Competition between internal AlF(4)(-) and receptor-mediated stimulation of dorsal raphe neuron G-proteins coupled to calcium current inhibition.

Intracellular aluminum fluoride (AlF(4)(-)), placed in a patch pipette, activated a G-protein, resulting in a "tonic" inhibition of the Ca(2+) current of isolated serotonergic neurons of the rat dorsal raphe nucleus. Serotonin (5-HT) also inhibits the Ca(2+) current of these cells. After external bath application and quick removal of 5-HT to an AlF(4)(-) containing cell, there was a reversal or transient disinhibition (TD) of the inhibitory effect of AlF(4)(-) on Ca(2+) current. A short predepolarization of the membrane potential to +70 mV, a condition that is known to reverse G-protein-mediated inhibition, reversed the inhibitory effect of AlF(4)(-) on Ca(2+) current and brought the Ca(2+) current to the same level as that seen at the peak of the TD current. With AlF(4)(-) in the pipette, the TD phenomenon could be eliminated by lowering pipette MgATP, or by totally chelating pipette Al(3+). In the presence of AlF(4)(-), but with either lowered MgATP or extreme efforts to eliminate pipette Al(3+), the rate of recovery from 5-HT on wash was slowed, a condition opposite to that where a TD occurred. The putative complex of AlF(4)(-)-bound G-protein (Galpha.GDP. AlF(4)(-)) appeared to free G-betagamma-subunits, mimicking the effect on Ca(2+) channels of the G.GTP complex. The ON-rate of the inhibition of Ca(2+) current, after a depolarizing pulse, by betagamma-subunits released by AlF(4)(-) in the pipette was significantly slower than that of the agonist-activated G-protein. The OFF-rate of the AlF(4)(-)-mediated inhibition in response to a depolarizing pulse, a measure of the affinity of the free G-betagamma-subunit for the Ca(2+) channel, was slightly slower than that of the agonist stimulated G-protein. In summary, AlF(4)(-) modified the OFF-rate kinetics of G-protein activation by agonists, but had little effect on the kinetics of the interaction of the betagamma-subunit with Ca(2+) channels. Agonist application temporarily reversed the effects of AlF(4)(-), making it a complementary tool to GTP-gamma-S for the study of G-protein interactions.