Beta1-adrenergic receptor activation stimulates cardiac L-type Ca2+ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio-]triphosphate (MANT-GTPgammaS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPgammaS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPgammaS on L-type Ca2+ currents (ICa,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5-/-) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPgammaS attenuated ICa,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77+/-13%; 100 nM MANT-GTPgammaS, +43+/-6%; 1 microM MANT-GTPgammaS, +21+/-9%; p<0.05). The leftward shift of current-voltage curves was abolished by 1 microM but not by 100 nM MANT-GTPgammaS. In myocytes from AC5-/- mice, the residual stimulation of ICa,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute beta-adrenergic stimulation in WT mice. Interestingly, basal ICa,L was lowered by 1 microM but not by 100 nM MANT-GTPgammaS. The decrease was less pronounced in myocytes from AC5-/- mice compared with wild types (-23+/-1 versus -40+/-7%), indicating basal ICa,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of ICa,L by MANT-GTPgammaS, both under basal conditions and following beta-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in ICa,L activation and that MANT-GTPgammaS predominantly acts via AC5 inhibition.