Targeted inactivation of alphai2 or alphai3 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 Gi2, Gi3, or Go). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive alpha 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 alphao, and in cells lacking the PT-insensitive G protein alphaq. In cells with targeted inactivation of alphai2 or alphai3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the alphai2- and alphai3-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 alphai2 or alphai3. Our results show that expression of two specific PT-sensitive alpha subunits (alphai2 and alphai3 but not alphao) is required for normal agonist-dependent activation of IK+Ach and suggest that both alphai2- and alphai3-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 alphai2 or alphai3.
Sowell
M O
MO
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA.
Ye
C
C
Ricupero
D A
DA
Hansen
S
S
Quinn
S J
SJ
Vassilev
P M
PM
Mortensen
R M
RM
D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism
D026902:Potassium Channel Blockers; D015221:Potassium Channels; D018047:Receptors, Purinergic P1; D002217:Carbachol; D019204:GTP-Binding Proteins