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
  11. Johannes W Hell

  • Curated by eLife

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    This study reports of a new signaling pathway in hippocampal neurons by which alpha-1 receptors for norepinephrine regulates Cav1.2 calcium channels; activation of alpha-1 receptors enhances a form of long-lasting synaptic plasticity that is dependent on L-type calcium channels. The experiments are comprehensive and well executed although additional data are warranted to compellingly support the main conclusions. The work has significance for the field of neuroscience in general and for cellular mechanisms of neuroregulation in particular.

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Abstract

The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca 2+ channel (LTCC) Ca V 1.2 as a principal target for G q -coupled α 1 -adrenergic receptors (ARs). α 1 AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with Ca V 1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of Ca V 1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α 1 AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that Ca V 1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α 1 AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of Ca V 1.2 activity via α 1 AR–Pyk2–Src signaling regulates synaptic strength.

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  1. Version published to 10.7554/elife.79648 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    In this manuscript, Man et al. describe a new signaling pathway for regulation of the voltage-gated calcium channel Cav1.2 and show that it can modulate synaptic plasticity in the hippocampus. Studies with specific inhibitors, phosphopecific antibodies, and gene knockdown show that activation of alpha-1 adrenergic receptors induces downstream activation of the serine/threonine protein kinase PKC and the tyrosine protein kinases Pyk2 and Src, which bind to the Cav1.2 channel through its large intracellular segment connecting domains II and III. This signaling complex leads to tyrosine phosphorylation of Cav1.2 and increased channel activity. Block of this novel signaling pathway in hippocampal slices with specific inhibitors of Pyk2 and Src reduced a specific component of long-term potentiation whose induction requires Cav1.2 channel activity.

    This work is an important advance, as it presents a novel signaling pathway through which the ubiquitous neurotransmitter norepinephine and the neurohormone epinephrine can regulate synaptic plasticity, attention, learning, and memory. The experiments are comprehensive, carefully done, and clearly presented. The authors should consider revisions and responses to the points below.

    1. Figure 2B, D. Inhibitors reduce Ica below control. Is there endogenous stimulation of this regulatory pathway under control conditions?

    We now explicitly state in the Discussion: “Inhibitors of PKC, Pyk2, and Src reduce under nearly all conditions Cav1.2 baseline activity and also tyrosine phosphorylation of Cav1.2, Pyk2, and Src even when activators for alpha1 AR and PKC were present. Especially notable is the strong reduction of channel activity way below the control conditions by the Src inhibitor PP2 as well as the PKC inhibitor chelerythrine in Figure 2C. This effect is consistent with PP2 strongly reducing down below control conditions tyrosine phosphorylation of Src (Figure 8J), Pyk2 (Figure 8L), and Cav1.2 (Figure 9E) even with the PKC activator PMA present. These findings suggest that Pyk2 and Src experience significant although clearly by far not full activation under basal conditions as reflected by their own phosphorylation status, which translates into tyrosine phosphorylation of Cav1.2 under such basal conditions.” Because there are multiple ways Pyk2 and Src can be activated including Ca influx and cell-matrix interactions, defining the cause of this baseline activity has to remain beyond the scope of the current work.

    1. As noted by the authors, it would be interesting to know if peptides from the linker between domains II and III block this signaling pathway. This would be an important result because, without this information, it is not clear if this is the correct functional site of interaction for this regulatory complex.

    Briefly, we were not able to identify shorter loopII/III-derived peptides that would constitute the Pyk2 binding site and thus cannot displace Pyk2 from loop II/III either acutely with peptides or through mutagenesis of the binding site.

    1. Figure 4B. The Brain IP for Src has a weak signal. The authors should replace this panel with a more convincing immunoblot.

    We provide now the uncropped version in the raw dataset, which clearly illustrates clean, monospecific detection of the Src band over the full length of the blot. Also, please note that earlier work already reported that showed that Src binds to the C-terminus of Cav1.2 (Bence-Hanulec et al., 2000).

