FUNCTIONAL-STUDIES OF SINGLE-SITE VARIANTS IN THE CALMODULIN-BINDING DOMAIN OF RC3 NEUROGRANIN IN XENOPUS OOCYTES/

Single-site variants in the calmodulin-binding domain of RC3/neurogran in were heterologously expressed in Xenopus oocytes to examine their e ffects on serotonin-evoked currents. RC3 variants serine(36)-->alanine (Ser(36)-->Ala), serine(36)-->glycine (Ser(36)-->Gly), and phenylalan ine(37)-->tryptophan (Phe(37)-->Trp), which bind calmodulin but are de ficient in protein kinase C (PKC) phosphorylation, display serotonin-e voked Ca2+-dependent Cl- currents in oocytes similar to control oocyte s. A serine(36)-->aspartate (Ser(36)-->Asp) variant, which does not bi nd calmodulin and mimics the PKC-phosphorylated state of RC3, signific antly enhances serotonin-evoked currents in a manner similar to wild-t ype. The results suggest that RC3 not only regulates the availability of free calmodulin in a dendritic spine but also, when phosphorylated, independently stimulates G-protein coupled second messenger pathways that generate inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG) and intracellular Ca2+.