RESUMO
Enhancement of cardiac L-type Ca2+ channel activity by norepinephrine via phosphorylation by protein kinase A (PKA) underlines the positive inotropic effect of this transmitter and is a classical example of an ion channel modulation. However, it is not clear whether the channel protein itself (and which subunit) is a substrate for PKA. We have expressed various combinations of the cardiac Ca2+ channel subunits in Xenopus oocytes by injecting subunit mR-NAs. Expression of beta or alpha 2/delta + beta subunits potentiated the native (endogenous) Ca2+ channel currents in the oocyte (similar to T or N but not L-type). This potentiated endogenous current was enhanced by intracellular injection of cAMP or of the catalytic subunit of PKA, and this effect was reversed by the injection of a PKA inhibitor suggesting the presence of basal phosphatase activity. When a cardiac channel of alpha 1 + beta, alpha 1 + alpha 2/delta or alpha 1 + alpha 2/delta + beta composition was expressed at levels high enough that the contribution of the endogenous current became negligible, cAMP and PKA failed to increase the Ca2+ channel current, whereas PKA inhibitors and the catalytic subunit of protein phosphatase 1 reduced the amplitude of the current. Reduction of the current by PKA inhibitors was observed regardless of the presence of the beta subunit, suggesting a major role for the alpha 1 subunit in this process. These results suggest that, like in the heart, when expressed in Xenopus oocytes, the cardiac L-type Ca2+ channels are phosphorylated in basal state and dephosphorylation reduces their activity. However, unlike the situation in the heart, the activity of the channel cannot be enhanced by PKA-catalyzed phosphorylation, suggesting that the channel is already fully phosphorylated in its basal state.