Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swellin g activated an anion channel current, when the phloretin-sensitive, volume- activated outwardly rectifying Cl- channel was eliminated. This current exh ibited ti ni e-de pendent inactivation at positive and negative voltages gr eater than around +/- 25 mV. The whole-cell current was selective for anion s and sensitive to Gd3+. In on-cell patches, single-channel events appeared with a lag period of similar to 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivati on similar to that of whole-cell current. Voltage-dependent activation pro le was bell-shaped with the maximum open probability at -20 to 0 mV The cha nnel in inside-out patches had the unitary conductance of similar to 400 pS , a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellula r ATP with art IC50 of 12.3 mm and an electric distance (delta) of 0.47, wh ereas the inward (but not outward) conductance was inhibited by intracellul ar ATP with an IC50 of 12.9 mM and delta of 0.40, Despite the open channel block by ATP, the channel was ATP-conductive with P-ATP/P-Cl of 0.09. The s ingle-channel activity was sensitive to Gd3+, SITS, and NPPB, but insensiti ve to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded t hat the volume- and voltage-dependent ATP-conductive large-conductance anio n channel serves as a conductive pathway for the swelling-induced ATP relea se in C127i cells.