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

Cardiac muscarinic receptors activate an inwardly rectifying K(+) channel, I(K(+(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 α subunits to determine which G proteins are required for receptor-mediated regulation of I(K(+(Ach))) in intact cells. The muscarinic agonist carbachol increased I(K(+(Ach))) activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α(o), and in cells lacking the PT-insensitive G protein α(q). In cells with targeted inactivation of α(i2) or α(i3), channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α(i2)- and α(i3)-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I(K(+(Ach))) in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α(i2) or α(i3). Our results show that expression of two specific PT-sensitive α subunits (α(i2) and α(i3) but not α(o)) is required for normal agonist-dependent activation of I(K(+(Ach))) and suggest that both α(i2)- and α(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 I(K(+(Ach))) or the expression of α(i2) or α(i3).