Camp-Dependent Protein Kinase Phosphorylation of the Acid-Sensing Ion Channel-1 Regulates Its Binding to the Protein Interacting with C-Kinase-1

cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1

A. Soren Leonard*†, Olena Yermolaieva*, Alesia Hruska-Hageman*†, Candice C. Askwith*†, Margaret P. Price*, John A. Wemmie‡, and Michael J. Welsh*†§¶

Departments of *Internal Medicine, ‡Psychiatry, and §Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, †Howard Hughes Medical Institute, University of Iowa, Iowa City, IA 52242

Contributed by Michael J. Welsh, December 20, 2002 The acid-sensing ion channel-1 (ASIC1) contributes to synaptic site for phosphorylation by protein kinase A (PKA) and because plasticity and may influence the response to cerebral ischemia and it is the most pH-sensitive subunit in the brain. acidosis. We found that cAMP-dependent protein kinase phosphorylated heterologously expressed ASIC1 and endogenous ASIC1 in Materials and Methods brain slices. ASIC1 also showed significant phosphorylation under Materials. PKA and protein kinase C (PKC) isoforms were basal conditions. Previous studies showed that the extreme C- purchased from Promega. Purified recombinant calcium͞ terminal residues of ASIC1 bind the PDZ domain of the protein calmodulin-dependent protein kinase II (CaMKII) was kindly interacting with C-kinase-1 (PICK1). We found that protein kinase provided by J. W. Hell (University of Iowa, Iowa City). [32P]ATP A phosphorylation of Ser-479 in the ASIC1 C terminus interfered (111 TBq͞mmol) was obtained from New England Nuclear͞ with PICK1 binding. In contrast, minimizing phosphorylation or Dupont. Protein A-Sepharose was purchased from Amersham mutating Ser-479 to Ala enhanced PICK1 binding. Phosphorylation- Pharmacia–Pharmacia. Forskolin and 3-isobutyl-1-methylxanthine dependent disruption of PICK1 binding reduced the cellular colo- (IBMX) were purchased from Sigma. Tetrodotoxin, microcystin- calization of ASIC1 and PICK1. Thus, the ASIC1 C terminus contains LR, the PKA inhibitor KT5720, and protein kinase A inhibitor two sites that influence its binding to PICK1. Regulation of this peptide were purchased from Calbiochem–Novabiochem. PICK1 interaction by phosphorylation provides a mechanism to control antibodies were purchased from Santa Cruz Biotechnology. the cellular localization of ASIC1. Secondary horseradish peroxidase-conjugated antibodies were received from Bio-Rad. Streptavidin-coated plates and ϩ he degenerin͞epithelial Na channel (DEG͞ENaC) cation all developing reagents were purchased from Pierce. ASIC1 ϩ Tchannel family includes five H -gated members: acid-sensing wild-type peptide (ASIC1͞PEP) (biotin–RRGKCQKEAK- ion channel (ASIC)1a, -1b, -2a, -2b, and -3 (‘‘a’’ and ‘‘b’’ refer to RSSADKGVALSLDD), ASIC1͞PEP-S478A peptide (biotin– alternatively spliced isoforms) (1–3). ASIC1a, -2a, and -2b share RRGKCQKEAKRASADKGVALSLDD), ASIC1͞PEP-S479A similar expression patterns in the central nervous system, with peptide (biotin–RRGKCQKEAKRSAADKGVALSLDD), and transcripts most abundant in the hippocampus, cortex, and ASIC3͞PEP (biotin–FWNRRSSQRRSGNTLLQE) were syn- Purkinje layer of the cerebellum (4–6). ASIC3 is predominately thesized at the Howard Hughes Medical Institute͞Keck Facility expressed in the peripheral nervous system (7). The molecular at Yale University. All other reagents were purchased from diversity of these channels is further expanded when individual commercial suppliers and were of standard biochemical quality. ASIC subunits heteromultimerize to generate cation channels with properties different from homomultimers. Fusion Proteins. For the production of polyhistidine fusion pro- ͞ NEUROSCIENCE Like other DEG ENaC subunits, ASICs have two transmem- teins containing the C-terminal region (ASIC͞C-term), oligo- brane sequences, a large extracellular domain containing several nucleotides flanking the indicated regions and containing ap- conserved cysteines, and intracellular N and C termini (1–3). propriate restriction