The cyclic AMP-dependent modulation of cardiac sarcolemmal slow calcium channels

The epinephrine-induced abbreviation of systole is well explained by the cAMP-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, and of the inhibitory component of the troponin complex. In contrast, the molecular mechanism of the β-agonist inotropic effects remained unclear until the recent finding that the depolarization-induced Ca²⁺ uptake by cardiac sarcolemmal vesicles, the in vitro equivalent of the slow Ca²⁺ channel, is activated upon cAMP-dependent phosphorylation of a sarcolemmal integral protein, calciductin [34]. This paper provides additional evidence for this mechanism by showing a linear correlation between calciductin phosphorylation and voltage-dependent Ca²⁺ uptake. At no detectable radiolabelling with (³²P)-phosphate, the latter is still ca 28% of its maximal value, suggesting the possibility of residual unlabeled protein-bound phosphate. Calciductin is an excellent substrate of cAMP-dependent protein kinase, since low levels of its catalytic subunit (ca 6% of the subunit liberated after complete dissociation of the enzyme) are capable of submaximally phosphorylating calciductin and activating Ca²⁺ uptake. The Ca²⁺ channels of phosphorylated vesicles are similar to those of non-phosphorylated vesicles and of other excitable cells in their inhibition by the calcium antagonist verapamil, lanthanum and other divalent cations. In contrast to the voltage-dependent Ca²⁺ uptake, $\text{Na}{}^{\mathrm{+}}\text{Ca}{}^{\mathrm{2+}}$ exchange was not modulated by cAMP-dependent phosphorylation. Sarcolemmal proteolipids were entirely extracted by acidic organic solvent mixtures and purified by high performance liquid chromatography. They made up 2% (w/w) of sarcolemmal proteins and contained essentially calciductin (97%) together with a 14 000 to 16 000 dalton component (3%). Sarcolemmal proteolipids were phosphorylated on seryl residues only. Their amino acid composition indicated the presence of a large number of acidic and hydrophobic residues, accounting for the behaviour of calciductin as an acidic integral proteolipid, similar to phospholamban.