Modulators of protein kinase C activity alter 5-hydroxytryptamine-3 receptor function

Date

1999-05

Journal Title

Journal ISSN

Volume Title

Publisher

Texas Tech University

Abstract

The 5-hydroxytryptamine3 (5-HT3) receptor is a member of the superfamily of ligand-gated ion channels. Other members of this family include the GABAA, glycine, and nicotinic acetylcholine receptors. The function of many members of this family is regulated by phosphorylation of the receptor by intracellular protein kinases. Protein kinase C (PKC) is a well-studied protein kinase, that phosphorylates proteins on serine or threonine residues and is stimulated by activation of many plasma membrane bound receptors. PKC phosphorylates and modulates the function of many ligand-gated ion channels. Several compounds exist that activate or inhibit PKC activity independent of receptor activation. The present study explores the modulation of the 5-HT3 receptor by several of these compounds. Phorbol 12-myristate, 13-acetate (PMA) stimulates PKC activity and enhances 5-HT3 receptor function. Bisindolylmaleimide I, is a selective PKC inhibitor that decreases 5-HT3 receptor function.

The goal of this study is to determine if the increase in 5-HT3 receptor mediated current following treatment with PMA is due to phosphorylation of the receptor by PKC and to determine if the inhibition of 5-HT3 receptor mediated current seen with bisindolylmaleimide I is due to inhibition of PKC activity. In order to answer these questions, recombinant murine 5-HT3 receptors were expressed in Xenopus laevis oocytes and studied by two-electrode voltage-clamp recordings. The enhancement in 5-HT3 receptor function by PMA follows a time and concentration dependency consistent with the involvement of PKC. Furthermore, the PKC inhibitor PKCI partially inhibits the PMA induced potentiation of current. Thus, the PMA effect is at least partially dependent on PKC activity. A 5-HT3 receptor was created in which all of the intracellular serine and threonine residues were mutated to non-phosphorlatable alanine residues (the XIST mutant receptor. The PMA induced increase in receptor function of the XIST mutant did not differ from the wild-type receptor. Howe\er. mutation of a t>Tosine residue, to a non-phosphorlatable phenylalanine residue, on the NTST mutant resulted in a reduction in the PMA effect. Paradoxically, mutation of the tyrosine residue alone had no effect on PMA dependent potentiation of current. Treatment with lavendustin A. a tyrosine kinase inhibitor, reduced the PMA enhancement of the MSI mutant, but not the wild-type receptor. Thus, PMA treatment leads to activation of PKC and a tyrosine kinase. Phosphorylation of the 5-HT3 receptor by a tyrosine kinase is likely, partially responsible for the increase in current. Some serine and/or threonine residues on the 5-HT3 receptor are also involved in the increase in receptor function, but it is not clear if they are substrates for PKC-dependent phosphorylation or part of a binding site for an associated phosphoprotein.

The PKC inhibitor, bisindolylmaleimide I. decreases 5-HT3 receptor function. However, the PKC inhibitors, chelerythrine and calphostin C, did not alter 5-HT3 receptor function. It is shown that the inhibition by bisindolylmaleimide I is due to competition with 5-HT at the agonist binding site rather than through a PKC dependent mechanism. Bisindohlmaleimide I decreased the potency, without altering the efficacy, of 5-HT3. A Schild plot was generated and the slope determined to be -1. Bisindolylmaleimide I also displaced binding of the selective 5-HT; receptor antagonist. [3^H]GR65630. All of these observations are consistent with bisindolylmaleimide I acting as a competitive entagonist at the 5-HT3 receptor.

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Rights Availability

Unrestricted.

Keywords

Protein engineering, Protein kinase C, Cellular signal transduction

Citation