Purinergic Signalling (2006) 2:545–557 553 Fig. 6 Voltage dependence of IACh and of IATP occlusion. A) At a Vh of j70 mV, co-application of ACh (200 mM) and ATP (100 mM) induced an inward current that was smaller in amplitude than the predicted sum of the individual IACh and IATP. B) At a Vh of +40 mV, the amplitude of the outward current induced by the co-application of ACh (500 mM) and ATP (200 mM) was equal to the predicted sum of the individual IACh and IATP. C) Co-application of ATP and ACh does not markedly change EACh or EATP. Representative currents induced by ACh, ATP, and by co-application of ACh and ATP at a Vh of +10 mV. Note that IACh + ATP is composed of early outward and delayed inward current, corresponding to the ACh component and the ATP component, respectively. explained by the desensitization kinetics of IACh or IATP alone, suggesting that IACh + ATP is mediated via both receptors. When Ca2+ was omitted from extracellular solution, the negative interaction between the two receptor channels was still obtained; with IACh + ATP being 77 T 1% (N = 5; P < 0.01) of the predicted sum of IACh and IATP. Hence negative interaction is not mediated by a Ca2+ inﬂux-dependent mechanism, although both nAChR and P2XR are Ca2+-permeable cation chan- nels (Fieber and Adams [35]; Rogers and Dani [36]). This result, however, does not necessarily rule out the possible involvement of [Ca]in in negative interaction. Occlusion was not only observed at a Vh of j70 mV; IACh + ATP at Vhs of j20, and j10 mV were 80 T 2% (N = 12; P < 0.01), and 76 T 2% (N = 8; P < 0.01), respectively, of the predicted sum of IACh and IATP. In sharp contrast, such occlusion was not observed at a positive potential; the outward current caused by co- application of ACh and ATP at a Vh of +40 mV was 100 T 1% (N = 10) of the predicted sum of individual current (Fig. 6B). We next investigated the possibility that the com- bined addition of ACh and ATP alters the driving force for Na+, by attempting to measure EACh + ATP. In these studies, currents in response to ACh, ATP and both ligands were measured at various ﬁxed potentials close to the reversal potential for IACh and IATP. The EACh and EATP were 8.7 T 1.5 mV (N = 5) and 21.0 T 1.6 mV (N = 7), respectively, and IACh + ATP measured at a potential between these two reversal potentials (e.g.10 mV, Fig. 6C) showed a combination of both the ACh-induced outward current and the ATP-induced inward current. These results suggest that the driving force for permeant ions during combined ACh and ATP is similar to that during the application of each ligand separately. The question arises as to whether occlusion is speciﬁc just for Na+ ions or whether inward currents carried by cations other than Na+ can also contribute to occlusion during co-activation of nAChRs and P2XRs. To address this question, we measured IACh, IATP, and IACh + ATP in the presence of Na+-free (NMDG+) saline containing 10 mM Ca2+ (Fig. 7). NMDG+ does not permeate through nAChR (Sorima- Fig. 7 Occlusion of IACh + ATP in the presence of Na+-free saline containing 10 mM Ca2+. IACh, IATP or IACh + ATP was recorded 20 s after switching to Na+-free (replaced by NMDG+) saline containing 10 mM Ca2+. The sequence of applications was repeated multiple times. Following each ligand application, the external solution was changed back to normal saline containing 150 mM Na+ before the next ligand application. Vh: j70 mV.