sites were used to amplify corresponding Previous studies have shown that the C termini of ASIC1, -2, and sequences from cDNA clones of hASIC1a (amino acids 459– -3 participate in protein interactions. ASIC1 and -2 C termini 528), hASIC2a (amino acids 466–512), and hASIC3 (amino acids share homology with type-II PDZ binding motifs and bind the 449–513) by PCR. The DNA products were ligated into pET-32a protein interacting with C-kinase-1 (PICK1) (8, 9). PICK1 and vector (Novagen). Fusion proteins were expressed in Escherichia ASIC1 are coexpressed throughout the brain, colocalize at coli (BL21-codon plus; Stratagene) and purified with Ni2ϩ synaptic sites in hippocampal neurons, and copurify from syn- chelate resin nickel–nitrolotriacetic acid (Sepharose, Qiagen, aptosomes (8, 9). The ASIC3 C terminus binds channel- Chatsworth, CA) according to the protocols suggested by the interacting PDZ domain protein (CIPP) (10). The function of manufacturer. these interactions is not well understood, although it has been suggested that they regulate the transport and distribution of the Phosphorylation Assays. The resins with protein bound were channels in nerve terminals. phosphorylated as described (14) with 0.5–1 ␮g of PKA, PKC, or ASIC1 contributes to the normal function of central neurons. ϩ CaMKII in 50 ␮l of basic phosphorylation buffer, and 0.2 ␮M Disrupting the mouse ASIC1 gene markedly reduced H -gated [32P]ATP. The pellets were extracted with SDS sample buffer currents in hippocampal neurons, impaired long-term potenti- and used directly for SDS͞PAGE. Phosphorylated protein bands ation of synaptic transmission, and produced learning and were visualized by autoradiography. For quantitative phosphor- memory deficits (11). The observation that extracellular acidosis ylation as required for the measurement of the stoichiometry of activates ASIC1 has suggested that it may also be activated in pathologic conditions, for example, when extracellular pH falls during cerebral ischemia (12, 13). Because protein kinases Abbreviations: ASIC1, acid-sensing ion channel-1; ASIC1͞PEP, ASIC1 peptide; PKA, protein regulate the function of many brain ion channels, we asked kinase A; IBMX, 3-isobutyl-1-methylxanthine; ASIC͞C-term, polyhistidine fusion proteins whether ASIC channels are phosphorylated. We focused spe- containing the C-terminal region; CaMKII, calmodulin-dependent protein kinase II. cifically on ASIC1, because it contains a conserved consensus ¶To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.252782799 PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 2029–2034 Downloaded by guest on September 28, 2021 32P phosphate incorporation by PKA or PKC, 10 ␮M unlabeled directed mutagenesis of ASIC1 was performed by PCR accord- ATP was added to the phosphorylation reaction. At this ATP ing to the QuikChange Mutagenesis Kit protocol (Stratagene). concentration, phosphorylation of ASIC subunits by PKA or hPICK1-DsRed was obtained by subcloning into pDsRed-N1 PKC was saturated. Extending the incubation time to 1 h also did (CLONTECH) as described (8). All constructs were driven by a not result in an increase in 32P phosphate incorporation by PKA cytomegalovirus promoter and were confirmed by sequencing. or PKC. Quantification of 32P incorporation into milligrams of After 24–48 h, cells were washed once with the recording ͞ ͞ ͞ ͞ ͞ ASIC1 C-term was through scintillation counting. medium (in mM: NaCl 140 KCl 5 MgCl2 1 CaCl2 1.5 glucose 5͞Hepes 10, pH 7.4) and were mounted in the recording Immunoprecipitation and Immunoblotting. COS-7 cells transfected chamber. Optical recordings were performed on a Nikon Eclipse by electroporation were solubilized in 1% Triton X-100͞0.5% TE200 microscope with an I-PentaMAX 512 ϫ 512 charge- deoxycholate͞0.1% SDS͞10 mM Tris⅐Cl, pH 7.4͞150 mM coupled device camera (Princeton Instruments, Trenton, NJ). A NaCl͞10 mM EDTA͞10 mM EGTA, with a glass͞Teflon ϫ100 oil SuperFluor objective (Nikon), a dichroic Quad 84000 homogenizer. Pepstatin