Calmodulin (CaM) and Ca 2؉ /CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca 2؉ cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca 2؉ pump (Ca 2؉ -ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca 2؉

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... ake, Ca 2؉ -stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca 2؉ uptake and Ca 2؉ -ATPase activities of SR (50% inhibition at ϳ2-3 M CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V max of Ca 2؉ uptake, unaltered K 0.5 for Ca 2؉ activation of Ca 2؉ transport, and a 10-fold decrease in the apparent affinity of the Ca 2؉ -ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca 2؉ -binding sites of the Ca 2؉ -ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca 2؉ -ATPase, phospholamban, and ryanodine receptor-Ca 2؉ release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca 2؉ pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 M). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca 2؉ transport. These findings suggest a critical role for membranebound CaM in controlling the velocity of Ca 2؉ pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca 2؉ pump function, CaM BP (1-2.5 M) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM. By regulating cytosolic Ca 2ϩ concentration, the sarcoplasmic reticulum (SR) 1 plays a central role in the contraction-relaxation cycle of heart muscle. Upon excitation of the cardiomyocyte, Ca 2ϩ is released from the SR through Ca 2ϩ -release channels (known as RYR-CRC) to initiate muscle contraction (1-5). Subsequent muscle relaxation occurs upon sequestration of Ca 2ϩ back into the SR lumen by a Ca 2ϩ -pumping ATPase (Ca 2ϩ -ATPase) present in the SR (1, 4, 6, 7) . A well known mechanism for the regulation of the cardiac SR Ca 2ϩ -ATPase involves phosphorylation of another intrinsic SR protein, phospholamban (8 -11). In its unphosphorylated state, phospholamban is thought to interact with the Ca 2ϩ -ATPase exerting an inhibitory effect; phosphorylation of phospholamban by cAMPdependent protein kinase or CaM kinase is thought to disrupt this interaction resulting in stimulation of Ca 2ϩ pump activity (8 -11). In cardiac SR, the RYR-CRC also undergoes phosphorylation by CaM kinase (12) (13) (14) , and this may result in stimulation of Ca 2ϩ release from the SR (12, (15) (16) (17) . Recent studies from this laboratory (14, 18 -22) and other laboratories (23-26) have demonstrated that in cardiac SR, a membrane-associated CaM kinase phosphorylates the Ca 2ϩ -ATPase in addition to RYR-CRC and phospholamban. The phosphorylation occurred at a serine residue and was specific for the cardiac/slow-twitch muscle isoform (SERCA2a) of the Ca 2ϩ -ATPase (18). Site-directed mutagenesis studies by Toyofuku et al. (23) resulted in the identification of Ser 38 as the site in SERCA2a that is phosphorylated by CaM kinase. Studies using native cardiac SR vesicles (14) , purified SR Ca 2ϩ -ATPase preparations (14, 18) , and SERCA2a expressed in HEK-293 cells (23) suggested that Ser 38 phosphorylation of the Ca 2ϩ -ATPase results in activation of the V max of Ca 2ϩ transport. Some studies have, however, questioned the physiological role of Ca 2ϩ -ATPase phosphorylation. Thus, a study by Odermatt et al. (24) showed CaM kinase-mediated phosphorylation of the Ca 2ϩ -ATPase in native rabbit cardiac SR as well as SERCA2a expressed in HEK-293 cells but failed to observe a significant stimulatory effect of phosphorylation on Ca 2ϩ -ATPase function. Another study by Reddy et al. (27) reported failure to observe phosphorylation of the Ca 2ϩ -ATPase in canine cardiac SR or purified Ca 2ϩ -ATPase reconstituted in lipid vesicles. These studies have attributed the stimulatory effect of CaM kinase to the phosphorylation of phospholamban and a consequent increase in Ca 2ϩ affinity of the Ca 2ϩ -ATPase. In native cardiac SR, analysis of the selective effect of Ca 2ϩ -ATPase phosphorylation on Ca 2ϩ -pumping activity of this enzyme is hampered by the concomitant phosphorylation of phospholamban and RYR-CRC by the membrane-bound CaM kinase. Recently, we achieved selective phosphorylation of the Ca 2ϩ -