G αi2, G αi3and G αoare all Required for Normal Muscarinic Inhibition of the Cardiac Calcium Channels in Nodal/Atrial-like Cultured Cardiocytes

The cardiac -type calcium current (ICa,L) is an important regulator of myocardial contractility. It is activated by sympathetic stimulation and inhibited by parasympathetic activity via muscarinic acetylcholine receptors. Muscarinic inhibition of ICa,Loccurs via activation of pertussis toxin (PTX)-sensitive heterotrimeric G-proteins. Although recent studies have shown that expression of Goαis important for this effect in adult mouse ventricular cells, two other PTX-sensitive G-proteins (Gi2and Gi3) are also expressed in cardiocytes and are activated. Their role in the regulation of ICa,Lhas not been examined. In addition, it is not known whether nodal/atrial cardiac cells use the same G-proteins. We show that gene inactivation of each of the three PTX-sensitive Gα-proteins (αi2, αi3, and αo) affects muscarinic inhibition of cardiac ICa,Lin embryonic stem (ES) cell-derived cardiocytes. Inactivation of eitherαi2 or αi3markedly slows the time course of muscarinic inhibition of ICa,L, and in cells where both αi2and αi3are inactivated the effects are not additive. We also establish an essential role for αoin this atrial/nodal-like cardiocyte system and show that αoacts proximal to NO generation. NO generation plays a critical role in ICa,Lregulation since the nitric oxide synthase (NOS) antagonist, -NMMA, blocked the inhibition of ICa,Lin WT and in αi2/αi3-null cells. In WT cells, the NO generating agent SIN-1 inhibited ICa,Land the addition of carbachol resulted in faster inhibition, suggesting that pathways in addition to NO are also activated. This study shows that αi2and αi3play a critical role in the normal inhibition of cardiocyte ICa,L. Thus, all muscarinic receptor activated G-proteins (Gi2, Gi3and Go) are necessary for normal inhibition and act through both NO and non-NO signaling pathways.