α1-Adrenergic receptor–PKC–Pyk2–Src signaling boosts L-type Ca2+ channel CaV1.2 activity and long-term potentiation in rodents

  1. Kwun Nok Mimi Man
  2. Peter Bartels
  3. Peter B Henderson
  4. Karam Kim
  5. Mei Shi
  6. Mingxu Zhang
  7. Sheng-Yang Ho
  8. Madeline Nieves-Cintron
  9. Manuel F Navedo
  10. Mary C Horne  Is a corresponding author
  11. Johannes W Hell  Is a corresponding author
  1. Department of Pharmacology, University of California, United States
  2. Department of Pharmacology, University of Iowa, United States
11 figures and 5 additional files

Figures

The α1AR agonist phenylephrine (PHE) augments NPo of L-type Ca2+ channels (LTCCs) in hippocampal neurons.

(A) Neurons were preincubated with vehicle, PHE and prazosin (PRAZ) before seal formation. (B) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs from …

Figure 2 with 1 supplement
The phenylephrine (PHE)-induced increase in NPo of L-type Ca2+ channels (LTCCs) in hippocampal neurons requires PKC, Pyk2, and Src.

(A) Neurons were preincubated with vehicle, PHE, and the indicated kinase inhibitors before seal formation. (B) Ten consecutive traces from representative cell-attached single-channel recordings of …

Figure 2—figure supplement 1
Group I mGluR and muscarinic receptor agonists did not change NPo of L-type Ca2+ channels (LTCCs) in hippocampal neurons.

(A) Ten consecutive traces from representative cell-attached single-channel recordings of LTCCs from cultured hippocampal neurons with vehicle (H2O; black), 100 µM dihydroxyphenylglycine (DHPG, …

Figure 3 with 1 supplement
The increase in NPo of L-type Ca2+ channels (LTCCs) in hippocampal neurons by PKC requires Pyk2 and Src.

(A) Neurons were preincubated with vehicle, the phorbol ester phorbol-12-myristate-13-acetate (PMA), and the indicated kinase inhibitors before seal formation. (B) Ten consecutive traces from …

Figure 3—figure supplement 1
L-type channel blocker isradipine completely blocks L-type single-channel currents in the presence of phorbol-12-myristate-13-acetate (PMA).

PMA (2 μM) in the bath solution potentiated the open probability of L-type channels during single-channel recording. Isradipine (10 μM) in the pipette solution completely blocked L-type currents in …

α1AR signaling augments Po of L-type Ca2+ channels (LTCCs) in hippocampal neurons.

(A, E) Seals were formed by the recording pipettes before application of phenylephrine (PHE) or norepinephrine (NE) and ultimately of either isradipine or nimodipine to ensure channel activity was …

Norepinephrine (NE) can augment Po of L-type Ca2+ channels (LTCCs) via α1AR signaling in hippocampal neurons.

(A) Neurons were preincubated with NE ± prazosin (PRAZ) before seal formation. (B, C) Sample diaries show time courses of Po recordings obtained after preincubation with either NE alone or NE + PRAZ …

Bradykinin (BK) signaling augments Po of L-type Ca2+ channels (LTCCs) in hippocampal neurons.

(A) Seals were formed by the recording pipettes before application of BK and ultimately of BayK8644 (BayK) to ensure channel activity was mediated by LTCCs. (B) Sample diary shows time course of Po …

Pyk2 binds to the loop between domains II and III of α11.2. Co-immunoprecipitation of Pyk2 and Src with CaV1.2 from brain (A) and heart (B).

Triton X-100 extracts were cleared from non-soluble material by ultracentrifugation before immunoprecipitation (IP) with antibodies against α11.2, Pyk2 itself, or non-immune control antibodies …

Figure 7—source data 1

Original files of the full raw unedited blots with bands labeled in red boxes.

https://cdn.elifesciences.org/articles/79648/elife-79648-fig7-data1-v2.zip
PKC activates interdependent Pyk2 and Src.

PC12 cells were pretreated with vehicle (0.02% DMSO), the Pyk2 inhibitor PF-431396 (3 μM), and the Src inhibitor PP2 (10 μM) or its inactive analogue PP3 (10 μM) for 5 min before application of …

Figure 8—source data 1

Original files of the full raw unedited blots with bands labeled in red boxes.

https://cdn.elifesciences.org/articles/79648/elife-79648-fig8-data1-v2.zip
Increase in α11.2 tyrosine phosphorylation by PKC is blocked by inhibitors or Pyk2 and Src. PC12 cells were treated as in Figure 8 for analysis of tyrosine phosphorylation by immunoprecipitation (IP) with 4G10 and immunoblotting (IB) with anti-α11.2.

IgG indicates control IP with non-immune mouse IgG. Vehicle (0.02% DMSO), PF-431396 (3 μM), PP2 (10 μM), PP3 (10 μM), or SU6656 (SU, 10 μM) were applied 5 min before phorbol-12-myristate-13-acetate …

Figure 9—source data 1

Original files of the full raw unedited blots with bands labeled in red boxes.

https://cdn.elifesciences.org/articles/79648/elife-79648-fig9-data1-v2.zip
Increase in α11.2 tyrosine phosphorylation by PKC is blocked by knockdown of Pyk2 and Src.

(A) Lysates from HEK293T/17 cells transfected with vectors encoding rat Pyk2 (rPyk2-GFP) and either the Pyk2-targeting FIV lentivirus-derived, pVETL-Sh1-GFP (pFV-Pyk2-Sh1) or control (empty) …

Figure 10—source data 1

Original files of the full raw unedited blots with bands labeled in red boxes.

https://cdn.elifesciences.org/articles/79648/elife-79648-fig10-data1-v2.zip
α1AR signaling augments LTPLTCC through L-type Ca2+ channel (LTCC) activity, Pyk2, and Src.

LTPLTCC was induced by four 200 Hz tetani, each 0.5 s long, in the CA3 Schaffer collateral projections to CA1 in acute hippocampal slices from 13- to 20-week-old mice. (A) LTPLTCC required …

Additional files

Supplementary file 1

Amino acid residues of fragments of intracellular loops of CaV1.2 α1-subunit used in GST pulldown studies.

https://cdn.elifesciences.org/articles/79648/elife-79648-supp1-v2.docx
Supplementary file 2

Antibody dilutions used for immunoblotting.

https://cdn.elifesciences.org/articles/79648/elife-79648-supp2-v2.docx
Supplementary file 3

shRNA sequences targeting rat Pyk2 and Src.

https://cdn.elifesciences.org/articles/79648/elife-79648-supp3-v2.docx
Supplementary file 4

Sequencing primers for validation of knockdown constructs.

https://cdn.elifesciences.org/articles/79648/elife-79648-supp4-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/79648/elife-79648-mdarchecklist1-v2.pdf

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