The Journal of Neurophysiology Vol. 83 No. 3 March 2000, pp. 1273-1282
Copyright ©2000 by the American Physiological Society

Competition Between Internal AlF₄ and Receptor-Mediated Stimulation of Dorsal Raphe Neuron G-Proteins Coupled to Calcium Current Inhibition

Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203-2098

Chen, Yuan and Nicholas J. Penington. Competition Between Internal AlF₄ and Receptor-Mediated Stimulation of Dorsal Raphe Neuron G-Proteins Coupled to Calcium Current Inhibition. J. Neurophysiol. 83: 1273-1282, 2000. 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·GDP·AlF₄) appeared to free G--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 -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--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 -subunit with Ca²⁺ channels. Agonist application temporarily reversed the effects of AlF₄, making it a complementary tool to GTP--S for the study of G-protein interactions.