Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Abstract

Cardiac muscarinic receptors activate an inwardly rectifying K+ channel, IK+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart Gi2, Gi3, or Go). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of IK+Ach in intact cells. The muscarinic agonist carbachol increased IK+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking αo, and in cells lacking the PT-insensitive G protein αq. In cells with targeted inactivation of αi2 or αi3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the αi2- and αi3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate IK+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking αi2 or αi3. Our results show that expression of two specific PT-sensitive α subunits (αi2 and αi3 but not αo) is required for normal agonist-dependent activation of IK+Ach and suggest that both αi2- and αi3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of IK+Ach or the expression of αi2 or αi3.