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. 2007 Feb 13;104(7):2519-24.
doi: 10.1073/pnas.0611031104. Epub 2007 Feb 6.

Exchange protein activated by cAMP (Epac) mediates cAMP activation of p38 MAPK and modulation of Ca2+-dependent K+ channels in cerebellar neurons

Jeanne Ster  1 , Frédéric De Bock, Nathalie C Guérineau, Andrea Janossy, Stéphanie Barrère-Lemaire, Johannes L Bos, Joël Bockaert, Laurent Fagni
Affiliations

Exchange protein activated by cAMP (Epac) mediates cAMP activation of p38 MAPK and modulation of Ca2+-dependent K+ channels in cerebellar neurons

Jeanne Ster et al. Proc Natl Acad Sci U S A. .

Abstract

The exchange factor directly activated by cAMP (Epac) is a newly discovered direct target for cAMP and a guanine-nucleotide exchange factor for the small GTPase Rap. Little is known about the neuronal functions of Epac. Here we show that activation of Epac by specific cAMP analogs or by the pituitary adenylate cyclase-activating polypeptide induces a potent activation of the Ca2+-sensitive big K+ channel, slight membrane hyperpolarization, and increased after-hyperpolarization in cultured cerebellar granule cells. These effects involve activation of Rap and p38 MAPK, which mobilizes intracellular Ca2+ stores. These findings reveal a cAMP Epac-dependent and protein kinase A-independent signaling cascade that controls neuronal excitability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Anti-Epac antibody characterization and endogenous Epac expression in neurons. (A) Western blots were obtained from HEK-293 cells nontransfected (NT) or transfected with Epac1 (T Epac1) or Epac2 (T Epac2) expression plasmids and revealed by using the anti-Epac1 [immunoblot (IB): Epac1] or anti-Epac2 (IB: Epac2) antibodies. (B and C) Western blots obtained from adult mouse cerebellar (B, Cereb), hippocampal (B, Hippo), or CGC extracts (C) by using the anti-Epac1 (Left) or anti-Epac2 (Right) antibodies. For each blot, S and P are soluble and particulate fractions, respectively. A–C are representative of independent experiments.
Fig. 2.
Fig. 2.
Epac and PACAP-38 affect resting membrane potential and AHP by activation of BK channels. (A) Action potential (upper traces) evoked by a brief current pulse (lower trace) in the absence (control) and presence of 8-pCPT (100 μM). These traces were obtained from the same neuron and were representative of 10 different neurons. (B) Resting membrane potential measured in the absence (control) and presence of the indicated drugs [iberiotoxin (IbTx), 0.5 μM; PACAP-38, 10 nM, + U73122, 1 μM]. In this and the following figures, each bar of a histogram represents the mean ± SEM, the number above or below each bar is the number of cells, and the asterisks indicate values significantly different from control. (C) Same as B, but for AHP.
Fig. 3.
Fig. 3.
Epac-induced activation of BK channels. (A and B) Cell-attached recordings of BK channels obtained from NT CGCs (A) or CGCs transfected with DN-Epac2 (B). (Inset) Expended trace of the BK channel activity recorded in the absence (control) and presence of 8-pCPT. (C) From left to right, effect on BK channels NPo of 10 μM and 100 μM 8-pCPT in NT and 100 μM 8-pCPT in DN-Epac2-transfected CGCs.
Fig. 4.
Fig. 4.
Epac mobilization of intracellular Ca2+ activates BK channels. (A) Representative trace of 8-pCPT-induced activation of a BK channel recorded in Ca2+-free external medium. (B) Inhibition of 8-pCPT-induced activation of BK channels by ryanodine (10 μM) in normal external medium. (C) BK channels activated by 8-pCPT after 30-min treatment with xestospongin-C (7.5 μM) in normal external medium. (D) Quantification of the 8-pCPT-induced activation of BK channels in normal, Ca2+-free, and ryanodine- and xestospongin-C-containing external medium.
Fig. 5.
Fig. 5.
Rap and p38 MAPK, but not p42/p44 MAPK or c-JunK, are involved in Epac-induced activation of BK channels. (A) BK channel activity recorded in a CGC transfected with Rap-N17. (B and C) BK channel activity recorded in CGCs treated with p38 MAPK (SB203580, 10 μM; B) or p42/p44 MAPK (PD98059, 25 μM; C) inhibitors. (D) Summary of the 8-pCPT effect on BK channel obtained in control neurons, in neurons transfected with Rap-N17, or in neurons treated with PD98059, SP600125, or SB203580 (10 μM).
Fig. 6.
Fig. 6.
Epac activates p38 MAPK and mobilizes intracellular Ca2+. (A) Western blots (Left) and quantitative histogram (n = 3) (Right) of p38 MAPK phosphorylation obtained from CGCs by using anti-phospho-p38 MAPK (Upper Left) or anti-p38 MAPK antibodies (Lower Left) in the absence of drug (control) or presence of SB203580, 8-pCPT (100 μM; 10 min), or SB203580 + 8-pCPT. (B) (Upper Left) Gray-level image illustrates a field of CGCs loaded with Ca2+-sensitive dye as described in Materials and Methods. 8-pCPT-induced Ca2+ increases in the cell marked with an arrowhead are shown in fluorescence micrographs a–g and plotted as a function of time in Lower. F/Fmin, fluorescence/minimum fluorescence. Color images correspond to an average of three successive confocal images taken at various times indicated by the letters in Lower. (Inset) The Ca2+ variation of this neuron in response to 10 mM caffeine.
Fig. 7.
Fig. 7.
PKA-independent activation of BK channels induced by cAMP and PACAP-38. (A) Activation of a BK channel by 8-Br-cAMP (100 μM), in the presence of the PKA inhibitor H-89 (10 μM; 5-min pretreatment). (B and C) The PACAP receptor agonist PACAP-38 (10 nM + 1 μM U73122) activated BK channel in nontransfected (B), but not DN-Epac2-transfected CGCs (C). (D) Quantification of BK channel activation induced by 8-pCPT or 8-Br-cAMP in NT and DN-Epac2-transfected CGCs. (E) Quantification of BK channel activation induced by PACAP-38, in control, H89-treated, and DN-Epac2-transfected neurons.
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