Cardiac troponin (Tn) I (CTnI), compared with skeletal TnI, contains extra amino acids (32 to 33) at its amino terminus, including two adjacent serine residues. These two serine residues are believed to be phosphorylated by protein kinase A (PKA) upon stimulation of the heart by beta-agonists. In this study, we found that phosphorylation of a cardiac skinned muscle preparation by PKA, mainly at CTnI, results in a decrease in the Ca2+ sensitivity of muscle contraction. The pCa50 decreased by approximately 0.27 +/- 0.06 pCa units upon phosphorylation. To study cardiac muscle relaxation, we used diazo-2, a photolabile Ca2+ chelator with a low Ca2+ affinity in its intact form that is converted to a high-affinity form after photolysis. We found that the rate of cardiac muscle relaxation increased from a time of half-relaxation (t1/2) = 110 +/- 10 milliseconds to t1/2 = 70 +/- 8 milliseconds after CTnI phosphorylation. This result demonstrates that CTnI phosphorylation can be linked with the increased rate of muscle relaxation in a relatively intact muscle preparation. Since CTnI phosphorylation has been shown previously to affect the Ca2+ affinity and Ca2+ off-rate of CTnC in vitro, it is likely that the faster relaxation seen here reflects faster dissociation of Ca2+ from cardiac TnC (CTnC). Model calculations show that increased dissociation of Ca2+ from CTnC, coupled with the faster uptake of Ca2+ by the sarcoplasmic reticulum stimulated by PKA phosphorylation of phospholamban, can account for the faster relaxation seen in the inotropic response of the heart to catecholamines.

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Summary.The pH dependence of the Ca 2+, Mg ztATPase pump of rabbit skeletal sarcoplasmic reticulum has been analyzed. Active uptake progress curves of the free luminal Ca 2+ concentration vs. time were obtained by fluorometric readout. The average rates (evaluated at t =2 sec) and steady-state maximal uptakes ([Ca2+~) These two parameters showed identical pH dependence, with a pH optimum between 6.0 and 6.5. These results are explained both qualitatively and quantitatively by a preliminary model of the steady-state behavior of the pump. The model assumes that enzyme dephosphorylation and countertranslocation represent the rate-limiting step of the cycle and that all other steps are at equilibrium. According to the model, the enzyme has two translocator sites, each bearing a doubly negative charge at pH 7.0 and above. Occupation of both sites by Ca 2* is necessary for transport. Partial or full protonation of the negative charges on the outwardly oriented translocators destroys their capacity for Ca 2+ transport. This process is responsible for the decrease in apparent translocator affinity with decreasing pH. A pK a of 7.2_+0.3 was determined for the outwardly oriented translocator. Transport of 2Ca 2+ is followed by their release to the lumen. Return of the carrier requires the loading of a charge-stoichiometric amount of alkali cation and H +.

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