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 (Gα·GDP·AlF₄/^-) appeared to free G- βγ-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 βγ-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-βγ-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 βγ-subunit with Ca²⁺ channels. Agonist application temporarily reversed the effects of AlF₄/^-, making it a complementary tool to GTP-γ-S for the study of G- protein interactions.