Targeted inactivation of alpha(i2) or alpha(i3) disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K+ channel, IK+ Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G(i2), G(i3), or G(o)). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive cu 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 alpha(o), and in cells lacking the PT-insensitive G protein alpha(q). In cells with targeted inactivation of alpha(i2) or alpha(i3), channel activation by both carbachol and adenosine was blocked, Carbachol-induced channel activation was restored in the alpha(i2)- and alpha(i3)-null cells by reexpressing the previously targeted gene and guanosine 5'-[gamma-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 alpha(i2) or alpha(i3). Our results show that expression of two specific PT-sensitive alpha subunits (alpha(i2) and alpha(i3) but not alpha(o)) is required for normal agonist-dependent activation of IK+ Ach and suggest that both alpha(i2)- and alpha(i3)-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 alpha(i2) or alpha(i3).