In cardiac tissue acetylcholine liberated from parasympathetic nerves acts via the m2 receptor to slow the heart (negative chronotropy) and decrease the force of contraction (negative inotropy). Adenosine, produced locally in the heart in response to ischemia, acts through A1 receptors to produce similar effects. These receptors can activate a number of different pertussis toxin sensitive G-proteins to directly and indirectly (via second messengers) regulate ion channels (inwardly rectifying potassium channels, acetylcholine activated potassium channels, L-type calcium channels, and pacemaker channels). Although some specificity in the signal transduction cascade has been defined, the exact role of these subtypes is unclear. The G-proteins in these pathways have been reported to be up-regulated in heart failure and pertussis toxin sensitive pathways play a role in decreased adrenergic responsiveness. In order to correlate physiological function and the function of the pathways activated, targeted disruption of alpha subunit genes (alpha-i2, alpha-i3, alpha-o) in mice and in embryonic stems cell has been performed. Inactivation of each alpha subunit has a specific disruption of some signaling pathways but not others. Alpha-o inactivation affects L-type Ca current and negative chronotropy whereas alpha-i1 and alpha-i3 disrupt activation of the acetylcholine activated potassium channel. This application proposes to define the specific role of these intracellular signaling cascades in the heart. The ionic channel, chronotropic and inotropic responses ro A1 adenosine and carbachol stimulation will be further defined in knockout mice and knockout cell lines. The mechanisms for these effects will be explored by characterizing receptor number and affinity, expression of other G proteins, and activation of second messenger cAMP. The structural basis for G-protein specificity in effector coupling will be studied by the production of mutant alpha-o molecules and testing their ability to restore functional coupling of effectors to receptors. These experiments should provide important information on the specificity of signal transduction by G proteins in heart.
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