A (1 ␮M), leupeptin, and aprotinin (10 filter set (Chroma Technology, Brattleboro, VT), and excitation ␮g͞ml), Pefabloc (0.2 mM), benzamidine (0.1 mg͞ml), p- filters 490͞20 for enhanced GFP and 555͞28 for DsRed were nitrophenyl phosphate (1 mM), and microcystin-LR (2 ␮M) used for all recordings. Recordings and image analysis including were present in all buffers. Unsolubilized material was sedi- region measurements were performed by using METAMORPH mented by ultracentrifugation (100,000 ϫ g, 30 min; 70.1 Ti software (Universal Imaging, Media, PA). rotor, 4°C), and the supernatant was collected and stored at Ϫ80°C. Results ASIC1 was precipitated by using antibody 6.4 (human The Intracellular C Termini of ASIC1 and ASIC3 Are Substrates for PKA. ASIC1a), which specifically binds to ASIC1 (11). For control To determine whether ASIC1 is a substrate for phosphorylation, immunoprecipitations, nonspecific rabbit IgG antibodies were we examined the affinity-purified C terminus of ASIC1 (ASIC1͞ obtained from Jackson Laboratories. Then 3–5 mg of protein A C-term). PKA phosphorylated the protein (Fig. 1A) with a Sepharose, preswollen and washed three times with TBS (150 stoichiometry of almost 1 mol of phosphate per mol of protein. mM NaCl͞10 mM Tris⅐Cl, pH 7.4) containing 1% Triton X-100, In contrast, there was little phosphorylation by PKC and none were added, and the samples were mixed for 2.5 h as before. The with CaMKII. immunocomplexes were sedimented by centrifugation and Both ASIC2 and ASIC3 complex with ASIC1 to form het- washed three times with 1% Triton X-100 in TBS and once with eromultimeric channels (6, 16–18). We found that ASIC2͞C- 10 mM Tris⅐Cl, pH 7.4, before being extracted with 20 ␮lofSDS term was not a substrate for PKA, whereas ASIC3͞C-term sample buffer (2% SDS͞20 mM DTT͞10% sucrose͞125 mM showed significantly less phosphorylation (Fig. 1A). Thus, Tris⅐Cl, pH 6.8) for 20 min at 60°C. ASIC1͞C-term showed a high substrate specificity for PKA. After separation by SDS͞PAGE, the proteins were trans- ferred onto nitrocellulose, which was blocked by incubation for PKA Phosphorylates Ser-479 of ASIC1. ASIC1͞C-term contains two 2 h with 3% BSA in TBS (TBS-BSA). Blots were incubated with potential PKA consensus phosphorylation sites, Ser-478 and ASIC1 or PICK1 antibody (1:1,000 in TBS-BSA) for 1–2h, -479 (Fig. 1B). To identify the PKA phosphorylated residues, we washed five times with TBS-BSA, incubated with horseradish– synthesized 24-aa peptides (residues 467–490, Fig. 1B) with the peroxidase (HRP)-labeled protein A or HRP-labeled donkey wild-type sequence or with the candidate Ser replaced by Ala. anti-goat respectively (diluted 1:1,000 in TBS-BSA), washed for PKA phosphorylated the wild-type peptide (Fig. 1C). Changing 3–4 h with 0.05% Tween 20 in TBS (8–10 changes), and Ser-479 to an Ala, ASIC1͞PEP-S479A, eliminated phosphory- developed with the enhanced chemiluminescence reagent lation, whereas replacing Ser-478 had no effect. These data (Pierce). suggest that Ser-479 is the PKA phosphorylation site. In parallel experiments, we found little PKC or CaMKII-dependent phos- Stimulation of Coronal Mouse Brain Slices. Acute hippocampal slices phorylation (Fig. 1C), confirming that this region is predomi- (0.4 mm) were prepared from 3-wk-old C57-black mice (Harlan nantly a PKA substrate. Breeders, Indianapolis) by using a vibratome and treated as To determine whether PKA phosphorylates full-length described (15). Supernatants were used immediately for immu- ASIC1, we expressed the channel in COS-7 cells, immunopuri- noprecipitation by using hASIC1 or control antibodies. fied it, and tested PKA phosphorylation in vitro. Fig. 2A shows that ASIC1 was a PKA substrate. Mutation of Ser-479 but not Association of Fusion Proteins. Two micrograms of affinity-purified Ser-478 abolished phosphorylation. ASIC3, but not ASIC2, ASIC1͞C–term polyhistidine fusion proteins [as estimated by showed slight PKA-dependent phosphorylation. These data SDS͞PAGE and subsequent