Article Figures & Data
Figures
- Fig. 1.
Disruption of PKA anchoring promotes a PP2B-mediated rundown of hippocampal AMPA receptor currents. Whole-cell recordings were performed from cultured hippocampal neurons. Summary of time course of AMPA receptor currents is presented. Responses were normalized to the peak amplitude from the initial current evoked in response to domoate (10 μm). A, Displacement of PKA by whole-cell dialysis of Ht31 (1 μm) leads to rundown of AMPA receptor currents. Insets show representative first and last responses from a control cell and one in which Ht31 was included in the pipette. The number of observations for each condition is indicated. B, The Ht31-induced rundown is prevented when experiments were performed in the absence of extracellular recording solution. C, Experiments performed in the presence of 200 μm cadmium indicate that voltage-gated calcium channels contribute to a late phase of the rundown. Dotted line depicts Ht31 data fromA for comparison. D, Bar graph summarizing the effect of phosphatase inhibitors on the rundown. Cyclosporin A (CsA; 1 μm) treatment prevents Ht31-induced rundown of AMPA receptor currents. *p < 0.01 compared with cyclosporin A alone. Inhibition of PP1/PP2A by okadaic acid (OA; 1 μm) partially reduced the Ht31 mediated rundown. **p < 0.01 compared with okadaic acid alone.
- Fig. 2.
Selective PP2B-mediated downregulation of GluR1 receptor currents by coexpression of AKAP79. A, Selective association of AKAP79 with endogenous PP2B. Extracts from HEK293 cells transfected with either AKAP18-GFP or AKAP79-GFP were immunoprecipitated with their respective antibodies or control IgG antibodies. Immunoprecipitates were blotted with anti-RII antibodies (top) or anti-PP2B antibodies (bottom). Although both AKAP18 and AKAP79 coimmunoprecipitated RII as expected, only AKAP79 coimmunoprecipitated PP2B. B, Basal phosphorylation of the PKA site, Ser845, of GluR1 is enhanced by coexpression of membrane-targeted AKAPs. GluR1 was expressed alone (control) or with either AKAP18 or AKAP79 in HEK293 cells. Extracts from HEK293 cells were immunoprecipitated with antibodies against the C-terminal tail of GluR1. Phosphorylation of Ser845 was determined by blotting with phospho-specific antibodies to Ser845 (top). Total amount of GluR1 was determined by blotting with the C-terminal GluR1 antibody (middle). Results from experiments are summarized (bottom). The relative amount of GluR1 phosphorylation was quantified by determining the ratio of signals from the phospho-Ser845 antibody to that from the C-terminal antibody. For each experiment, data were normalized to the GluR1 alone (group control). The number of experiments is indicated inparentheses. C, Whole-cell recordings were performed from transfected HEK293 cells. Summary of time course of recombinant GluR1 receptor currents. Responses were normalized to the peak amplitude from the initial current evoked in response to glutamate (1 mm) in the presence of cyclothiazide (100 μm). HEK293 cells were transfected with GluR1 alone (control) or with either AKAP18 or AKAP79. GluR1 currents selectively rundown when cotransfected with AKAP79. The pipette solution contained 1 mm BAPTA. The number of observations for each condition is indicated. Unless otherwise indicated, all subsequent figures are presented in the same manner. Insets show representative first and last responses from control, AKAP18, and AKAP79 cells.D, Blocking rises in intracellular calcium by elevating the concentration of BAPTA in the pipette solution to 10 mmprevents the AKAP79-dependent rundown of GluR1 receptor currents.E, Experiments performed in the absence of extracellular calcium indicate that the AKAP-dependent rundown is calcium dependent and suggest that calcium entry is required for the rundown.F, Bar graph summarizing effects of phosphatase inhibitors on the Ht31-induced rundown. Inhibition of PP2B activity by cyclosporin A (1 μm) but not PP1/PP2A by okadaic acid (1 μm) prevents the AKAP79-dependent rundown. *p < 0.05 compared with control. **p < 0.05 compared with okadaic acid alone.
- Fig. 3.
GluR1 is regulated by AKAP79-anchored PKA. A, Top, Extracts from HEK293 cells transfected with AKAP79 and AKAP79(1–360) were immunoprecipitated with antibodies to AKAP79 and blotted with antibodies against the RII subunit. AKAP79(1–360) does not coimmunoprecipitate RII and thus is unlikely to bind PKA inside cells.Bottom, Parallel electrophysiology experiments demonstrate that PKA anchoring is required for the AKAP79-dependent rundown. Ex, Extract; IP, immunoprecipitate; wt, wild type.B, HEK293 cells were transfected with GluR1 alone or with either AKAP18 or AKAP79. Acute application of 8-bromo-cAMP, a cell-permeant cAMP analog, prevents the AKAP79-dependent rundown but does not affect the time course of current responses from control or AKAP18 cells (compare with Fig. 2C). C, The effect of cAMP on GluR1 receptor currents, from cells cotransfected with AKAP79, was blocked by inclusion of the PKA-anchoring inhibitory peptide Ht31 (1 μm) or the specific PKA inhibitory peptide PKI (1 μm) in the whole-cell recording pipette solution. These results indicate that prevention of AKAP79-dependent rundown by 8-bromo-cAMP was attributable to activation of a pool of anchored PKA. AKAP79 plus cAMP data from Figure 3B are shown for comparison.
- Fig. 4.
AKAP79-dependent regulation of GluR1 receptor currents occurs selectively at Ser845. HEK293 cells were transfected with GluR1 phosphorylation site mutants either alone (control) or with AKAP79. GluR1 receptor currents were evoked as in Figure2C. A, AKAP79-dependent rundown still occurs when Ser831 is mutated to alanine (S831A), indicating that Ser831 is not involved in the rundown. B, Ser845 is the site of regulation for the AKAP79-dependent rundown because GluR1 receptor currents from an alanine mutant of Ser845 (S845A) do not rundown when cotransfected with AKAP79.
- Fig. 5.
AKAP79-dependent rundown requires a PDZ-domain interaction at the C terminal of GluR1. A, HEK293 cells were transfected with GluR1 and AKAP79. Extracts were immunoprecipitated with antibodies to SAP97 and blotted for SAP97, AKAP79, and GluR1. Endogenous SAP97 is present in HEK293 cells (top). Transfected GluR1 and AKAP79 are coimmunoprecipitated with the endogenous SAP97, suggesting that a GluR1/SAP97/AKAP79 complex forms during cotransfection of GluR1 and AKAP79. IP, Immunoprecipitating antibody.B, HEK293 cells were transfected with an alanine mutant of Thr887 of GluR1 (T887A) in the presence or absence of AKAP79. Time course of GluR1 receptor currents indicate that a PDZ-domain interaction between GluR1 and endogenous SAP97 is likely required for the AKAP79-dependent rundown because it did not occur with the T887A mutant.
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