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Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis

Erik Norberg1, Marie Karlsson, Olga Korenovska

  • 1Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

The EMBO Journal
|November 2, 2010
PubMed

Insights

Protein kinase C (PKC) inhibitors trigger cell death by increasing calcium (Ca²⁺) influx via the HCN2 channel. Dephosphorylation of HCN2 at Thr⁵⁴⁹ is crucial for this calcium-mediated apoptosis.

Area of Science:

  • Cell Biology
  • Neuroscience
  • Molecular Biology

Background:

  • Cellular calcium uptake is a regulated process involving ion channels.
  • Protein kinase C (PKC) inhibitors like staurosporine (STS) and PKC412 can induce cell death via calcium influx, but the mechanisms are unclear.

Purpose of the Study:

  • To elucidate the molecular mechanisms by which PKC inhibitors trigger calcium influx and subsequent apoptosis.
  • To investigate the role of hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in PKC inhibitor-induced cell death.

Main Methods:

  • Utilized lung carcinoma cells and primary cortical neurons.
  • Investigated calcium influx using PKC inhibitors (STS, PKC412).
  • Employed gene silencing (downregulation) and heterologous expression of HCN2 in HEK293 cells.
  • Performed site-directed mutagenesis of putative PKC phosphorylation sites in HCN2.

Main Results:

  • PKC inhibitors induced prolonged calcium influx through HCN2 in lung carcinoma cells and neurons, leading to apoptosis-inducing factor (AIF)-mediated apoptosis.
  • HCN2 downregulation abolished drug-induced calcium increase and apoptosis.
  • HEK293 cells lacking HCN2 did not exhibit calcium entry upon inhibitor exposure, but HCN2 expression sensitized them to apoptosis.
  • Dephosphorylation of Thr⁵⁴⁹ in HCN2 was essential for prolonged calcium entry and AIF-mediated apoptosis.

Conclusions:

  • HCN2 acts as a critical mediator of calcium influx induced by PKC inhibitors.
  • The findings reveal a novel role for HCN2 as an upstream regulator in PKC inhibitor-triggered cell death pathways.
  • Specific dephosphorylation of HCN2 at Thr⁵⁴⁹ is a key regulatory step in this process.

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