staining with Coomassie brilliant indicate that full-length ASIC1 is a PKA substrate and that blue in addition to quantitative bicinchoninic acid- protein assay Ser-479 is the phosphorylation site. (Pierce)] were immobilized for binding with 1.5 ␮g of purified PICK1 GST fusion protein. Fusion proteins were combined with PKA-Dependent Phosphorylation of ASIC1 in COS Cells and Brain the indicated conditions in TBS with 0.1% TX-100 for 30 min, Slices. To test ASIC1 phosphorylation in cells, COS cells were while tilting at 4°C. Samples were rapidly washed three times treated with cAMP agonists (10 ␮M forskolin and 100 ␮M with the same buffer and once with 10 mM Tris⅐Cl, pH 7.4, and IBMX) or a specific PKA inhibitor (1 ␮M KT5720) (19) (Fig. then extracted with SDS sample buffer for immunoblotting as 2B). To detect phosphorylation, immunopurified ASIC1 was described. The IC50 value and the curve fitting for the ASIC1 assayed via back-phosphorylation, which is a measure of in vivo peptide to inhibit this interaction were obtained from a dose phosphorylation through protection of sites in vitro (15). PKA response of experimental data performed in PRISM software activation increased ASIC1 phosphorylation in vivo, as indicated (Ver. 3.02, GraphPad Software, San Diego). by decreased in vitro phosphorylation, whereas PKA inhibition reduced phosphorylation in vivo (Fig. 2B). These results indicate Fluorescence Imaging. COS-7 cells were transfected by electropo- that ASIC1 is a PKA substrate in cells, and that the channel is ration, and rat E18 hippocampal neurons (G. Brewer, Southern phosphorylated under basal conditions. There was no PKA- Illinois University School of Medicine, Springfield) in primary dependent phosphorylation of ASIC1-S479A, even after appli- culture for 2–3 days were transfected as described (8) with cation of KT5720 to reduce the possibility that an additional hASIC1a subcloned into pEGFP-C2 (CLONTECH). Site- PKA site was constitutively phosphorylated in cells (Fig. 2B).

2030 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.252782799 Leonard et al. Downloaded by guest on September 28, 2021 NEUROSCIENCE

Fig. 1. The C termini of ASIC1 and ASIC3 are substrates for PKA. (A) Affinity-purified ASIC1͞C-term was immobilized on Ni2ϩ 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 (ASIC2͞C-term) and ASIC3 Fig. 2. PKA phosphorylates Ser-479 of ASIC1. (A) The indicated full-length (ASIC3͞C-term) were also tested with PKA (lanes 6 and 7). Autoradiography ASIC1 proteins were solubilized from transfected COS-7 cells, immunopuri- (Upper) and Coomassie staining (Lower) are shown. (B) The intracellular C fied, tested as a substrate for PKA with [32P]ATP, and separated by SDS͞PAGE. terminus of ASIC1 contains the conserved PKA phosphorylation site Ser-479 GFP served as an additional control. (A–C Upper) Autoradiograms. (A–C and the PICK1 interaction site at the extreme C terminus. The second trans- Lower) immunoblots with ASIC-specific antibodies. (B) Cells transfected with membrane domain is indicated. The location of the 24-aa ASIC1͞PEP is indi- indicated constructs were treated with 10 ␮M forskolin and 100 ␮M IBMX, 1 cated. (C) For in vitro kinase assays with PKA, PKC, or CaMKII, 24-aa peptides ␮M KT5720, or vehicle and assayed via back-phosphorylation to detect PKA- were synthesized with Ser-478 (ASIC1͞PEP-S478A) or Ser-479 (ASIC1͞PEP- dependent phosphorylation (n ϭ 3). (C) ASIC1 was immunopurified from S479A) mutated to alanine. Peptides were biotinylated at the N terminus and mouse brain sections that were treated with vehicle, 10 ␮M forskolin, and 100 immobilized on streptavidin-coated plates for the in vitro kinase assays. After ␮M IBMX, 1 ␮M KT5720, or 10 ␮M dideoxy-forskolin and assayed via quantification of 32P incorporation through scintillation counting, results back-phosphorylation to detect PKA-dependent phosphorylation. Control were plotted as absolute cpm (n ϭ 9, error bars are SD). immunoprecipitations used preimmune sera (lanes 7–8) (n ϭ 3).