    1. Scatter plots are provided for the electrophysiological results but not immunoblots. For immunoblots that are quanitified, it would be valuable to add a scatter plot of the replicates.

    We now also provide scatter plots for the biochemical analysis.

  3. eLife

    eLife assessment

    This study reports of a new signaling pathway in hippocampal neurons by which alpha-1 receptors for norepinephrine regulates Cav1.2 calcium channels; activation of alpha-1 receptors enhances a form of long-lasting synaptic plasticity that is dependent on L-type calcium channels. The experiments are comprehensive and well executed although additional data are warranted to compellingly support the main conclusions. The work has significance for the field of neuroscience in general and for cellular mechanisms of neuroregulation in particular.

  4. eLife

    Reviewer #1 (Public Review):

    This study shows that activation of α1-adrenergic receptors in hippocampal neurons in culture increases nPo of single L-type calcium channels. This pathway is dissected using a large number of activating agents and blockers to involve PKC, Pyk2 and src. The pathway is further examined using PC12 cells, where it is activated by bradykinin. Finally, a form of LTP which is dependent on L-type calcium channels is augmented in young mice by use of the α1-AR agonist, phenylephrine.

    1. My main critique would be that the study, while very well executed and rigorous, is fragmented, consisting of three parts that each feel incomplete: i, hippocampal neuron studies, mainly single channel recordings; ii, biochemical studies mainly in PC12 cells, using a different agonist bradykinin, and iii, the examination of LTP in young mice.
  5. eLife

    Reviewer #2 (Public Review):

    The authors demonstrated that noradrenaline regulates Cav1.2 through PKC, which phosphorylates and activates Pyk2. Pyk2, in turn, autophosphorylates itself at Y402, which serves as a binding site for Src SH2 domain. Src will then phosphorylate Pyk2 at Y579 for full activation. Src also autophosphorylates itself at Y416. In this way, these two proteins generate a self-activating complex where Pyk activate Src, which then activates Pyk. Overall, this leads to an an activation of Cav1.2 and mediates noradrenaline-mediated augmentation of LTCC-mediated LTP.

  6. eLife

    Reviewer #3 (Public Review):

    In this manuscript, Man et al. describe a new signaling pathway for regulation of the voltage-gated calcium channel Cav1.2 and show that it can modulate synaptic plasticity in the hippocampus. Studies with specific inhibitors, phosphopecific antibodies, and gene knockdown show that activation of alpha-1 adrenergic receptors induces downstream activation of the serine/threonine protein kinase PKC and the tyrosine protein kinases Pyk2 and Src, which bind to the Cav1.2 channel through its large intracellular segment connecting domains II and III. This signaling complex leads to tyrosine phosphorylation of Cav1.2 and increased channel activity. Block of this novel signaling pathway in hippocampal slices with specific inhibitors of Pyk2 and Src reduced a specific component of long-term potentiation whose induction requires Cav1.2 channel activity.

    This work is an important advance, as it presents a novel signaling pathway through which the ubiquitous neurotransmitter norepinephine and the neurohormone epinephrine can regulate synaptic plasticity, attention, learning, and memory. The experiments are comprehensive, carefully done, and clearly presented. The authors should consider revisions and responses to the points below.

    1. Figure 2B, D. Inhibitors reduce Ica below control. Is there endogenous stimulation of this regulatory pathway under control conditions?

    2. As noted by the authors, it would be interesting to know if peptides from the linker between domains II and III block this signaling pathway. This would be an important result because, without this information, it is not clear if this is the correct functional site of interaction for this regulatory complex.

    3. Figure 4B. The Brain IP for Src has a weak signal. The authors should replace this panel with a more convincing immunoblot.

    4. Scatter plots are provided for the electrophysiological results but not immunoblots. For immunoblots that are quanitified, it would be valuable to add a scatter plot of the replicates.

  7. Version published to 10.1101/2022.07.01.498400v1 on bioRxiv