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A critical protein kinase C phosphorylation site on the 5-HT(1A) receptor controlling coupling to N-type calcium

X Wu1, N Kushwaha, P R Albert

  • 1Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Box 29, 450 Clarkson Avenue, Brooklyn, NY 11203-2098, USA.

The Journal of Physiology
|January 5, 2002
PubMed

Insights

Specific protein kinase C (PKC) sites on serotonin 5-HT(1A) receptors modulate inhibitory coupling to N-type Ca(2+) channels. The T149 site is crucial for uncoupling by sub-maximal PKC activation, while maximal activation affects downstream sites.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Pharmacology

Background:

  • Serotonin 5-HT(1A) receptors are G protein-coupled receptors involved in various physiological processes.
  • Protein kinase C (PKC) is a family of enzymes that regulate cellular signaling pathways.
  • Inhibitory coupling of 5-HT(1A) receptors to N-type Ca(2+) channels is a key mechanism in neuronal excitability.

Purpose of the Study:

  • To investigate the role of specific PKC phosphorylation sites on 5-HT(1A) receptors in modulating their inhibitory coupling to N-type Ca(2+) channels.
  • To determine how different concentrations of a PKC activator affect this signaling pathway.

Main Methods:

  • Patch-clamp electrophysiology was used to measure Ca(2+) currents in F11 hybrid cells.
  • Site-directed mutagenesis was employed to alter putative PKC phosphorylation sites on the 5-HT(1A) receptor.
  • Stable transfection of modified 5-HT(1A) receptors into F11 cells.

Main Results:

  • Sub-maximal PKC activation (10 nM PMA) selectively reduced 5-HT-mediated inhibition of Ca(2+) current, while maximal activation (500 nM PMA) inhibited both 5-HT and GTP-gamma-S-induced inhibition.
  • Mutation of the T149A site in the second intracellular loop largely uncoupled the receptor from Ca(2+) channel modulation and reduced sensitivity to sub-maximal PMA.
  • Mutation of three PKC sites in the third intracellular loop (i3 mutant) did not alter sensitivity to PMA compared to wild-type receptors.

Conclusions:

  • The T149 site on the 5-HT(1A) receptor is critical for receptor uncoupling mediated by sub-maximal PKC activation.
  • Maximal PKC activation leads to uncoupling of G proteins from N-type Ca(2+) channels via downstream sites.
  • These findings elucidate the specific roles of PKC phosphorylation sites in regulating 5-HT(1A) receptor signaling and neuronal function.

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