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1 regulates its binding to the protein interacting with C

The C termini of ASIC1 and ASIC3 are substrates for PKA. (A) Affinity purified ASIC1/C term was immobilized on Ni2+ agarose beads for in vitro kinase assays with [32P]ATP and PKA, PKC, or CaMKII (lanes 1 3), or with kinase omitted (lane 4). The C termini of ASIC2 (ASIC2/C term) and ASIC3

(ASIC3/C term) were also tested with PKA (lanes 6 and 7). Autoradiography (Upper) and Coomassie staining (Lower) are shown. (B) The intracellular C terminus of ASIC1 contains the conserved PKA phosphorylation site Ser 479 and the PICK1 interaction

mutated to alanine. Peptides were biotinylated at the N terminus and immobilized on streptavidin coated plates for the in vitro kinase assays. After quantification of 32P incorporation through scintillation counting, results were plotted as absolute cpm (n = 9, error bars are SD).

PKA phosphorylates Ser 479 of ASIC1. (A) The indicated full length ASIC1 proteins were solubilized from transfected COS 7 cells, immunopurified, tested as a substrate

for PKA with [32P]ATP, and separated by SDS/PAGE. GFP served as an additional control. (A C Upper) Autoradiograms. (A C Lower) immunoblots with ASIC specific antibodies. (B) Cells transfected with indicated constructs were treated with 10 M forskolin and 100 M IBMX, 1 M KT5720, or vehicle and

assayed via back phosphorylation to detect PKA dependent phosphorylation (n = 3). (C) ASIC1 was immunopurified from mouse brain sections that were treated with vehicle, 10 M forskolin, and 100 M IBMX, 1 Mused preimmune sera (lanes 7 8) (n = 3).

PKA phosphorylation of ASIC1 reduces PICK1 binding. (A) Full length ASIC1 and PICK1 were coexpressed in COS 7 cells and treated with 1 M KT5720 or 10 M forskolin and 100 M IBMX.

ASIC1 was immunoprecipitated and then gels were immunoblotted for PICK1 (Upper) or ASIC1 (Lower). Control immunoprecipitations were performed with preimmune sera. (B) Quantification of immunosignal for PICK1 (n = 3, error bars are SD). (C) Affinity purified ASIC1/C term (2 g) was incubated with PKA in the absence () or presence (+) of 1 mM ATP and then tested

for binding to 1 M purified PICK1. ASIC1/PEP (10 M) was included in the PICK1 binding assay, as indicated. Samples were

immunoblotted with a PICK1 (Upper) or ASIC1 (Lower) antibody. (D) Sequence alignment between GluR2 and ASIC1. Boxed regions show conserved amino acids, and arrowhead indicates PKA phosphorylation

site S479. (E) The affinity purified ASIC1/C term was immobilized for in vitro binding assays to purified PICK1. ASIC1/PEP was added as an inhibitor of PICK1 binding (filled squares). As a control, ASIC1/PEP

was prephosphorylated with PKA and the PKA inactivated and then added at 10 M (open square). As an additional control, ASIC3/PEP

was added at 10 M (filled circle).

PKA phosphorylation of ASIC1 reduces colocalization with PICK1. (A) ASIC1 (Upper) or ASIC1 S479A (Lower) were coexpressed with PICK1 in COS 7 cells and imaged live by using fluorescence microscopy. (B) Percent colocalization was calculated before () and after (+) 20 min treatment with 10 M forskolin and 100 M IBMX in

the same cell. Cells were chosen by a blinded observer and percent colocalization was determined by METAMORPH software with

parameters set for overlapping area (n = 50, error bars are SD).

PKA phosphorylation of ASIC1 reduces clustering in hippocampal neurons. (A) Rat hippocampal neurons were transfected with ASIC1 (Left) or ASIC1 S479A (Right). Neurons were imaged live with fluorescence microscopy before (Upper) and 20 min after (Lower) addition of 1 M KT5720. (Insets) Enlargements of boxed areas. (B) Relative cluster intensity was calculated before () and 20 min after (+) adding 1 M KT5720 in the same cell. A total of

30 clusters per neuron were analyzed with METAMORPH software (n = 3, error bars are SD). Images were thresholded to define clusters. Relative cluster intensity was a ratio of cluster intensity.

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