We speculated that this unexpected effect of PTX may have been caused by inhibiting an upregulated Gai3. Recently, Zuberi et al. showed that Gai2 knockout leads to increased L-VDCC mRNA expression and a propensity towards ventricular arrhythmia. Muscarinic receptor-mediated inhibition of L-VDCC activity has been reported to Tomtovok depend on Gai2 but not Gai3 . Though strongly suggested by these data, subtypespecific effects on cardiac L-VDCC by the highly homologous Gai2 and Gai3 isoforms remain unclear so far. Therefore, the present work was undertaken to elucidate whether the effects of these Gai proteins are redundant or distinct. Using cardiomyocytes from mice lacking Gai2 or Gai3 and wild-type control animals, we determined structural and functional changes. Further, we examined specific signalling pathways implicated in cardiac L-VDCC modulation by Gai protein. In this work, we provide evidence that the L-VDCC activity and kinetics are regulated in a non-redundant manner and we support this idea by demonstrating subtype-specific activation of the extracellular signal-regulated kinases 1/2 signalling cascade. The two inhibitory G protein isoforms Gi2 and Gi3 are both upregulated in heart failure . One functionally important target of Gi protein signalling is the L-VDCC, the crucial trigger of cardiac excitation-contraction coupling. Gi-protein-mediated inhibition of L-VDCC has been demonstrated for b2-adrenergic and muscarinic receptor signalling. In this context, we previously provided single-channel evidence that Gai2 does not confer the L-VDCC inhibition observed in mice with chronic overexpression of the b2-adrenergic receptor . On the other hand, cardiac Gai2 seems necessary and sufficient to mediate the muscarinic receptor-mediated L-VDCC inhibition , presumably through the classical adenylyl cyclase pathway. So far, no isoform-specific function could be assigned to cardiac Gai3; however, Gi3 has been shown to be an exclusive and specific regulator of autophagy in the liver . There are no changes of cardiac L-VDCC composition regarding the main cardiac L-VDCC subunits, Cava1 and Cavb2 that would explain the obtained effects on current density.