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, New York, 11203.
APStracts 4:0031N, 1997.
ABSTRACT
1. Phorbol esters activating protein kinase C (PKC) partially uncouple the
inhibitory effect of 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 using the
patch clamp technique to study the site of action of PKC. 2. Activation of the
G-protein with internal GTP-_-S occluded the response to 5-HT, but
unexpectedly this effect was not reversed by the addition of the phorbol ester
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. 3. The rate of re-inhibition of the Ca2+
current (related to the concentration of activated G-proteins) by GTP-_-S
after the addition of PMA at -80mV was identical to the rate when only GTP-_-S
was present. 4. By contrast, when cells were exposed first to PMA, then GTP-_-
S was perfused into the cell, GTP-_-S lost about one half of its ability to
activate the G-protein. 5. The rate of re-inhibition of the Ca2+ current by
internal GTP-_-S was also reduced in cells pretreated with PMA. 6. 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 by-passed, after prior PKC activation, it was
found that direct activation of the G-protein by a non-hydrolyzable analog of
GTP was reduced; taken together 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.
Received 4 Novebmer 1996; accepted in final form 14 January 1997.
APS Manuscript Number J869-6.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1997 The American Physiological Society.
Published in APStracts on 5 February 1997