G-protein activators induce a potassium conductance in murine macrophages

The whole-cell patch clamp technique was used to test whether intracellular application of G-protein activators affect ionic currents in murine macrophages. Both the J774.1 macrophage-like cell line and primary bone marrow derived macrophages were used. Cells were bathed in Na Hanks' solution and intracellularly dialyzed (via the patch pipette) with K Hanks (145 mM KCl, < 100 nM Ca) plus or minus the G-protein activators GTP gamma S (10 microM), GppNHp (10 microM), or AIF4- (200 microM AlCl3 + 5 mM KF). In the absence of G-protein activators, only two K currents, an inwardly rectifying K current (Kir) and an outward, inactivating K current (Ko) were observed. In the presence of protein activators, two effects were observed: (i) the Kir conductance, which is stable for up to 30 min under control conditions, decayed twice as fast and (ii) an outwardly rectifying, noninactivating current appeared. The induced outward current appeared < 2 min after attaining the whole-cell patch clamp configuration. The current could be distinguished from the Kir and Ko currents on the basis of its direction of rectification (outward), barium sensitivity (> 1 mM), and kinetics (no time-dependent inactivation). Intracellular application of GTP (500 microM), GDP (500 microM), cAMP (100 microM + 0.5 mM ATP), or IP3 (20 microM) did not induce the current; 100 microM ATP gamma S activated a half-maximal amount of current. Induction of outward current by 10 microM GTP gamma S could be prevented by pre-exposing cells to pertussis toxin but not cholera toxin. This current is K selective since (i) its induction was accompanied by hyperpolarization of the cell toward EK, even after Kir had "washed out", (ii) it was present after > 90% of both intracellular and extracellular Cl were replaced by isethionate, and (iii) the induced outward conductance was absent when Ki was completely replaced by Cs, and was reduced by approximately 1/3 when [K]i was reduced by 1/3. Quinidine (1 mM) and 4-aminopyridine (10 mM) inhibited the current, but apamin (1 microM) and charybdotoxin (1 microM) did not.