Control immunoprecipitations from GFP-transfected cells and neurons, we treated mouse brain slices with agents to activate immunoblotting with anti-ASIC1 antibody indicate that the or inhibit PKA, immunopurified ASIC1, and used back- results were specific. phosphorylation to assess phosphorylation status. The cAMP To determine whether ASIC1 is a PKA substrate in intact agonist forskolin, but not the inactive isomer 1,9-dideoxyfor-

Leonard et al. PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 2031 Downloaded by guest on September 28, 2021 cAMP agonists reduced coprecipitation by 40%. To ensure that it was the phosphorylation of ASIC1 that influenced PICK1 binding, we used the affinity-purified ASIC1͞C-term and examined binding of purified GST-tagged PICK1. When ASIC1͞C- term was prephosphorylated with PKA, binding of PICK1 fell (Fig. 3C, lanes 1 and 2). These findings suggest a novel mechanism in which phosphorylation regulates PICK1 binding. Recent studies demonstrate that the coiled-coiled domain of PICK1 contributes to its binding to ␣-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptors in a juxtamembrane region of GluR2 (amino acids 842–853) (20). This sequence in GluR2 shares some sequence similarity with the juxtamembrane region of ASIC1 (Fig. 3D); importantly, this portion of ASIC1 includes the PKA phosphorylation site, Ser-479. To test whether this juxtamembrane region of ASIC1 binds PICK1, we assayed the ability of an ASIC1 wild-type peptide (ASIC1͞PEP, Fig. 1B) to inhibit binding. Adding 10 ␮M ASIC1͞PEP reduced the ability of ASIC1͞C-term to pull down PICK1 (Fig. 3C). Pre- phosphorylating ASIC1͞C-term did not further reduce PICK1 binding, suggesting that these interventions were not additive (Fig. 3C). Varying the concentration of ASIC1͞PEP showed that Ϸ ␮ inhibition was saturable with an IC50 of 0.5 M (Fig. 3E). If ASIC1͞PEP was phosphorylated with PKA, it lost the ability to inhibit PICK1 binding to ASIC1͞C-term (Fig. 3E). The specificity of the effect was confirmed by the inability of an ASIC3 peptide (ASIC3͞PEP) from the corresponding location to inhibit the interaction. These data suggest that two regions in the ASIC1 C terminus bind PICK1 and offer a mechanism for how phosphorylation regulates this interaction.

PKA Phosphorylation of ASIC1 Reduces Colocalization with PICK1. When coexpressed in heterologous cells, PICK1 and ASIC1 colocalize in intracellular clusters (8, 9). Our finding that ASIC1 phosphorylation reduced PICK1 binding suggested that the phosphorylation status of ASIC1 would influence colocalization. To test this hypothesis, we cotransfected COS-7 cells with PICK1 and ASIC1 or ASIC1-S479A and examined the protein distribution in the presence and absence of cAMP agonists. Fig. 4A shows that ASIC1 and PICK1 colocalized in clusters, but ASIC1 Fig. 3. PKA phosphorylation of ASIC1 reduces PICK1 binding. (A) Full-length ␮ was also present in the periphery of the cells not colocalized with ASIC1 and PICK1 were coexpressed in COS-7 cells and treated with 1 M PICK1. In contrast, ASIC1-S479A colocalized almost com- KT5720 or 10 ␮M forskolin and 100 ␮M IBMX. ASIC1 was immunoprecipitated and then gels were immunoblotted for PICK1 (Upper) or ASIC1 (Lower). pletely with PICK1. A quantitative analysis is shown in Fig. 4B; Control immunoprecipitations were performed with preimmune sera. (B) colocalization with PICK1 was greater with ASIC1-S479A than Quantification of immunosignal for PICK1 (n ϭ 3, error bars are SD). (C) with wild-type ASIC1, and cAMP agonists further reduced Affinity-purified ASIC1͞C-term (2 ␮g) was incubated with PKA in the absence PICK1 colocalization with ASIC1 but not with ASIC1-S479A. (Ϫ) or presence (ϩ) of 1 mM ATP and then tested for binding to 1 ␮M purified When expressed alone, the localization of ASIC1 and ASIC1- PICK1. ASIC1͞PEP (10 ␮M) was included in the PICK1-binding assay, as indi- S479A are diffuse and unaffected by PKA activation (data not cated. Samples were immunoblotted with a PICK1 (Upper) or ASIC1 (Lower) shown). antibody. (D) Sequence alignment between GluR2 and ASIC1. Boxed regions show conserved amino acids, and arrowhead indicates PKA phosphorylation PKA Phosphorylation of ASIC1 Reduces Clustering in Hippocampal site S479. (E) The affinity-purified ASIC1͞C-term was immobilized for in vitro Neurons. binding assays to purified PICK1. ASIC1͞PEP was added as an inhibitor of PICK1 In hippocampal neurons, ASIC1 immunostaining is binding (filled squares). As a control, ASIC1͞PEP was prephosphorylated with detected in a synaptic pattern and colocalizes with PSD95 (8, 11). PKA and the PKA inactivated and then added at 10 ␮M (open square). As an To test the effect of phosphorylating ASIC1, we compared live additional control, ASIC3͞PEP was added at 10 ␮M(filled circle). fluorescent images of ASIC1 to that of ASIC1-S479A. Consis- tent with previous results, we found ASIC1 in a punctate and diffuse pattern (Fig. 5A Upper Left). Because our results indi- skolin, increased ASIC1 phosphorylation (Fig. 2C). Inhibiting cated that ASIC1 showed substantial phosphorylation under PKA with KT5720 had the opposite effect, indicating that some baseline conditions, we applied KT5720 and 20 min later found ASIC1 is phosphorylated in brain under basal conditions. an increase in the punctate distribution of ASIC1 (Fig. 5A Lower Left). ASIC1-S479A showed a similar appearance to ASIC1 with PKA Phosphorylation of ASIC1 Reduces PICK1 Binding. Previous a punctate and diffuse appearance in the cell soma and proximal studies showed that the PDZ domain of PICK1 interacts with the dendrites, but KT5720 did not alter its distribution (Fig. 5A extreme C terminus of ASIC1 (8, 9). We tested the hypothesis Right). A quantitative analysis indicated that inhibiting PKA that phosphorylation of ASIC1 would alter this interaction. We increased the intensity of ASIC1 in clusters (Fig. 5B). cAMP coexpressed PICK1 and ASIC1 and treated the cells with agonists had no measurable effect, consistent with significant KT5720 to reduce the basal level of ASIC1 phosphorylation. basal phosphorylation. In contrast, neither stimulating nor in- Immunoprecipitating ASIC1 specifically coprecipitated PICK1 hibiting PKA altered the distribution of ASIC1-S479A. (Fig. 3 A and B; see also ref. 8). However, treating cells with To test whether phosphorylating ASIC1 influences Hϩ-gated

2032 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.252782799 Leonard et al. Downloaded by guest on September 28, 2021 Fig. 4. 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 (Ϫ) and after (ϩ) 20 min treatment with 10 ␮M Fig. 5. PKA phosphorylation of ASIC1 reduces clustering in hippocampal forskolin and 100 ␮M IBMX in the same cell. Cells were chosen by a blinded neurons. (A) Rat hippocampal neurons were transfected with ASIC1 (Left)or ASIC1-S479A (Right). Neurons were imaged live with fluorescence microscopy observer and percent colocalization was determined by METAMORPH software ␮ with parameters set for overlapping area (n ϭ 50, error bars are SD). before (Upper) and 20 min after (Lower) addition of 1 M KT5720. (Insets) Enlargements of boxed areas. (B) Relative cluster intensity was calculated before (Ϫ) and 20 min after (ϩ) adding 1 ␮M KT5720 in the same cell. A total ϭ currents, we recorded whole-cell currents from cultured hip- of 30 clusters per neuron were analyzed with METAMORPH software (n 3, error bars are SD). Images were thresholded to define clusters. Relative cluster pocampal neurons and COS-7 cells expressing ASIC1 and intensity was a ratio of cluster intensity and immediate background.

