A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium channels.
The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels was examined using patch-clamp techniques in F11 rat dorsal root ganglion x mouse neuroblastoma hybrid cells. The PKC activator phorbol 12-myristate 13-acetate (PMA, 10 nM) reduced by 28.6 +/- 6.8 % 5-HT-mediated, but not GTP-gamma-S-induced, inhibition of Ca(2+) current, whereas a higher concentration of PMA (500 nM) inhibited both the actions of 5-HT and GTP-gamma-S. 5-HT(1A) receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A-S253G-T343A) were stably transfected into F11 cells. The T149A 5 HT(1A) receptor inhibited forskolin-stimulated cyclic AMP levels but was largely uncoupled from Ca(2+) channel modulation. In one (i2) clone a response rate to 5-HT of 31.6 % was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nM) compared to wild-type 5-HT(1A) receptors, with only a 13.4 +/- 3 % reduction in 5-HT-induced channel inhibition; when exposed to 500 nM PMA, reductions in the action of 5-HT were comparable to those of the wild-type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild-type 5-HT(1A) receptor to either concentration of PMA. PMA at 10 nM exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist D-alanine-5-leucine-enkephalin (DADLE) in wild-type and T149A mutant-expressing clones. The T149 site of the 5-HT(1A) receptor is crucial for receptor uncoupling by sub-maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N-type Ca(2+) channel.
Wu
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Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Box 29, 450 Clarkson Avenue, Brooklyn, NY 11203-2098, USA.
Kushwaha
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N
Albert
P R
PR
Penington
N J
NJ
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D015220:Calcium Channels / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006822:Hybrid Cells; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D009154:Mutation / Q000502:physiology; D009994:Osmolar Concentration; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D013755:Tetradecanoylphorbol Acetate / Q000008:administration & dosage / Q000494:pharmacology
D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; C081021:Go 6976; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016308:Enkephalin, Leucine-2-Alanine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate