β-blockers augment L-type Ca2+ channel activity by targeting spatially restricted β2AR signaling in neurons
- Guangzhou Medical University, China
- University of California Davis, United States
- VA Northern California Health Care System, United States
Figures
Figure 1 with 1 supplement
Carvedilol and alprenolol selectively promote phosphorylation of β2AR at PKA sites.
HEK293 cells stably expressing FLAG-tagged β2AR were either directly stimulated for 5 min with the βAR agonist ISO or different β-blockers at indicated concentrations (A) n = 4), or pretreated for …
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Figure 1—source data 1
Excel spreadsheet containing the individual numeric values of phosphorylated β2AR / total β2AR relative density analyzed in Figure 1.
- https://doi.org/10.7554/eLife.49464.004
Figure 1—figure supplement 1
Uncropped blots for Figure 1.
(A) Uncropped blots corresponding to Figure 1A. (B) Uncropped blots corresponding to Figure 1B. (C) Uncropped blots corresponding to Figure 1C. Red box indicates the crop region displayed in main …
Figure 2 with 3 supplements
Carvedilol and alprenolol induce concentration-dependent PKA phosphorylation of β2AR in HEK293 and hippocampal neurons.
HEK293 cells stably expressing FLAG-tagged β2AR were treated with increasing concentrations of CAR (A) n = 4) and ALP (B) n = 3), or pretreated for 15 min with 10 μM β2AR antagonist ICI118551 (C) n =…
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Figure 2—source data 1
Excel spreadsheet containing the individual numeric values of phosphorylated β2AR / total β2AR relative density analyzed in Figure 2A-D.
- https://doi.org/10.7554/eLife.49464.009
Figure 2—figure supplement 1
Uncropped blots for Figure 2.
(A) Uncropped blots corresponding to Figure 2A. (B) Uncropped blots corresponding to Figure 2B. (C) Uncropped blots corresponding to Figure 2C. (D) Uncropped blots corresponding to Figure 2D. Red …
Figure 2—figure supplement 2
Phosphorylation of ERK and β2AR at different drug concentrations.
HEK293 cells stably expressing FLAG-tagged β2AR were stimulated by isoproterenol (ISO) or carvedilol (CAR) for 30 min with indicated concentrations. Cell lysates were analyzed for PKA-phosphorylated …
Figure 2—figure supplement 3
GRK-phosphorylation of β2AR at different carvedilol treated times.
HEK293 cells stably expressing FLAG-tagged β2AR were stimulated by 1 μM isoproterenol (ISO) or carvedilol (CAR) for indicated times. Cell lysates were analyzed for PKA-phosphorylated β2AR, …
Figure 3 with 3 supplements
Carvedilol and alprenolol promote Gsα recruitment to β2AR and increase spatially restricted cAMP signal.
(A) HEK293 cells co-expressing FLAG-tagged β2AR, HA-tagged Gsα and EGFP were stimulated with 100 nM ISO or indicated β-blockers for 5 min. In proximity ligation assay (PLA), cells were …
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Figure 3—source data 1
Excel spreadsheet containing the individual numeric values of PLA dots / cell number in each raw image analyzed in Figure 3A, and the individual numeric values for maximum FRET responses in Figure 3B-E.
- https://doi.org/10.7554/eLife.49464.014
Figure 3—figure supplement 1
Carvedilol-induced β2AR phosphorylation is AC-dependent.
HEK293 cells stably expressing FLAG-tagged β2AR were pretreated with the Gi inhibitor pertussis toxin (PTX, 200 ng/ml, 16 hr) or the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (ddA, 50 μM, …
Figure 3—figure supplement 2
Carvedilol- and alprenolol-induced cAMP can be abolished by β2AR or AC inhibition.
The cAMP biosensor ICUE3 and FLAG-β2AR were co-expressed in HEK293 cells. Cells were treated with 100 nM βAR agonist ISO or different β-blockers, and changes in cAMP FRET ratio were measured. In …
Figure 3—figure supplement 3
Carvedilol- and alprenolol-induced cAMP are highly restricted.
HEK293 cells expressing either CAAX-ICUE3 targeted to non-rafts regions of the plasma membrane (A) or LYN-ICUE3 targeted to rafts regions of the plasma membrane (B) were treated with 1 μM ISO or …
Figure 4 with 1 supplement
Carvedilol promotes endogenous β2AR-dependent phosphorylation of LTCC α11.2 by PKA in neurons.
(A) Rat neurons on 10–14 days in vitro (DIV) were treated for 5 min with 1 μM indicated drugs. The phosphorylation of endogenous LTCC α11.2 subunit was determined with phospho-specific antibodies, …
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Figure 4—source data 1
Excel spreadsheet containing the individual numeric values of phosphorylated a11.2 or GluA1 / total a11.2 or GluA1 relative density analyzed in Figure 4.
- https://doi.org/10.7554/eLife.49464.017
Figure 4—figure supplement 1
Uncropped blots for Figure 4.
(A) Uncropped blots corresponding to Figure 4A. (B) Uncropped blots corresponding to Figure 4B. (C) Uncropped blots corresponding to Figure 4C. Red box indicates the crop region displayed in main …
Figure 5 with 2 supplements
Carvedilol augments LTCC CaV1.2 channel activity in neurons.
(A) Representative single channel recordings of LTCC CaV1.2 currents using 110 mM Ba2+ as charge carrier in rat hippocampal neurons on 7–10 days in vitro (DIV) after depolarization from −80 (hp) to …
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Figure 5—source data 1
Excel spreadsheet containing the individual numeric values of Po, availability and current analyzed in Figure 5C-G.
- https://doi.org/10.7554/eLife.49464.021
Figure 5—figure supplement 1
Over-time effect of carvedilol on LTCC of neurons recorded in the cell attached configuration without using BayK.
Cells were depolarized with 110 mM Ba2+ for 2 s from a holding potential of −80 mV to 0 mV and the NPopen was determined over time. (A) Diagram reflecting our patch statistics without using BayK in …
Figure 5—figure supplement 2
Inhibition of β2AR or LTCC counteracts carvedilol-induced cell death of cultured cortical neurons.
Rat cortical neurons on 7 days in vitro (DIV) were incubated overnight in fresh medium with or without 2 mM Ca2+ (no Ca2+), cells were then either mock treated (NT), or treated with 1 μM indicated …
Figure 6 with 1 supplement
A mutant β2AR is selectively activated by carvedilol but not isoproterenol.
(A) Schematic of an engineered β2AR with S204/207A double serine mutations that loses high affinity binding to ISO but not CAR at nanomolar range. (B) cAMP biosensor ICUE3 and β2AR wild-type (WT) or …
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Figure 6—source data 1
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 6B, and the individual numeric values of phosphorylated β2AR / total β2AR relative density analyzed in Figure 6C-D.
- https://doi.org/10.7554/eLife.49464.024
Figure 6—figure supplement 1
Uncropped blots for Figure 6C and D.
(A) Uncropped blots corresponding to Figure 6C. (B) Uncropped blots corresponding to Figure 6D. Red box indicates the crop region displayed in main figure.
Figure 7 with 1 supplement
The β2AR mutant selectively supports carvedilol-induced augmentation of LTCC activity in neurons.
(A–B) β1AR/β2AR double knockout (DKO) mouse hippocampal neurons on 7–10 days in vitro (DIV) were cotransfected with FLAG-tagged β2AR WT (A) or mutant (B) and HA-tagged LTCC α11.2 subunit, 24 hr …
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Figure 7—source data 1
Excel spreadsheet containing the individual numeric values of Po, availability and current analyzed in Figure 7E-G.
- https://doi.org/10.7554/eLife.49464.027
Figure 7—figure supplement 1
The mutant β2AR is selectively activated by carvedilol and promotes LTCC phosphorylation in neurons.
β1AR/β2AR double knockout (DKO) hippocampal mouse neurons at 7–10 days in vitro (DIV) were cotransfected with FLAG-tagged β2AR WT (A) or mutant (B) and HA-tagged LTCC α11.2 subunit, 24 hr later …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Mus musculus) | β1AR/β2AR double knockout | Jackson Laboratories | Stock # 003810 | |
| Strain (Rattus norvegicus) | Sprague Dawley | Charles River Laboratories | ||
| Cell line (Homo sapiens) | HEK293/β2AR-WT | De Arcangelis et al., 2009 | HEK293 cells stably expressing FLAG-β2AR | |
| Cell line (Homo sapiens) | HEK293/β2AR-S204/207A | This paper | HEK293 cells stably expressing FLAG-β2AR-S204/207A | |
| Antibody | Phospho-β2AR (Ser261/262) (mouse monoclonal) | Dr. Richard Clark (UT Huston) | Clone 2G3 | IF (1 μg/ml), WB (1:1000) |
| Antibody | Phospho-β2AR (Ser355/356) (mouse monoclonal) | Dr. Richard Clark (UT Huston) | Clone 10A5 | WB (1:1000) |
| Antibody | β2AR (rabbit polyclonal) | Santa Cruz Biotechnology | sc-570 RRID:AB_2225412 | PLA (1:100), WB (1:1000) |
| Antibody | Phospho-β2AR (Ser355/356) (rabbit polyclonal) | Santa Cruz Biotechnology | sc-16719R RRID:AB_781609 | WB (1:1000) |
| Antibody | α11.2 (rabbit polyclonal) | Patriarchi et al., 2016 | FP1 | WB (1:1000) |
| Antibody | Phospho-α11.2 (Ser1928) (rabbit polyclonal) | Patriarchi et al., 2016 | CH3P | IF (1 μg/ml), WB (1:1000) |
| Antibody | Phospho-α11.2 (Ser1700) (rabbit polyclonal) | Patriarchi et al., 2016, Originally from Dr. William Catterall (U of Washington) | WB (1:1000) | |
| Antibody | GluA1 (rabbit polyclonal) | Patriarchi et al., 2016 | WB (1:1000) | |
| Antibody | Phospho-GluA1 (Ser831) (rabbit polyclonal) | Patriarchi et al., 2016 | WB (1:1000) | |
| Antibody | Phospho-GluA1 (Ser845) (rabbit polyclonal) | Patriarchi et al., 2016 | WB (1:1000) | |
| Antibody | FLAG-M1 | Sigma-Aldrich | F3040 RRID:AB_439712 | IF (1 μg/ml), WB (1:1000) |
| Antibody | HA | Covance | MMS-101R RRID:AB_291262 | PLA (1:1000) |
| Recombinant DNA reagent | β2AR-mutant | This paper | FLAG-tagged human β2AR with S204/207A double mutations | |
| Recombinant DNA reagent | β2AR-ICUE3 | This paper | ICUE3 fused to the C-terminal of human β2AR | |
| Commercial assay or kit | Duolink in situ detection reagents | Sigma-Aldrich | DUO92007 | PLA |
| Software, algorithm | pCLAMP10 | Molecular Devices | electrophysiology | |
| Software, algorithm | MetaFluor | Molecular Devices | FRET |
Additional files
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Supplementary file 1
Biophysical properties of L-type Ca2+ currents in the neurons recorded in Figure 5A–5E.
Values are mean ± SEM. *p<0.05 with Kruskal Wallis – Dunn’s multiple comparison test.
- https://doi.org/10.7554/eLife.49464.028
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Supplementary file 2
Biophysical properties of L-type Ca2+ currents in the neurons recorded in Figure 7C–7G.
Values are mean ± SEM. *p<0.05 with Kruskal Wallis – Dunn’s multiple comparison test.
- https://doi.org/10.7554/eLife.49464.029
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Transparent reporting form
- https://doi.org/10.7554/eLife.49464.030
