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cAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca(2+) signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca(2+) handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca(2+)](i) transients were evoked by electrical stimulation and Ca(2+) sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT). Patch-clamp was used to record the L-type calcium current (I(Ca)), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca(2+)](i) transients were slightly reduced by 10 microm 8-CPT (F/F(0): decreased from 4.7 +/- 0.5 to 3.8 +/- 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. I(Ca) was unaltered by Epac activation, so this cannot explain the decreased [Ca(2+)](i) transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca(2+) load underlies the decrease in the [Ca(2+)](i) transients. This decrease in the SR Ca(2+) load was provoked by the increase in the SR Ca(2+) leak induced by Epac activation. 8-CPT significantly increased Ca(2+) spark frequency (Ca(2+) sparks s(-1) (100 microm)(-1): from 2.4 +/- 0.6 to 6.9 +/- 1.5, P < 0.01) while reducing their amplitude (F/F(0): 1.8 +/- 0.02 versus 1.6 +/- 0.01, P < 0.001) in a Ca(2+)/calmodulin kinase II (CaMKII)-dependent and PKA-independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca(2+) release and signalling in cardiac myocytes.

The Ca(V)beta subunits of voltage-gated calcium channels regulate the trafficking and biophysical properties of these channels. We have taken advantage of mutations in the tyrosine residue within the alpha interaction domain (AID) in the I-II linker of Ca(V)2.2 which reduce, but do not abolish, the binding of beta1b to the AID of Ca(V)2.2. We have found that the mutation Y388S decreased the affinity of Ca(V)beta1b binding to the Ca(V)2.2 I-II linker from 14 to 329 nm. However, the Y388S mutation had no effect on current density and cell surface expression of Ca(V)2.2/alpha2delta-2/beta1b channels expressed in human embryonic kidney tsA-201 cells, when equivalent proportions of cDNA were used. Furthermore, despite the 24-fold reduced affinity of Ca(V)beta1b for the Y388S I-II linker of Ca(V)2.2, all the key features of modulation as well as trafficking by Ca(V)beta subunits remained intact. This is in contrast to the much more marked effect of the W391A mutation, which abolished interaction with the Ca(V)2.2 I-II linker, and very markedly affected the trafficking of the channels. However, using the Xenopus oocyte expression system, where expression levels can be accurately titrated, when Ca(V)beta1b cDNA was diluted 50-fold, all evidence of interaction with Ca(V)2.2 Y388S was lost, although wild-type Ca(V)2.2 was still normally modulated by the reduced concentration of beta1b. These results indicate that high affinity interaction with the alpha1 subunit is not necessary for any of the modulatory effects of Ca(V)beta subunits, but occupancy of the interaction site is important, and this will occur, despite the reduced affinity, if the Ca(V)beta subunit is present in sufficient excess.