PICK1/calcineurin suppress ASIC1-mediated Ca2+ entry in rat pulmonary arterial smooth muscle cells

Abstract

Acid-sensing ion channel 1 (ASIC1) contributes to Ca(2+) influx and contraction in pulmonary arterial smooth muscle cells (PASMC). ASIC1 binds the PDZ (PSD-95/Dlg/ZO-1) domain of the protein interacting with C kinase 1 (PICK1), and this interaction is important for the subcellular localization and/or activity of ASIC1. Therefore, we first hypothesized that PICK1 facilitates ASIC1-dependent Ca(2+) influx in PASMC by promoting plasma membrane localization. Using Duolink to determine protein-protein interactions and a biotinylation assay to assess membrane localization, we demonstrated that the PICK1 PDZ domain inhibitor FSC231 diminished the colocalization of PICK1 and ASIC1 but did not limit ASIC1 plasma membrane localization. Although stimulation of store-operated Ca(2+) entry (SOCE) greatly enhanced colocalization between ASIC1 and PICK1, both FSC231 and shRNA knockdown of PICK1 largely augmented SOCE. These data suggest PICK1 imparts a basal inhibitory effect on ASIC1 Ca(2+) entry in PASMC and led to an alternative hypothesis that PICK1 facilitates the interaction between ASIC1 and negative intracellular modulators, namely PKC and/or the calcium-calmodulin-activated phosphatase calcineurin. FSC231 limited PKC-mediated inhibition of SOCE, supporting a potential role for PICK1 in this response. Additionally, we found PICK1 inhibits ASIC1-mediated SOCE through an effect of calcineurin to dephosphorylate the channel. Furthermore, it appears PICK1/calcineurin-mediated regulation of SOCE opposes PKA phosphorylation and activation of ASIC1. Together our data suggest PKA and PICK1/calcineurin differentially regulate ASIC1-mediated SOCE and these modulatory complexes are important in determining downstream Ca(2+) signaling.

Keywords: DEG/ENaC; Duolink; FSC231; PKA; PKC; store-operated calcium entry.

Fig. 1.

Protein interacting with C kinase 1 (PICK1) is expressed in rat pulmonary arterial smooth muscle cells (PASMC). A: PCR analysis of PICK1 (top: 201 bp) and β-actin (bottom: 244 bp) mRNA expression in PASMC and rat brain tissue as a positive control. B: immunoblot showing protein expression of PICK1 (∼52 kDa) in PASMC. C: immunofluorescence in PASMC showing smooth muscle 22 α (SM22α; red), PICK1 (green), and the nuclear dye SYTOX (blue).

Fig. 2.

Colocalization of PICK1 and acid-sensing ion channel 1 (ASIC1) in PASMC. A: representative confocal images of the Duolink PLA interaction between goat anti-ASIC1 and rabbit anti-PICK1 (red puncta). For negative controls, PASMC were incubated with each primary alone (B and C) and both PLA probes. Actin is labeled with Alexa Fluor 647 phalloidin (blue) and the nuclei are labeled with SYTOX (green). Zoomed Duolink (D) and traditional colocalization (E) images showing ASIC1-PICK1 localize at the cell edge. Endogenous PICK1 and ASIC1 were immunoprecipitated (IP) from PASMC lysates with anti-ASIC1 and anti-PICK1-labeled beads. Coimmunoprecipitated ASIC1 and PICK1 were detected by Western blotting (WB) using either anti-PICK1 (F) or anti-ASIC1 (G) antibodies.

Fig. 3.

Inhibition of PICK1 diminishes colocalization and clustering with ASIC1. Representative confocal images (A) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 (A; red puncta) in the absence or presence of the PICK1 inhibitor FSC231 (50 μM for 30 min). Actin is labeled with Alexa Fluor 647 Phalloidin (blue) and the nuclei are labeled with SYTOX (green). Summary data for average number of puncta per cell (B) and puncta size (C). Values are means ± SE; n = 9–11 images from 5 separate experiments/group; *P < 0.05 vs. vehicle; analyzed by unpaired t-test.

Fig. 4.

PICK1 does not alter ASIC1 plasma membrane (PM) localization in PASMC. Representative Western blots (A and B) and summary data (D) showing the ratio of cell surface biotinylated (A) to total (B) ASIC1 expression following 24-h treatment with the PICK1 inhibitor FSC231 (50 μM). To verify specificity of biotin labeling for cell surface proteins, a separate blot of cell surface biotinylated proteins was probed for SM22α (23kDa; C). The blot also contains a total protein sample (+) as a positive control. Values are means ± SE; n = 6/group; analyzed by unpaired t-test.

Fig. 5.

