The Lurcher Mutant of δ2 Ionotropic Glutamate Receptor is Regulated by Phosphoinositides

Abstract

The δ2 glutamate receptor (GluD2) is an orphan glutamate-like receptor, since no specific ligand capable of activating it has been identified. Nevertheless, it is increasingly appreciated that GluD2 is an important part of the physiology of the cerebellum. It is highly expressed in the parallel fiber-Purkinje cell (PF-PC synapse) and it has been linked to presynaptic terminal differentiation and the induction of long-term depression (LTD). Although, the wild-type receptor is non-conductive, a mutation in the third transmembrane domain (A654T), named lurcher (GluD2Lc), exhibits constitutive activity. Motivated by our previous work showing that NMDA receptor activity was dependent on phosphoinositides (PIPs) indirectly through the PIP2-sensitive and NMDA receptor-associated protein a-actinin, we asked whether GluD2 behavior was also dependent on PIPs and used GluD2Lc as our model to address this question.Our results in X. laevis oocytes have shown that GluD2Lc currents were potentiated upon activation of Gq-coupled receptors (M1R and mGluR1). Pretreatment with the PI4K inhibitor phenylarsine oxide (PAO) led to potentiated GluD2Lc currents as well. Moreover, pretreatment with the PI3K inhibitors wortmannin (at low μM concentrations) and LY294002 inhibited the GluD2Lc current. GluD2Lc co-expression with PIP5K or PI3K yielded consistent results with PIP5K inhibiting, while PI3K stimulating activity. These results are consistent with a dependence of GluD2Lc activity on PIPs.In order to distinguish between direct versus indirect phosphoinositide effects on GluD2 we are pursuing two strategies: 1) we are testing effects of PIPs on the activity of GluD2Lc in inside-out macropatches, and 2) we utilize site-directed mutagenesis to probe for critical residues in the proximal C-terminus that alter GluD2Lc dependence on PIPs. The proximal C-terminus could be responsible for direct interactions with phosphoinositides but could also couple to PIPs indirectly through PIP-sensitive proteins that interact with GluD2.