Abstract
1. In order to study the effect of intracellular free Ca2+ concentration ([Ca2+]i) on the activity of ATP-activated, GTP-dependent Ca2+ channels in rat macrophages, experiments were performed using the inside-out configuration of the patch-clamp technique. 2. Channel activity was observed in the cell-attached mode when 100 microM ATP was added to the pipette solution containing 105 mM Ba2+, but it disappeared rapidly after patch excision. The activity could be restored by the application of 100 microM GTP or GTP gamma S onto the internal surface of the plasma membrane. 3. The properties of the GTP gamma S-evoked channels are identical to those of channels activated by extracellular application of ATP. The channels exhibited four current sublevels with conductances of about 3.5, 7, 10 and 15 pS when 105 mM Ba2+ was the only permeant cation. The extrapolated reversal potentials were similar for all the sublevels and averaged about +40 mV. 4. Elevation of [Ca2+]i within the range 0.01-1 microM resulted in a decrease in mean inward current. The half-maximal value of the mean current was about 0.08 microM. 5. This decreases in mean current resulted from a redistribution of sublevel occupancies: the 1st sublevel tended to be come more abundant with elevation of [Ca2+]i, while the relative weights of the high-conductance 3rd and 4th sublevels decreased. 6. The open-channel current fell with an increase in [Ca2+]i as quickly as the mean current did, indicating that the sublevel redistribution alone is sufficient to produce the revealed decrease in net inward current. 7. It is concluded that [Ca2+]i elevation does not fix the channel in a closed state but rather decreases the ability of the channel to operate in high-conductance states.