Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes

We recently showed that colchicine treatment of rat ventricular myocytes increases the L-type Ca2+ current (I-Ca) and intracellular Ca2+ concentration ([Ca2+] (i)) transients and interferes with adrenergic signaling. These actions were ascribed to adenylyl cyclase (AC) stimulation after G(s) activation by alpha,beta-tubulin. Colchicine depolymerizes microtubules into alpha,beta-tubulin dimers. This study analyzed muscarinic signals in myocytes with intact or depolymerized microtubules. Myocytes were loaded with the Ca2+ indicator fluo 3 and were field stimulated at 1 Hz or voltage clamped. In untreated cells, carbachol (CCh; 1 muM) induced ACh-activated K+ current [I-K(ACh)], which happens via betagamma-subunits from the activation of G(i). Carbachol also reduced [Ca2+](i) transients and contractions. Once G(i) is activated by muscarinic agonist, the alpha(i)-subunit is released from the betagamma-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 [Ca2+](i) transients and contractions than in untreated cells. The alpha(i)-subunit became effective in signaling through the AC/cAMP cascade and reduced I-Ca without changing its voltage-dependence. Isoproterenol (Iso) regained its efficacy and reversed I-Ca inhibition by CCh. Stimulation of I-Ca by forskolin persisted in colchicine-treated cells when Iso was ineffective. The effect of CCh on I-K(ACh) was occluded in colchicine- treated cells. Colchicine treatment, per se, may increase I-K(ACh) by betagamma-subunits released from G(s) to mask this effect of CCh. Microtubules suppress I-Ca regulation by alpha(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.