Targeted In Vivo Mutations of the AMPA Receptor Subunit GluR2 and Its Interacting Protein PICK1 Eliminate Cerebellar Long-Term Depression

Abstract

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.

Synaptic AMPA receptor composition in development, plasticity and disease

“…Knock-in mice expressing GluA1 or GluA2 subunits mutated to non-phosphorylatable or phosphomimetic residues have demonstrated that phosphorylation of GluA1 by CaMKII or PKA is necessary for full hippocampal LTP expression 134 , while dephosphorylation of the PKA site in GluA1 is required for LTD 46 . Furthermore, PKC phosphorylation of GluA2 is required for cerebellar LTD 135 . Beyond GluA1 and GluA2, transient expression of GluA4 in CA1 pyramidal neurons during early development underpins the switch in kinase signaling in LTP from PKA to CaMKII-dependent mechanisms 136 .…”

Reassessment of long-term depression in cerebellar Purkinje cells in mice carrying mutated GluA2 C terminus

“…The other is the GluA2 Δ7 KI (hereafter, Δ7) genotype, which lacks the last 7 amino acids of the C-terminal tail of GluA2. The region lacking the 7 amino acids is essential for the interaction of GluA2 with PICK1 and GRIP1/2 on the synaptic membrane (15)(16)(17). Because of the mutations, these mice are believed to be devoid of LTD; however, we found in this study that LTD is still inducible under certain stimulation conditions.…”

“…3A) (19,20). The patch pipette was filled with a Cs + -based internal solution to improve space clamping along PC dendrites by blocking K + channels (15,21). The mean Rm (membrane resistance in MΩ, measured by passing square currents through the patch pipette) in cells recorded with the pipette containing the Cs + -based internal solution was three-to four-times larger than those recorded with a pipette containing a K + -based solution (Table S1).…”

“…This protocol was originally used by Steinberg et al (15), with five PF stimulations at 100 Hz concomitant with somatic depolarization (100-ms duration) applied at 2-s intervals for 1 min. This protocol induced LTD in slices obtained from young mice [postnatal day (P) [20][21][22][23][24][25] in WT, but in neither K882A nor Δ7 PCs (15). In the present study, however, when applied to mature WT mice (3-6 mo), the original protocol of Steinberg et al (15) was ineffective for inducing LTD (98.0 ± 5.7%, n = 4, P = 0.78, paired t test).…”

“…Normally, PKCα phosphorylates Ser880 and thereby detaches the AMPA-subtype glutamate receptors (AMPARs) from a synaptic scaffold protein, GRIP1/2; consequently, AMPARs are internalized. On the other hand, PDZ ligands and phosphorylation by other kinases remain functionally intact (13)(14)(15)(16)(17). The other is the GluA2 Δ7 KI (hereafter, Δ7) genotype, which lacks the last 7 amino acids of the C-terminal tail of GluA2.…”