Anti-Kv1.2 Immunoprecipitation Identifies shared mGluR1-Associated Signalosome Complex proteins and PKC-Mediated Regulation of Kv1.2 in the Cerebellum

Abstract

The voltage gated potassium channel Kv1.2 plays a key role in the central nervous system and mutations in Kv1.2 leads to neurological disorders such as epilepsies and ataxias. In the cerebellum regulation of Kv1.2 is coupled to learning and memory. We have previously shown that blocking Kv1.2 by infusing its specific inhibitor Tityustoxin-k (TsTX) into the lobulus simplex of the cerebellum facilitates eyeblink conditioning (EBC) and that EBC modulates Kv1.2 surface expression in cerebellar interneurons. The metabotropic glutamate receptor mGluR1 is required for EBC although the molecular mechanisms are not fully understood. We have previously shown that infusion of the mGluR1 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the lobulus simplex of the cerebellum mimics the faciliatory effect of TsTX on EBC. We therefore hypothesize that mGluR1 could act, in part, through suppression of Kv1.2. Earlier studies have shown that Kv1.2 suppression involves channel tyrosine phosphorylation and its endocytocytic removal from the cell surface. In this study we report that an excitatory chemical stimulus (50mM K+-100{micro}M glutamate) applied to cerebellar slices enhanced Kv1.2 tyrosine phosphorylation and that this increase was lessened in the presence of the mGluR1 inhibitor YM298198. More direct evidence for mGluR1 modulation of Kv1.2 comes from our finding that selective activation of mGluR1 with DHPG reduced the amount of Kv1.2 detected by cell surface biotinylation in cerebellar slices. To determine the molecular pathways involved we used an unbiased mass spectrometry-based proteomics approach to identify Kv1.2-protein interactions that are modulated by mGluR1. Among the interactions enhanced by DHPG were those with PKC-{gamma}, CaMKII and Gq/G11, each of which had been shown in other studies to co-immunoprecipitate with mGluR1 and contribute to its signaling. Of particular note was the interaction between Kv1.2 and PKC-{gamma} since in HEK cells and hippocampal neurons Kv1.2 endocytosis is elicited by PKC activation. Here we show that activation of PKCs with PMA reduced surface Kv1.2, while the PKC inhibitor Go6983 attenuated the reduction in surface Kv1.2 levels elicited by DHPG, suggesting that the mechanism by which mGluR1 modulates cerebellar Kv1.2 likely involves PKC.