beta(1)-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-GTP gamma S) as a potent competitive AC inhibitor. Intriguingly, MANT-GTP gamma S inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTP gamma S on L- type Ca2+ currents (I-Ca,I-L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5(-/-)) mice by whole-cell recordings. In wild-type myocytes, MANT-GTP gamma S attenuated I-Ca,I-L stimulation following isoproterenol application in a concentration- dependent manner (control, +77 +/- 13%; 100 nM MANT-GTP gamma S, +43 +/- 6%; 1 mu M MANT-GTP gamma S, +21 +/- 9%; p < 0.05). The leftward shift of current-voltage curves was abolished by 1 mu M but not by 100 nM MANT-GTP gamma S. In myocytes from AC5(-/-) mice, the residual stimulation of I-Ca,I-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 I-Ca,I-L was lowered by 1 mu M but not by 100 nM MANT-GTP gamma S. The decrease was less pronounced in myocytes from AC5(-/-) mice compared with wild types (+23 +/- 1 versus -40 +/- 7%), indicating basal I-Ca,I-L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of I-Ca,I-L by MANT-GTP gamma S, 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 I-Ca,I-L activation and that MANT-GTP gamma S predominantly acts via AC5 inhibition.