ORDER OF APPLICATION DETERMINES THE INTERACTION BETWEEN PHORBOL ESTERS AND GTP-GAMMA-S IN DORSAL RAPHE NEURONS - EVIDENCE THAT THE EFFECT OF 5-HT IS MODIFIED UPSTREAM OF THE G-PROTEIN CA CHANNEL INTERACTION

Phorbol esters activating protein kinase C (PKC) partially uncouple th e inhibitory effect of serotonin (5-HT) from serotonergic neuron Ca2current. Presently the site of action of PKC is not known and may be t he 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 inte rnal guanosine 5'-O(3-thiotriphosphate) (GTP-gamma-S) occluded the res ponse to 5-HT, but unexpectedly this effect was not reversed by the ad dition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) desp ite the voltage-dependent reversal of the effect of GTP-gamma-S by lon g depolarizing steps to +80 mV. PMA was, however, able to partially re verse 5-HT-induced inhibition of Ca2+ current. The rate of reinhibitio n of the Ca2+ current (related to the concentration of activated G pro teins) by GTP-gamma-S after the addition of PMA at -50 mV was identica l to the rate when only GTP-gamma-S was present. By contrast, when cel ls were exposed first to PMA, and then GTP-gamma-S was perfused into t he cell, GTP-gamma-S lost about half of its ability to activate the G protein. The rate of reinhibition of the Ca2+ current by internal GTP- gamma-S was also reduced in cells pretreated with PMA. The original re sult in which PMA did not reverse the action of GTP-gamma-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 r ule out the receptor. When the receptor was bypassed, after prior PKC activation, it was found that direct activation of the C protein by a nonhydrolyzable analogue of GTP was reduced; taken as a whole, this in dicates that in dorsal raphe, and perhaps other neurons, the site of t he critical phosphorylation may be on the G protein and possibly at th e GTP binding site.