PICK1. We applied cAMP agonists (forskolin and IBMX) and NEUROSCIENCE PKA inhibitor (KT5720); neither intervention significantly altered acid-evoked currents (data not shown). Thus, PKA- second PICK1-binding site is similar to the observation that dependent phosphorylation appeared to have little direct effect GluR2 binding to PICK1 involves both a PDZ interaction and an ϩ on H -gated ASIC1 current, as expected from previous results interaction between a sequence in the GluR2 C terminus and the characterizing the channel’s interaction with PICK1 (8, 9). coiled-coiled domain in PICK1 (20, 26). Interestingly, the PICK1-interacting sequence we identified in ASIC1 shows some Discussion sequence similarity to that in GluR2. Our data show that PKA phosphorylated ASIC1 at Ser-479 in The physiologic significance of ASIC1 phosphorylation was the intracellular C-terminal tail. In the absence of an added shown by its influence on PICK1 binding. The phosphorylation stimulus, there was significant phosphorylation of ASIC1 ex- site, Ser-479, lies in the sequence involved in binding to PICK1. pressed in heterologous cells and endogenous ASIC1 in brain Minimizing Ser-479 phosphorylation or mutating Ser-479 to slices. cAMP agonists further increased phosphorylation. Al- Ala enhanced ASIC1 binding to PICK1. Conversely, phos- though ASIC1 is the first member of the DEG͞ENaC family phorylating Ser-479 reduced ASIC1 binding to PICK1. These demonstrated to be a direct substrate for PKA, phosphorylation data are consistent with other reports of phosphorylation- of associated proteins has been shown to indirectly regulate the dependent regulation of protein binding to coiled-coiled activity and trafficking of the ENaC family members (21–23), domains (27). and ASIC1 and ASIC2 have been reported to be phosphorylated Earlier studies showed that PICK1 transfected into hippocam- by PKC (24, 25). Whereas the C terminus of ASIC1 was an pal neurons localized at glutamatergic synapses (28). PICK1 excellent PKA substrate, ASIC2 was not phosphorylated, and participated in glutamate receptor trafficking by clustering the ASIC3 showed less phosphorylation. receptor to internalized pools (26, 29, 30). Previous work has also Previous studies demonstrated that the last four amino acids shown that ASIC1 colocalized with PICK1 at synapses (8, 11). of ASIC1 bind the PDZ domain of PICK1 (8, 9). Our results Our present data show that this interaction is regulated by indicated that an additional site in the ASIC1 C terminus phosphorylation of ASIC1. PKA-dependent phosphorylation of contributes to PICK1 binding. Finding that ASIC1 contains a ASIC1 altered the channel’s subcellular distribution and reduced

Leonard et al. PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 2033 Downloaded by guest on September 28, 2021 its colocalization with PICK1. Thus, much like the trafficking of signal-transduction cascade that may connect stimuli from both glutamate receptors to the synapse, the delivery of ASIC1 external and internal sources. subunits is likely regulated by a combination of protein interactions and posttranslational modifications. A.S.L., A.H.-H., and C.C.A. are Postdoctoral Associates, and M.J.W. The phosphorylation of ASIC1 also contributes to our under- is an Investigator of Howard Hughes Medical Institute (HHMI). We standing of these channels as signal integrators (31). ASIC thank the University of Iowa DNA Core Facility (supported by National Institutes of Health Grant DK25295) for assistance with channel activity is regulated by extracellular protons (2), neu- sequencing and oligonucleotide synthesis and the University of Iowa ropeptides (32, 33), extracellular calcium (34–36), zinc (37), Central Microscopy Research Facility. This work was supported temperature (38), and mechanical stimuli (39, 40). Our current by HHMI. J.A.W. was supported by the Veterans Administration data indicate that ASIC1 is also a target for an intracellular Research Career Development Award.

