]i transients and contractions. Once Gi is activated by muscarinic agonist, the ␣ i-subunit is released from the ␥-subunits, but it is silent, and its inhibition of the AC/cAMP cascade, manifested by I Ca reduction, is not seen unless AC has been previously activated. In colchicine-treated cells, CCh caused greater reductions of [Ca 2ϩ ]i transients and contractions than in untreated cells. The ␣i-subunit became effective in signaling through the AC/cAMP cascade and reduced ICa without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed ICa inhibition by CCh. Stimulation of I Ca by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on IK(ACh) was occluded in colchicine-treated cells. Colchicine treatment, per se, may increase I K(ACh) by ␥-subunits released from Gs to mask this effect of CCh. Microtubules suppress I Ca regulation by ␣i; their disruption releases restraints that unmask muscarinic inhibition of I Ca. Summarily, colchicine treatment reverses regulation of ventricular excitation-contraction coupling by autonomic agents.