Store depletion increases the interaction and clustering between PICK1 and ASIC1. Representative confocal images (A) of the Duolink interaction between goat anti-ASIC1 and rabbit anti-PICK1 (A; red puncta) upon stimulation of store depletion [SD; Ca²⁺-free plus cyclopiazonic acid (CPA) and diltiazem] and store-operated Ca²⁺ entry (SOCE: Ca²⁺-repletion plus CPA and diltiazem). Actin is labeled with Alexa Fluor 647 Phalloidin (blue) and the nuclei are labeled with SYTOX (green). Summary data for average number of puncta per cell (B) and puncta size (C). Baseline (BL) values are the same data from Fig. 3 vehicle. Values are means ± SE; n = 3–5 images from 7 separate experiments/group; *P < 0.05 vs. baseline; #P < 0.05 vs. store depletion (SD), analyzed with one-way ANOVA and individual groups compared with the Student-Newman-Keuls test.

Fig. 6.

PICK1 inhibits ASIC1-dependent SOCE. A: SOCE responses as determined by area under the curve (AUC) for PASMC pretreated with increasing concentrations of the PICK1 inhibitor, FSC231 (10–100 μM) for 24 h. Values are means ± SE; n = 4–6/group; *P < 0.05 vs. vehicle. B: SOCE responses (AUC) for PASMC pretreated for 30 min with FSC231 (50 μM) with or without pretreatment with the ASIC1 inhibitor psalmotoxin 1 (PcTX1; 20 nM). Values are means ± SE; n = 4–6/group; *P < 0.05 vs. vehicle; #P < 0.05 vs. control; analyzed with one-way (A) and two-way (B) ANOVA and individual groups compared with the Student-Newman-Keuls test.

Fig. 7.

PICK1 inhibits SOCE. Fold change in PICK1 mRNA (A) and protein (B) expression in PASMC following treatment with PICK1 shRNA compared with the negative (NEG) control shRNA. Values are means ± SE; n = 4/group; *P < 0.05 vs. NEG shRNA, analyzed by one sample t-test. C: SOCE responses as determined by AUC in PASMC treated with NEG or PICK1 shRNA. Values are means ± SE; n = 8–10/group; *P < 0.05 vs. NEG shRNA, analyzed by unpaired t-test.

Fig. 8.

PKC inhibits SOCE. SOCE responses as determined by area under the curve (AUC) for PASMC pretreated with the PKC activator VII CGK062 (30 μM) or myristoylated-PKC inhibitor (myr-PKC, 10 μM; A). Responses in the presence of CGK062 were additionally assessed in the presence of FSC231 (B) and the percent PKC-induced inhibition of SOCE under vehicle and FSC231 treatments was determined (C). Values are means ± SE; n = 5–6/group; *P < 0.05 vs. vehicle; #P < 0.05 vs. non-CGK062, analyzed with t-test (C), one-way (A) or two-way (B) ANOVA, and individual groups compared with the Student-Newman-Keuls test.

Fig. 9.

PICK1/Calcineurin inhibit PKA-stimulated SOCE. SOCE responses were determined by AUC in PASMC pretreated with the PICK1 inhibitor FSC231 (50 μM), calcineurin inhibitor cyclosporin A (CsA; 1 μM), ASIC1 inhibitor psalmotoxin 1 (PcTX1; 20 nM), PKA activator forskolin (10 μM), or PKA inhibitor KT 5720 (300 nM) as indicated on graphs. Values are means ± SE; n = 4–6/group. A: *P < 0.05 vs. vehicle; #P < 0.05 vs. FSC231/CsA. B: *P < 0.05 vs. non-FSC231/CsA treatment and #P < 0.05 vs. non-forskolin or -KT 5720 treatment; analyzed with one-way (A) or two-way (B) ANOVA and individual groups compared with the Student-Newman-Keuls test.

Fig. 10.

PICK1 and calcineurin inhibit ASIC1 phosphorylation. PASMC were pretreated with vehicle; PKA activator forskolin (10 μM), PKA inhibitor KT 5720 (300 nM), PICK1 inhibitor FSC231 (50 μM), or calcineurin inhibitor CsA (1 μM). Endogenous ASIC1 was immunoprecipitated from PASMC lysates with anti-ASIC1-labeled beads. Phosphorylation of ASIC1 was detected by Western blotting using anti-phosphoserine antibody (A). PASMC lysates were additionally probed for total ASIC1 (B). Summary data showing the fold change in ratio of co-IP ASIC1-phosphoserine to total ASIC1 relative to vehicle-treated samples (dotted line; C). Values are means ± SE; n = 3/group; *P < 0.05 vs. vehicle, analyzed with one-way ANOVA and individual groups compared with the Student-Newman-Keuls test.

Fig. 11.

Summary of major findings. Ca²⁺ depletion of the sarcoplasmic reticulum (SR) stimulates ASIC1-dependent SOCE (solid line) in PASMC. ASIC1-mediated SOCE is augmented by PKA-mediated phosphorylation and inhibited by PKC-mediated phosphorylation. Store depletion also increases the association between ASIC1 and PICK1 which limits SOCE by facilitating calcineurin (Cn)-dependent dephosphorylation of ASIC1.