Calcineurin inhibits desensitization of cloned rat 5-HT1C receptors
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H W Boddeke1, B J Hoffman, J M Palacios
1Sandoz Pharma Ltd, Basle, Switzerland.
Abstract:
Functional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp recording technique. Stimulation of 5-HT1C receptors with serotonin (5-HT) evoked calcium-dependent outward currents of 109 pA in cells clamped at -50 mV. Pretreatment with the protein kinase C (PKC) activator phorbol myristic acetate (PMA) reduced the 5-HT-induced current amplitude by 46% of the control value. Inclusion of inositol triphosphate (IP3) in the pipette solution induced an outward current of 84 pA. The IP3-induced response was not affected by 60 min pretreatment with PMA. In the presence of the PKC antagonist calphostin C, 60 min treatment with PMA (10(-6) mol/l) reduced the 5-HT response only by 8%. In cells preincubated with PMA, injection of the calcium/calmodulin dependent serine proteinphosphatase calcineurin gradually increased the 5-HT-induced responses by 34%. In A9 cells which were incubated 24 h with the 5-HT1C receptor agonist meta chlorophenylpiperazine hydrochloride (mCPP), 5-HT-induced responses were reduced by 23% of the vehicle pretreated control value. Injection of calcineurin in mCPP treated cells enhanced the 5-HT-induced response by 24%. The results suggest that in A9 cells rat 5-HT1C receptors are desensitized after phosphorylation by PKC. This desensitization can be counteracted by calcineurin-induced dephosphorylation.
Insights
Serotonin (5-HT) stimulation of 5-HT1C receptors causes calcium-dependent outward currents. Protein Kinase C (PKC) activation desensitizes these receptors, but calcineurin can reverse this effect.
Area of Science:
- Neuroscience
- Molecular Biology
- Cellular Signaling
Background:
- The 5-HT1C receptor is a G protein-coupled receptor involved in various physiological processes.
- Understanding the regulation of 5-HT1C receptor function is crucial for developing targeted therapeutics.
Purpose of the Study:
- To investigate the functional responses of rat 5-HT1C receptors expressed in A9 cells.
- To elucidate the role of protein kinase C (PKC) and calcineurin in modulating 5-HT1C receptor activity.
Main Methods:
- Whole-cell voltage clamp recording technique was employed to measure functional responses.
- Cells were stimulated with serotonin (5-HT) and treated with PKC activators (PMA) and antagonists (calphostin C).
- Inositol triphosphate (IP3) and calcineurin were introduced to assess their effects on receptor currents.
Main Results:
- Serotonin (5-HT) stimulation evoked calcium-dependent outward currents.
- PKC activation by PMA significantly reduced 5-HT-induced currents, indicating receptor desensitization.
- Calcineurin injection counteracted the PKC-mediated desensitization, restoring receptor responsiveness.
- Prolonged exposure to mCPP also led to desensitization, which was partially reversed by calcineurin.
Conclusions:
- Rat 5-HT1C receptors in A9 cells undergo desensitization mediated by PKC-dependent phosphorylation.
- Calcineurin-induced dephosphorylation can reverse this desensitization, highlighting a regulatory mechanism for 5-HT1C receptor function.