Purinergic Signalling (2006) 2:545–557 551 Fig. 4 Inhibition of IACh and IATP, respectively, in the presence of ATP and ACh. A) Sample currents in response to ACh (100 mM) in the presence of ATP (50 mM). The responses to ACh applied in the presence of ATP were reduced. The extent of occlusion was normalized to the control ACh responses recorded before and after ATP application. This sequence was routinely repeated twice or three times, and the averaged amount of occlusion was plotted in B. B) Relative amplitude of the ACh current amplitude in the presence of ATP (ordinate) plotted against the peak amplitude of IATP obtained in the same neuron (abscissae). Note the negative correlation between these two variables. The line was drawn using KaleidaGraph (Synergy Software). Vh: j70 mV. C) The IATP was recorded in the presence of 100 mM ACh and this response was ﬂanked by control ATP alone responses. This sequence was routinely obtained twice or three times in the same cell, and the averaged amount of occlusion was plotted in D. D) The ratio of the ATP current amplitude in the presence and absence of ACh (ordinate) is negatively correlated with the peak amplitude of IACh (abscissae). Vh : j70 mV. due to the reduced amplitude of the IACh at the time of ATP application. To examine this possibility, IATP was measured in the presence of both ACh and d-tubocurarine (dTc, 10 mM) or serotonin (5HT, 50 mM), two experimental conditions which change the time course of the IACh response. The former agent, dTc, has been shown to be both an open channel blocker and a competitive antagonist of nAChR (Manalis [31]), and, as expected, a low concentration (10 mM) slightly reduced the peak amplitude of IACh (79 T 4% of control, N = 11) and caused the IACh to be terminated within 0.5 s (Fig. 5A, insert). Once the IACh response had returned to baseline (in the presence of ACh and 10 mM dTc) and ATP was applied, there was no inhibition of IATP (95 T 1% of control, N = 5; Fig. 5A). The IATP in the presence of ACh alone was 77 T 4% of control (N = 5, P < 0.01; Fig. 5A). When we measured the current (IACh + ATP + dTc) induced by a combination of ACh, ATP and dTc, the peak amplitude of IACh + ATP + dTc was occluded under these conditions, being 74 T 2% (N = 10) of the predicted sum of IATP and IACh + dTc (Fig. 5B). The level of IACh + ATP + dTc at 0.5 s following ligand application, which corresponds to a time when the nAChR channels are completely blocked (Fig. 5A, insert), was 96 T 3% (N = 8) of the control IATP, measured immediately before and after (Fig. 5C). These results suggest that the inhibition of IATP by IACh disappears immediately after the IACh is abol- ished, although ACh is still present and bound to the nAChRs. 5HT has been shown to also accelerate the decay of IACh (Grassi et al. [32]; Garcia-Colunga and Miledi [33]; Sorimachi and Wakamori [34]), but not to the same extent as dTc. 5HT at 50 mM decreased the peak amplitude of IACh slightly, to 88 T 4% (N = 6) of control, and reduced the time constant (tau) of IACh decay from 2.02 T 0.18 to 1.06 T 0.19 s (N = 6). As shown in Fig. 5D, the extent of inhibition of IATP by