550 suramin-sensitive, Na+-independent current supports the previous suggestion that the P2XR channel is permeable to glucosamine (Nakazawa [30]). To con- ﬁrm this, ATP did not induce a current at all when sucrose (0.25 M) was substituted for NaCl (again in the ab- sence of external Ca2+, N = 5). Addition of Ca2+ to the NMDG+- substituted saline further increased IATP. The currents induced by 500 mM ATP in the presence of 2 and 10 mM Ca2+, but in the absence of external Na+ (replaced by NMDG+) were 18 T 2% (N = 11) and 29 T 2% (N = 14, Fig. 3B) of the control IATP, res- pectively. Similarly, the currents induced by 500 mM ATP in the presence of 2 and 10 mM Ca2+ added to the sucrose-substituted saline were 6 T 1% (N = 6), and 13 T 2% (N = 16; Fig. 3C) of the control IATP, respectively. We also measured the EATP of IATP in the presence of 1 mM external Ca2+ and 150 mM NaCl or 110 mM external Ca2+ and 0 mM NaCl. The EATP at 110 mM Fig. 3 ATP-induced current in Na+- and NaCl-free saline containing 0 and 10 mM Ca2+. A) A representative IATP recorded in Na+-free (Na+ replaced by NMDG+) and Ca2+-free saline (containing 0.5 mM EGTA) at a Vh of j70 mV. B) A representative IATP in Na+-free (NMDG+) saline containing 10 mM Ca2+. C) A representative IATP in NaCl-free (NaCl replaced by sucrose) saline containing 10 mM Ca2+. Purinergic Signalling (2006) 2:545–557 Ca2+ was 21.1 T 1.0 mV (N = 18), from which we calculated a PCa/PCs of 6.3. The EATP at 1 mM Ca2+ was 20.2 T 1.0 mV (N = 13), from which we calculated a PNa/PCs ratio of 2.1. From these, we obtained a PCa/ PNa ratio of 3.0, conﬁrming the substantial Ca2+ permeability of P2XRs. Negative interaction between P2XR and nAChR channels Requirement of actual current ﬂow through receptor channels for cross-inhibition. It has previously been reported that there is mutual occlusion between P2XR and nAChR in some neurons (Nakazawa et al. [18]; Nakazawa [30]; Searl et al. [19]; Zhou and Galligan [20]; Barajas-Lopez et al. [21]; Khakh et al. [22, 23]; Boue-Grabot et al. [24]). To examine whether there were negative interactions between the ACh-activated current (IACh) and IATP, one receptor agonist was added in the presence of the other. As shown in Fig. 4A, the IACh (100 mM) was markedly reduced when activated in the presence of ATP (50 mM). We next examined IACh (100 mM) in the presence of various concentrations of ATP. With 2, 10, 20, and 100 mM ATP, IACh was reduced to 95 T 1% (N = 20), 84 T 2% (N = 24), 60 T 4% (N = 15), and 46 T 6% (N = 7), respectively, of control (P < 0.01 except at 2 mM ATP). Thus, the IACh inhibition became stronger as the concentration of ATP was increased and with a higher agonist-receptor occupancy. In fact, when IATP at 100 mM ATP was markedly inhibited in the presence of 200 mM PPADS (8 T 2% of control, N = 6), IACh was not occluded (97 T 1% of control; N = 6). When the peak amplitude of IATP, evoked by various concen- trations of ATP, was plotted against the ratio of IACh in the presence and absence of ATP, there was an inverse correlation between the amplitudes of these responses (Fig. 4B). Conversely, when ATP (50 mM) was applied in the presence of ACh (100 mM), IATP was also occluded (Fig. 4C). Again, when IACh at 200 mM ACh was nulliﬁed in the presence of 1 mM hexamethonium, a nAChR antagonist (0.2 T 0.2% of control, N = 6), IATP was 96 T 2% (N = 6) of control. IACh at 10, 20, 50, and 200 mM ACh were 6 T 2% (N = 5), 20 T 4% (N = 5), 61 T 8% (N = 6), and 139 T 8% (N = 6) of IACh at 100 mM. There was also an inverse correlation between the peak amplitude of IACh, evoked by various concentrations of ACh, and the ratio of IATP in the presence and absence of ACh (Fig. 4D). However, the inhibition of IATP by nAChR activation was weaker than that of IACh by P2XR activation. As the IACh desensitized faster than the IATP, the weaker inhibition of IATP by ACh could be