ATP-OPERATED CALCIUM-PERMEABLE CHANNELS ACTIVATED VIA A GUANINE NUCLEOTIDE-DEPENDENT MECHANISM IN RAT MACROPHAGES

1. To elucidate the possible involvement of a G protein in ATP-evoked Ca2+-permeable channel activity, membrane currents of rat peritoneal m acrophages were recorded using inside-out and cell-attached configurat ions of the patch clamp technique. 2. In inside-out experiments with a pipette solution containing 105 mM Ba2+, application of 100 mu M GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches i nvestigated (n = 69). The mean value of NPo (N, number of open channel s; P-o, channel open probability) was equal to 0.49 +/- 0.27 (mean +/- S.E.M.; n = 16). The delay in the activity development was 21 +/- 8 s (n = 18) with 200 mu M ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtai ned with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S-evoked channels were identical to those of channels activated by extracellular application of ATP The channels exhibited at least fo ur conductance sublevels, the 4th one being the least frequent. With 1 05 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 an d 17 pS. Extrapolated reversal potential (E(r)) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channel s with similar characteristics could be induced by the extracellular a pplication of fluoride in cell-attached experiments without any agonis t in the pipette solution. 5. Currents through non-selective cationic channels with properties much different from ATP-activated GTP-depende nt Ca2+-permeable channels were recorded with GTP gamma S added to the intracellular solution (inside-out experiments) and fluoride applied to the bath solution (cell-attached conditions). With isomolar concent rations of Ba2+ used extracellularly and K+ intracellularly, the E(r) appeared to be about 0 mV. These channels did not show determinable cu rrent sublevels. They were observed rarely (6 out of more than 100 exp eriments) and could not be attributed to activation by an agonist. 6. The data obtained suggest that an ATP-activated receptor is coupled wi th a Ca2+-permeable channel via a GTP-dependent mechanism, presumably via a G protein.