Lin Y , Jover-Mengual T , Wong J , Bennett MV , Zukin RS .

NS 07412 NINDS NIH HHS , NS 20752 NINDS NIH HHS , R01 NS020752 NINDS NIH HHS , T32 NS007412 NINDS NIH HHS

	Fig. 1. PSD-95 expression partially occludes PKC potentiation of NMDA-elicited currents. NMDA-elicited currents (100 μM NMDA with 10 μM glycine) were recorded in Ca2+ Ringer's solution at a holding potential of −60 mV from oocytes expressing NR1–4b/NR2A receptors in the absence or presence of PSD-95. (A and B) PKC activated by TPA potentiated NMDA-elicited currents in oocytes expressing NR1/NR2A receptors (Left). Expression of PSD-95 increased the initial or basal NMDA-elicited currents by ≈2-fold, but PKC potentiated NMDA currents to similar final values in the presence and absence of PSD-95 (Right). (C) Quantitation of these data. (D) Because PSD-95 increased the basal response but not the response after TPA, the potentiation ratio was reduced from ≈10- to ≈5-fold. ∗∗, P < 0.01; ∗∗∗, P < 0.001. n.s., not significant.
	Fig. 2. PSD-95 partially occludes PKC potentiation of NMDA single channel activity. Representative traces of NMDA single channel activity recorded from outside-out patches excised from oocytes expressing NR1–4b/NR2A receptors in the absence or presence of PSD-95. (A–C) PDS-95 expression enhanced NMDA channel number times open probability, np o by ≈1.3-fold. (B and C) PKC activation by TPA markedly increased np o to nearly the same final value in cells expressing NMDARs in the absence or presence of PSD-95. (D) Neither PSD-95 nor PKC activation, nor the two together, detectably altered single channel conductance; the slope of single channel currents was linear from −100 to + 80 mV in all four conditions (white circles denoting NMDARs + PSD-95 obscure underlying black circles for NMDARs alone). NMDA activated single channels with a unitary conductance (γ = 50 ± 1 pS; E rev ≈ 0 mV) that was the same in the absence or presence of PSD-95 before and after PKC activation; n = 5 per group). (E and F) Mean open time distributions were the same in all four conditions. Responses were elicited by application of NMDA (10 μM with 10 μM glycine) at a holding potential of −60 mV. To activate PKC, TPA (100 nM) was bath-applied. ∗, P < 0.05; ∗∗, P < 0.01.
	Fig. 3. PSD-95 partially occludes PKC potentiation of NMDA channel number and opening rate. (A and B) Sample records of NMDA-elicited currents recorded from oocytes expressing NR1–4b/NR2A receptors in the absence or presence of PSD-95 and before and after TPA application in Ca2+- free (Ba2+) Ringer's. (C and D) Responses in the absence and presence of PSD-95 in the continuous presence of the open channel blocker MK-801 applied shortly before NMDA (5 μM) to control (Left) and TPA-treated (Right) oocytes. Charge transfer is indicated by cross-hatching. Different oocytes were used for each condition. Coexpression of PSD-95 increased the initial (basal) NMDA-elicited current (compare A with B) and the number of functional NMDA channels, N, at the cell surface under basal conditions (compare C with D). TPA increased N to the same final value in cells expressing NMDARs alone vs. NMDARs with PSD-95, but the potentiation ratio was smaller in cells coexpressing PSD-95 (compare C with D). (E–H), Responses in C and D were normalized to the same peak amplitude for pairwise comparison of the time course of response decay in MK-801, a measure of opening rate, k β. (E) TPA increased the rate of decay of NMDA currents recorded from cells expressing NMDARs in the absence of PSD-95, indicating an increase in the channel opening rate, k β. (F) TPA did not detectably alter k β in cells coexpressing PSD-95. (G) The rate of decay of NMDA currents was greater in the presence than the absence of PSD-95. (H) After TPA treatment, the rate of decay of NMDA currents did not differ in the absence and presence of PSD-95. Increase in k β caused by coexpression of PSD-95 occludes PKC potentiation of k β. (I–L) Summary of data in experiments illustrated in A–H. In the presence of PSD-95, I, N, and k β, but not p o, were greater than in its absence, but the final values after TPA potentiation were not significantly different. Thus, PSD-95 expression occludes the TPA potentiation ratios. Currents were elicited by application of NMDA (1 mM NMDA with 50 μM glycine) at a holding potential of −60 mV.
	Fig. 4. PSD-95 partially occludes PKC potentiation of surface NMDAR expression. Representative tangential (left column) and cross-sectional (right column) images of oocytes expressing NR1–4b/NR2A receptors in the absence (A and B) or presence (C and D) of PSD-95. (E–H) TPA increased NMDAR surface expression to nearly the same final value in the absence or presence of PSD-95. (I and J) Oocytes expressing NMDARs labeled with secondary antibody in the absence of primary antibody (I) or water-injected oocytes labeled as in A--H (J) showed negligible fluorescence. (K) Summary of data in A--J. ∗, P < 0.05; ∗∗, P < 0.01.
	Fig. 5. PSD-95-induced occlusion of PKC potentiation is not regulated by NR1 splicing. (A–C) NMDA-elicited currents recorded from oocytes expressing NR1–1a/NR2A (A), NR1–1b/NR2A (B), or NR1–2b/NR2A (C) receptors alone or together with PSD-95 before and after PKC activation. Responses were elicited by NMDA (300 μM) with glycine (10 μM) and recorded in Ca2+ Ringer's solution at a holding potential of −60 mV. For each splice variant, PKC potentiated NMDA-elicited currents to nearly the same final responses in the absence or presence of PSD-95. Thus, the PKC potentiation ratio was reduced in cells expressing PSD-95.
	Fig. 6. Ser-1462 in the ESDV PDZ binding motif of NR2A is required for PSD-95 potentiation. The ESDV motif of NR2A was either deleted (NR2A-δESDV) or Ser-1462 was mutated to Ala (NR2A-S1462A) or Glu (NR2A-S1462E). These constructs were coexpressed with NR1–4b, which has the C2′ cassette containing a putative C-terminal PDZ binding motif (tSXV). (A–D) NMDA-elicited currents recorded from oocytes expressing NR1–4b with (A) NR2A wild type, (B) NR2A-δESDV, (C) NR2A-S1462A, or (D) NR2A-S1462E in the absence or presence of PSD-95. Mean values of current and PKC potentiation ratios are shown in the bar graphs to the right. (A) As in Figs. 1–5, coexpression of PSD-95 increased the basal responses but did not affect the final current after PKC potentiation, thus, reducing the PKC potentiation ratio. (B–D) All three mutants prevented the increase in response caused by PSD-95 expression and had no effect on PKC potentiation. See SI Table 2 for detailed values. Methods are as in Fig. 1.
	Fig. 7. PKC promotes NR1 and NR2 assembly but not NMDAR/PSD-95 association in neurons. (A) Representative Western blots of Input (Left) and immunoprecipitate (IP, Right) probed with an anti-NR1 antibody. Lysates (protein samples) were prepared from cortical neurons at 9 days in vitro. TPA application did not significantly alter total NR2A or PSD-95 protein abundance. TPA increased the abundance of NR2A, but not of PSD-95, that coimmunoprecipitated with an anti-NR1 antibody. TPA effects were blocked by the highly specific PKC blocker GF109203X (GF). (B) Summary of data.

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