554 Purinergic Signalling (2006) 2:545–557 Fig. 8 Lack of inhibition in response to co-application of ACh and ATP under two experimental conditions designed to virtually eliminate IACh. A) Control IACh recorded at a Vh of j70 mV in standard saline. B) Current responses recorded at j70 mV from 20 s after exchanging the standard saline for a Na+-free (replaced by NMDG+), Ca2+-free saline. This sequence was repeated twice or three times. Note the small amplitude of IACh, and the lack of occlusion of IACh + ATP. C) Control IACh recorded at a Vh of j70 mV in another neuron. D) Representative traces of IACh, IATP, and IACh + ATP recorded at a Vh of +7 mV, which is very close to EACh. Note the lack of occlusion at this potential, when current ﬂow through the AChR is negligible. chi and Wakamori [16]), and hence IACh would be mediated by only Ca2+ inﬂux, whereas IATP would be mediated via both NMDG+ and Ca2+ inﬂux (Fig. 3). Under these conditions, IACh + ATP was again occluded, being 70 T 3% of the predicted sum of IACh and IATP (N = 11; P < 0.01; Fig. 7). In a further attempt to distinguish whether channel activation or ion permeation was primarily responsible for occlusion of one channel by the other, we com- pared IATP with IACh + ATP under two experimental conditions, in which a higher concentration of ACh should activate nAChR but produce very little current. In the absence of Na+ and Ca2+ (replaced by NMDG+) application of ACh induces a negligible current (Sorimachi and Wakamori [16]), while ATP induces a substantial current under the same conditions (Fig. 3A). As shown in Fig. 8B, there was no occlusion in these conditions; the amplitude of IATP + ACh was 105 T 1% (N = 10) of the predicted sum of IATP and IACh. We also investigated currents in response to the ligands at a potential close to the EACh (7 T 2 mV in this ex- periment, N = 13), taking advantage of the more positive EATP (22.7 T 0.9 mV), so that at this poten- tial only the IATP was observed. The amplitude of IATP + ACh was 105 T 3% (N = 13) of that of IATP, and IACh was close to zero (Fig. 8D). These results are, however, in contrast to that obtained in sympathetic neurons (Nakazawa[30]), in which IATP in the absence of both Na+ (replaced by glucosamine) and Ca2+ was inhibited in the presence of ACh, which caused no current (Nakazawa [30]). In these experiments, we only included data in which IATP was large enough to be clearly resolved, greater than 50 pA (corresponding to a control IATP at a Vh of j70 mV larger than 1.5 nA). These results suggest that actual current ﬂow through both nAChR and P2XR is responsible for occlusion. Discussion In this study, we found that ATP induced an inward current and increased [Ca]in in isolated rat AP neurons. The ATP-induced current and [Ca]in increase were mimicked by ATPgS and 2-methylthio-ATP, but not by a, b-methylene-ATP, b, g-methylene-ATP nor ADP, and was inhibited by suramin and PPADS, suggesting that it was mediated by P2XRs. These