1. Mano, I. & Driscoll, M. (1999) BioEssays 21, 568–578. 20. Hanley, J. G., Khatri, L., Hanson, P. I. & Ziff, E. B. (2002) Neuron 34, 53–67. 2. Waldmann, R. & Lazdunski, M. (1998) Curr. Opin. Neurobiol. 8, 418–424. 21. Snyder, P. M. (2002) Endocr. Rev. 23, 258–275. 3. Welsh, M. J., Price, M. P. & Xie, J. (2002) J. Biol. Chem. 277, 2369–2372. 22. Volk, K. A., Snyder, P. M. & Stokes, J. B. (2001) J. Biol. Chem. 276, 4. Waldmann, R., Champigny, G., Bassilana, F., Heurteaux, C. & Lazdunski, M. 43887–43893. (1997) Nature 386, 173–177. 23. Shimkets, R. A., Lifton, R. & Canessa, C. M. (1998) Proc. Natl. Acad. Sci. USA 5. Garcı´a-An˜overos, J., Derfler, B., Neville-Golden, J., Hyman, B. T. & Corey, 95, 3301–3305. D. P. (1997) Proc. Natl. Acad. Sci. USA 94, 1459–1464. 24. Berdiev, B. K., Xia, J., Jovov, B., Markert, J. M., Mapstone, T. B., Gillespie, 6. Lingueglia, E., de Weille, J. R., Bassilana, F., Heurteaux, C., Sakai, H., G. Y., Fuller, C. M., Bubien, J. K. & Benos, D. J. (2002) J. Biol. Chem. 277, Waldmann, R. & Lazdunski, M. (1997) J. Biol. Chem. 272, 29778–29783. 45734–45740. 7. Waldmann, R., Bassilana, F., de Weille, J. R., Champigny, G., Heurteaux, C. 25. Baron, A., Deval, E., Salinas, M., Lingueglia, E., Voilley, N. & Lazdunski, M. & Lazdunski, M. (1997) J. Biol. Chem. 272, 20975–20978. (2002) J. Biol. Chem. 277, 50463–50468. 8. Hruska-Hageman, A. M., Wemmie, J. A., Price, M. P. & Welsh, M. J. (2001) 26. Xia, J., Zhang, X., Staudinger, J. & Huganir, R. L. (1999) Neuron 22, 179–187. Biochem. J. 361, 443–450. 27. Szilak, L., Moitra, J. & Vinson, C. (1997) Protein Sci. 6, 1273–1283. 9. Duggan, A., Garcia-Anoveros, J. & Corey, D. P. (2002) J. Biol. Chem. 277, 28. Boudin, H. & Craig, A. M. (2001) J. Biol. Chem. 276, 30270–30276. 5203–5208. 29. Perez, J. L., Khatri, L., Chang, C., Srivastava, S., Osten, P. & Ziff, E. B. (2001) 10. Anzai, N., Deval, E., Schaefer, L., Friend, V., Lazdunski, M. & Lingueglia, E. J. Neurosci. 21, 5417–5428. (2002) J. Biol. Chem. 277, 16655–16661. 30. Kim, C. H., Chung, H. J., Lee, H. K. & Huganir, R. L. (2001) Proc. Natl. Acad. 11. Wemmie, J. A., Chen, J., Askwith, C. C., Hruska-Hageman, A. M., Price, M. P., Sci. USA 98, 11725–11730. Nolan, B. C., Yoder, P. G., Lamani, E., Hoshi, T., Freeman, J. H. J. & Welsh, 31. Lopez, J. C. (2002) Nat. Rev. Neurosci. 3, 763. M. J. (2002) Neuron 34, 463–477. 32. Askwith, C. C., Cheng, C., Ikuma, M., Benson, C. J., Price, M. P. & Welsh, M. J. 12. Obrenovitch, T. P., Garofalo, O., Harris, R. J., Bordi, L., Ono, M., Momma, (2000) Neuron 26, 133–141. F., Bachelard, H. S. & Symon, L. (1988) J. Cereb. Blood Flow Metab. 8, 866–874. 33. Catarsi, S., Babinski, K. & Seguela, P. (2001) Neuropharmacology 41, 592–600. 13. Johnson, M. B., Jin, K., Minami, M., Chen, D. & Simon, R. P. (2001) J. Cereb. 34. Immke, D. C. & McCleskey, E. W. (2001) Nat. Neurosci. 4, 869–870. Blood Flow Metab. 21, 734–740. 35. Berdiev, B. K., Mapstone, T. B., Markert, J. M., Gillespie, G. Y., Lockhart, J., 14. Leonard, A. S. & Hell, J. W. (1997) J. Biol. Chem. 272, 12107–12115. Fuller, C. M. & Benos, D. J. (2001) J. Biol. Chem. 276, 38755–38761. 15. Leonard, A. S., Lim, I. A., Hemsworth, D. E., Horne, M. C. & Hell, J. W. (1999) 36. Zhang, P. & Canessa, C. M. (2002) J. Gen. Physiol. 120, 553–566. Proc. Natl. Acad. Sci. USA 96, 3239–3244. 37. Baron, A., Schaefer, L., Lingueglia, E., Champigny, G. & Lazdunski, M. (2001) 16. Bassilana, F., Champigny, G., Waldmann, R., de Weille, J. R., Heurteaux, C. J. Biol. Chem. 276, 35361–35367. & Lazdunski, M. (1997) J. Biol. Chem. 272, 28819–28822. 38. Askwith, C. C., Benson, C. J., Welsh, M. J. & Snyder, P. M. (2001) Proc. Natl. 17. Babinski, K., Catarsi, S., Biagini, G. & Seguela, P. (2000) J. Biol. Chem. 275, Acad. Sci. USA 98, 6459–6463. 28519–28525. 39. Price, M. P., Lewin, G. B., McIlwrath, S. L., Cheng, C., Xie, J., Heppenstall, 18. Benson, C. J., Xie, J., Wemmie, J. A., Price, M. P., Henss, J. M., Welsh, M. J. P. A., Stucky, C. L., Mannsfeldt, A. G., Brennan, T. J., Drummond, H. A., et al. & Snyder, P. M. (2002) Proc. Natl. Acad. Sci. USA 99, 2338–2343. (2000) Nature 407, 1007–1011. 19. Weisskopf, M. G., Castillo, P. E., Zalutsky, R. A. & Nicoll, R. A. (1994) Science 40. Price, M. P., McIllwrath, S. L., Xie, J., Cheng, C., Qiao, J., Tarr, D. E., Sluka, 265, 1878–1882. K. A., Brennan, T. J., Lewin, G. R. & Welsh, M. J. (2001) Neuron 32, 1071–1083.

2034 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.252782799 Leonard et al. Downloaded by guest on September 28, 2021