(a) Isolated DRG neurons were treated with PTX (red trace, n = 319 neurons, 14 recordings) or not treated (black trace, n = 211 neurons, seven recordings) and tested as indicated with PS (25 µM), DAMGO (3 µM) and capsaicin (2 µM). Only PS-sensitive neurons were analyzed (all cells were from three mice). (b) Statistical summary: PTX treatment significantly reduced the inhibitory effect of DAMGO. (c) In similar experiments, treatment with PTX (red trace; n = 159 cells, three recordings) abolished the inhibition of TRPM3 channels in HEK cells overexpressing TRPM3 and µORs (control black trace, n = 178 cells, three recordings). The violet trace represents additional control cells untreated with PTX that expressed TRPM3, but not µORs (n = 163 cells, same three recordings as for the black trace), the green trace represents cells treated with PTX but not transfected with µORs (n = 142 cells, same three recordings as for the red trace). (d) Statistical summary of the experiments shown in (c). (e, f) Application of forskolin (10 µM) together with IBMX (200 µM) did not prevent the action of DAMGO (0.3 µM) in isolated DRG neurons (green trace: n = 67 neurons, treated with IBMX +forskolin, three recordings; black trace: n = 114 neurons, untreated, from three recordings). In this panel, all cells are from one mouse. Example traces of individual cells of the experiments shown in (a) and (e) are shown in Figure 5—figure supplement 2. (g, h) The same result was obtained in HEK cells overexpressing TRPM3 and µORs (green trace: n = 76 treated cells; black trace: n = 91 untreated cells, five recordings for each condition). (i) In whole-cell patch-clamp recordings of HEK cells overexpressing TRPM3 and µORs, the inhibition of PS-activated TRPM3-dependent currents (25 µM PS) by 3 µM DAMGO was almost complete, even when intracellular ATP was replaced by the non-hydrolyzable analog AMP-PNP. (j) However, removal of intracellular Mg2+ abolished the inhibition by DAMGO. two exemplary recordings are shown in (i, j), an exemplary control measurement is shown in Figure 5—figure supplement 1a. In these recordings, the break-in to the whole-cell configuration occurred 200 s before the beginning of the traces shown (i.e. 300 s before the application of DAMGO). (k) Quantitative analysis of the inhibition of 9 cells under control (with ATP and with Mg2+) conditions, 7 cells with AMP-PNP (and Mg2+) and 8 cells without Mg2+ (but with ATP) at membrane potentials of +80 mV. Figure 5—figure supplement 1b demonstrates that the series resistance of the recordings was not statistically different between the experimental groups analyzed here. In (b), (f) and (k), each individual symbol represents the value obtained from a single cell.