before and 5 min after I_ACh+ATP. Error bars show half-s.e.m. In the outer two bars, the downward half-s.e.m. is for each individual current and the upward half-s.e.m. is for the expected currents (I_expected =I_ACh+I_ATP). Just as it was at a membrane potential of −60 mV, at +10 mV I_ACh+ATP was significantly lower (P < 0.05) than I_expected. At +40 mV, however, no difference was observed between these currents.

Figure 6. Whole-cell currents induced by simultaneous application of ACh and ATP (I_ACh+ATP) appear to be mediated by the opening of both nicotinic and P2X channels

A, currents induced by ACh (I_ACh) and ATP (I_ATP). B, I_ACh+ATP before (Control), in the presence of 1 mm hexamethonium (a nicotinic channel blocker) or in the presence of 60 μm PPADS (a P2X receptor antagonist). In the presence of hexamethonium I_ACh+ATP (B) had a similar amplitude and kinetics to the current induced by ATP alone (A), whereas in the presence of PPADS I_ACh+ATP (B) had a similar amplitude and kinetics to the current induced by ACh alone (A). All recordings are from the same submucosal neuron and were taken at a holding potential of −60 mV.

Figure 7. No cross-desensitization was observed between nicotinic and P2X receptors

Control I_ACh (A and D) and I_ATP (B and C) were recorded 5 min before (left recordings) and immediately after (∼5 s) a prolonged application of the other agonist. E and F, simultaneous application of both agonists desensitized both receptor populations. I_ACh (E) and I_ATP (F) recorded 5 min before (control currents; left recordings) and immediately after (∼5 s) a prolonged application of ACh + ATP. G, mean amplitude of I_ACh and I_ATP recorded after the prolonged application of ATP, ACh or ACh + ATP, as a percentage of control response. Error bars are s.e.m. A and B, C and D, and E and F are from three different submucosal neurons and the holding potential was −60 mV.

Figure 8. Mean amplitude of inward currents induced by application of ACh (I_ACh), ATP (I_ATP) or ACh + ATP (I_ACh+ATP) in seven different experimental groups of submucosal neurons

Results for each group are represented by a pair of bars. The first bar of each pair is a combined bar and shows the mean I_ATP and I_ACh before application of ACh + ATP. This combined bar represents the expected current (I_expected =I_ACh+I_ATP). The second bar represents I_ACh+ATP. In the combined bars, downward error bars show s.e.m. for I_ACh and I_ATP, while upward error bars show s.e.m. for I_ACh+ATP. Ca²⁺-free experiments were carried out in 0 Ca²⁺ plus 50 μm EGTA extracellular media and standard intracellular solution plus 5 mm BAPTA. In the α,β-methylene ATP (α,β-meATP) and GTP-γ-S experiments the pipette solution contained these substances instead of ATP and GTP, respectively. K-252a, staurosporine and genistein experiments were carried out using standard intracellular solution plus 10 μm of these agonists. During the N-ethylmaleimide (NEM) experiments, recorded neurons were pretreated for 10 min with standard extracellular solution plus 30 mm NEM. During these experiments, only one cell from each coverslip was recorded and coverslips were discarded after a neuron had been exposed to NEM. The holding potential was −60 mV in all these experiments.