Figure 2.

Knockdown of PICK1 abolishes NMDAR-induced intracellular accumulation of AMPARs. A, Knockdown of PICK1 does not affect the surface expression of GluA1 as shown in images of representative cells (A1), and after quantitation (A2) (control, 1 ± 0.04, n = 37; shPICK1, 0.9 ± 0.06, n = 44; shPICK1 plus GFP-PICK1, 0.9 ± 0.07, n = 20). B, shPICK1-infected cells exhibit an increase in surface expression of GluA2, an effect that is reversed by replacement of endogenous receptors with GFP-PICK1 (shPICK1 plus GFP-PICK1). B1, Representative cells. B2, Quantitation (control, 1 ± 0.07, n = 38; shPICK1, 1.5 ± 0.15, n = 47; shPICK1 plus GFP-PICK1, 0.6 ± 0.05, n = 24). C, PICK1 knockdown causes an increase in extrasynaptic, but not synaptic GluA2. C1, Examples of dendritic staining for surface GluA2 (red) and Bassoon (blue) in control or shPICK1-infected cells. Scale bar, 10 μm. C2, Quantitation of number of GluA2 clusters/50 μm dendrite (control, 104.4 ± 3.7, n = 15; shPICK1, 136.8 ± 4.6, n = 12). C3, Quantitation of number of synapses/50 μm dendrite (control, 88.1 ± 6.5, n = 15; shPICK1, 97.3 ± 4.5, n = 12). C4, Quantitation of percentage of synapses (defined by Bassoon staining) that express detectable surface GluA2 puncta (control, 60.7 ± 1.9%, n = 15; shPICK1, 56.4 ± 3.1%, n = 12). D, E, PICK1 knockdown inhibits NMDAR-induced intracellular accumulation of surface GluA1-containing AMPARs (D) and surface GluA2-containing AMPARs (E). Representative images (D1, E1) and quantitation (D2, E2) of NMDAR-triggered intracellular accumulation of surface AMPARs in cells expressing GFP, shPICK1, or shPICK1 plus GFP-PICK1. (D2, Control, 1.0 ± 0.07, n = 24; control plus NMDA, 1.7 ± 0.06, n = 21; shPICK1 plus NMDA, 1.2 ± 0.08, n = 24; shPICK1 plus GFP-PICK1 plus NMDA, 1.7 ± 0.09, n = 21.) (E2, Control, 1.0 ± 0.07, n = 32; control plus NMDA, 2.7 ± 0.27, n = 31; shPICK1 plus NMDA, 1.2 ± 0.12, n = 27; shPICK1 plus GFP-PICK1 plus NMDA, 2.6 ± 0.27, n = 24.)