Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number

Abstract

Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). However, mechanisms involved in recruiting AMPARs to synapses are unknown. Here we use hippocampal slice cultures and biolistic gene transfections to study the targeting of AMPARs to synapses. We show that AMPARs are localized to synapses through direct binding of the first two PDZ domains of synaptic PSD-95 (postsynaptic density protein of 95 kDa) to the AMPAR-associated protein, stargazin. Increasing the level of synaptic PSD-95 recruits new AMPARs to synapses without changing the number of surface AMPARs. At the same time, we show that stargazin overexpression drastically increases the number of extra-synaptic AMPARs, but fails to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, we make compensatory mutations to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs.

Figure 1

Slice culture recording configuration. Each experiment involved simultaneous whole-cell voltage-clamp recordings from both a transfected cell (PSD-95 GFP in this example, cell on right, gold particle in nucleus) and an immediately adjacent untransfected cell (left). (Left) The differential interference contrast (DIC) transmitted light image. (Center) Clusters of PSD-95 GFP in the spines of the transfected cell when viewed under fluorescence. (Right) The two images are overlaid. (Scale bar: 20 μm.)

Figure 2

PSD-95 and stargazin have differential effects on synaptic and surface AMPAR number. (A) Confocal image of a pyramidal cell expressing PSD-95 GFP showing its localization to synaptic spines. (Scale bar: 5 μm.) (B) Averaged EPSCs recorded simultaneously from a pair of cells, showing the responses at −70 mV and +40 mV for a PSD-95-transfected cell and a neighboring untransfected (control) cell. (Scale bars: 10 pA, 20 ms.) (C) Bar graph representations of data from the PSD-95 transfections. AMPAR EPSCs are significantly enhanced (P < 1 × 10⁻⁶, n = 27 pairs), whereas NMDAR EPSCs are unchanged (P = 0.81, n = 23 pairs). (D) Confocal image of a cell expressing stargazin-GFP shows that it also localizes to synaptic spines (same scale as A). (E and F) Overexpression of stargazin has no effect on evoked synaptic responses (AMPAR EPSCs, n = 26 pairs, P = 0.48; NMDAR EPSCs, n = 14 pairs, P = 0.99). (G) PSD-95-expressing cells do not show a change in the response to bath-applied AMPA (1 μM, n = 3 pairs). (H) Overexpression of stargazin dramatically increases responses to bath application of AMPA (500 nM, n = 7 pairs, P = 0.003).

Figure 3

The stargazin PDZ-binding region is required for synaptic, but not extra-synaptic, AMPAR trafficking. (A) Confocal image of a pyramidal cell expressing stargazinΔC-GFP shows its diffuse localization. (Scale bar: 5 μm.) (B and C) Overexpression of stargazinΔC strongly reduces AMPAR EPSCs (n = 54 pairs, P < 1 × 10 ⁻⁹) while having no effect on NMDAR EPSCs (n = 37 pairs, P = 0.43). (Scale bars, traces: 10 pA, 20 ms.) (D) In contrast, stargazinΔC still dramatically increases responses to bath application of 500 nM AMPA (n = 9 pairs, P = 0.0006).

Figure 4

PSD-95 palmitoylation and PDZ domains are needed to enhance synaptic AMPAR EPSCs. (A) Diagrams showing the domain structure of the various PSD-95 and MAGUK constructs. SH3, Src homology 3; GK, guanylate kinase. (B) Summary graph showing the effects of overexpressing the various constructs on the evoked AMPAR EPSC in simultaneously recorded pairs. For each construct, the normalized response was obtained by dividing the average AMPAR EPSC amplitude in the transfected cells by the average amplitude in paired, untransfected cells. Statistically significant values are marked with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001); the number of paired recordings is listed for each construct. (C) Summary graph showing the effects of these constructs on the NMDAR EPSC.

Figure 5

Compensatory mutations to PSD-95 and stargazin reconstitute binding and clustering. (A) GST-fusion proteins show that stargazin with a point mutation in its PDZ-binding region (T321F) does not bind WT PSD-95, but does bind PSD-95 bearing a compensatory point mutation (H225V) in its second PDZ domain. (B) Confocal images of pyramidal cells expressing stargazin T321F GFP. Stargazin T321F GFP alone expressed diffusely (Top). Coexpression of untagged PSD-95 did not alter the diffuse localization of stargazin T321F (Middle). Cotransfection of an untagged compensatory PSD95 (PSD-95 H225V) caused robust synaptic localization of stargazin T321F GFP (Bottom). (Scale bar: 5 μm.)

Figure 6

PSD-95 and stargazin directly interact to mediate synaptic AMPAR delivery. (A) Stargazin T321F overexpression dramatically reduces AMPAR EPSCs (n = 14 pairs, P < 0.00001) but does not change NMDAR EPSCs (n = 13 pairs, P = 0.52). (B) Coexpression of stargazin T321F with PSD-95 still results in a significant reduction of the AMPAR EPSC (n = 30 pairs, P = 0.035). (C) Coexpression of stargazin T321F with a PSD-95 construct bearing a compensatory mutation rescues and significantly enhances the AMPAR EPSC. PSD-95 mutants bearing a complementary mutation in either PDZ1 (H130V) or PDZ2 (H225V) were cotransfected with the stargazin mutant. In the bar graphs, results from both cotransfections were combined as the results were identical (see text) (AMPAR EPSCs, P = 0.00001, n = 35 pairs; NMDAR EPSCs, P = 0.43, n = 31 pairs). (Scale bars: 10 pA, 20 ms.)