The Journal of Neurophysiology Vol. 77 No. 5 May 1997, pp. 2697-2703
Copyright ©1997 The American Physiological Society

Order of Application Determines the Interaction Between Phorbol Esters and GTP--S in Dorsal Raphe Neurons: Evidence That the Effect of 5-HT Is Modified Upstream of the G Protein Ca Channel Interaction

Yuan Chen and Nicholas J. Penington

Department of Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York, 11203

Chen, Yuan and Nicholas J. Penington. Order of application determines the interaction between phorbol esters and GTP--S in dorsal raphe neurons: evidence that the effect of 5-HT is modified upstream of the G protein Ca channel interaction. J. Neurophysiol. 77: 2697-2703, 1997. Phorbol esters activating protein kinase C (PKC) partially uncouple the inhibitory effect of serotonin (5-HT) from serotonergic neuron Ca²⁺ current. Presently the site of action of PKC is not known and may be the receptor, G protein, or ion channel. We recorded Ca²⁺ current from acutely isolated neurons with the use of the patch-clamp technique to study the site of action of PKC. Activation of the G protein with internal guanosine 5-O-(3-thiotriphosphate) (GTP--S) occluded the response to 5-HT, but unexpectedly this effect was not reversed by the addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) despite the voltage-dependent reversal of the effect of GTP--S by long depolarizing steps to +80 mV. PMA was, however, able to partially reverse 5-HT-induced inhibition of Ca²⁺ current. The rate of reinhibition of the Ca²⁺ current (related to the concentration of activated G proteins) by GTP--S after the addition of PMA at 50 mV was identical to the rate when only GTP--S was present.By contrast, when cells were exposed first to PMA, and thenGTP--S was perfused into the cell, GTP--S lost about half of its ability to activate the G protein. The rate of reinhibition of the Ca²⁺ current by internal GTP--S was also reduced in cells pretreated with PMA. The original result in which PMA did not reverse the action of GTP--S suggested that the channel was not the functional site of action of PMA, nor was the site on the G protein that binds to the channel, but it did not rule out the receptor. When the receptor was bypassed, after prior PKC activation, it was found that direct activation of the G protein by a nonhydrolyzable analogue of GTP was reduced; taken as a whole, this indicates that in dorsal raphe, and perhaps other neurons, the site of the critical phosphorylation may be on the G protein and possibly at the GTP binding site.

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