Competition between internal AlF4- and receptor-mediated stimulation of dorsal raphe neuron G-proteins coupled to calcium current inhibition

Intracellular aluminum fluoride (AlF4-), placed in a patch pipette, activated a G-protein, resulting in a "tonic" inhibition of' the Ca2+ current of isolated serotonergic neurons of the rat dorsal raphe nucleus. Serotonin (5-HT) also inhibits the Ca2+ current of' these cells. After external bath application and quick removal of 5-HT to an AlF4- containing cell, there was a reversal or transient disinhibition (TD) of the inhibitory effect of AlF4- on Ca2+ 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 AlF4- on Ca2+ current and brought the Ca2+ current to the same level as that seen at the peak of the TD current. With AlF4- in the pipette, the TD phenomenon could be eliminated by lowering pipette MgATP, or by totally chelating pipette Al3+. In the presence of AlF4-, but with either lowered MgATP or extreme efforts to eliminate pipette Al3+, the rate of recovery from 5-HT on wash was slowed, a condition opposite to that where a TD occurred. The putative complex of AlF4--bound G-protein (G alpha . GDP . AlF4-) appeared to free G-beta gamma-subunits, mimicking the effect on Ca2+ channels of the G.GTP complex. The ON-rate of the inhibition of Ca2+ current, after a depolarizing pulse, by py-subunits released by AlF4- in the pipette was significantly slower than that of the agonist-activated G-protein. The OFF-rate of the AlF4--mediated inhibition in response to a depolarizing pulse, a measure of the affinity of the free G-beta gamma-subunit for the Ca2+ channel, was slightly slower than that of the agonist stimulated G-protein. In summary, AlF4- modified the OFF-rate kinetics of G-protein activation by agonists, but had little effect on the kinetics of the interaction of the beta gamma-subunit with Ca2+ channels. Agonist application temporarily reversed the effects of AlF4-, making it a complementary tool to GTP-gamma-S for the study of G-protein interactions.