Two major functional membrane proteins, Ca²⁺ pump ATPase (SERCA), and phospholamban (PLN) play major roles in the control of calcium cycling during excitation-contraction (EC) coupling. This lecture focused on the Ca pump ATPase/PLN system, important for muscle relaxation by pumping intracellular Ca²⁺ions. Release of Ca²⁺ from the lumen of sarcoplasmic reticulum (SR) to the intracellular space caused by the Ca²⁺ release channel/ryanodine receptor triggers contraction. (Figure 1).

Tada reviewed most of the molecular biological aspects of the PLN-SERCA2 system, which governs the Ca cycling mechanism underlying the EC coupling of the myocardium, and the inhibitory effects of PLN and SERCA2 that can cause a "calcium cycling defect" that results in cardiac hypertrophy and even failure. Notably, further understanding of the data is required to determine causal relationships.

The intracellular interplay between cyclic AMP (cAMP) and Ca²⁺ comprises ß-adrenergic stimulation of cardiomyocytes, after which the ß-receptor/adenylate cyclase system produces cAMP to activate cAMP-dependent protein kinase (A kinase) A kinase catalyzes phosphorylation of at least three functional proteins in cardiac myocytes, which are essential for Ca cycling mechanisms: the -subunit of the voltage-sensitive Ca channel; PLN in the SR; and troponin I (Tn-I) in the myofibrillar apparatus. Interestingly, the former two increase Ca²⁺ in and out of the cytoplasm, while Tn-I phosphorylation protects myofibrils from overreacting to an increased flow of intracellular Ca²⁺.

Early data from Tada and colleagues identified the presence of PLN in cardiac SR. The three genes for Ca²⁺-ATPase (SERCA, Sarco Endoplasmic Reticulum Calcium ATPases) are: SERCA1, expressed in fast-twitch skeletal muscle SR; SERCA2, expressed in cardiac, slow-twitch skeletal and smooth muscles; SERCA3, expressed in a variety of muscle SR and non-muscle cell ER and considered a housekeeping gene Figure 2). PLN and SERCA2 are always co-expressed, because only one PLN gene has been identified to date, which is expressed in the SR of cardiac, slow-twitch skeletal and smooth muscle cells. SERCA2a is expressed in cardiac and slow-skeletal muscles and SERCA2b in smooth muscle and non-muscle cells. Figure 3 summarizes the characteristics of PLN.

PLN phosphorylation, phosphoester in nature, occurs at the Ser and Thr residues, whereas the phosphoprotein intermediate of ATPase is acyl phosphate in nature and occurs at the Asp residue (Figure 4). CaM kinase catalyzes phosphorylation of the Thr residue in addition to the A kinase-catalyzed phosphorylation at the Ser residue. The formed phosphoprotein is nearly immediately dephosphorylated by PP1 (protein phosphatase type 1). Tada and colleagues showed that upon PLN phosphorylation, the rates of formation and decomposition of the ATPase intermediate E-P are enhanced and thus increase the turnover rate of the ATPase reaction. The tight coupling of ATP hydrolysis and Ca²⁺ translocation across the SR membrane enhances the rate of Ca uptake by PLN phosphorylation. The affinity of the Ca pump for Ca²⁺ is increased.

Further work by Tada and colleagues identified 4 intermediary steps in the formation and decomposition of E-P. Of these steps, the rates of conversion from E₂ to E₁ and the rate from E₁-P to E₂-P are enhanced when PLN is phosphorylated by A kinase. Similar results were obtained with CaM kinase phosphorylation of PLN. Hence, they proposed the working hypothesis that PLN would act directly on the Ca pump ATPase through a protein-protein interaction.

The molecular mechanisms of protein-protein interactions between PLN and SERCA2a were explored by Tada and colleagues via cross-linking experiments employing the cross-linker Denny-Jaffe reagent, with Iodine-125 (I-125) labeled on one end with an N=N bond and on the other end Lys protein residues. Figure 5 illustrates the trial design. Labeled PLN was incubated with purified SERCA Ca pump ATPase in the presence of a non-ionic detergent and the two proteins were cross-linked under UV light. Two conditions were required for cross-linking: 1) PLN in a dephospho state, and 2) Ca pump ATPase in a Ca²⁺ -free E₂ state. The double N=N bond was broken by adding sodium dithionite to obtain I-125-labeled Ca ATPase. The labeled Ca ATPAse was fragmented by cyanogen bromide to identify the I-125-labeled ATPase segments. Two significant features were identified: 1) SERCA1 and SERCA2 have I-125 labeled sequences at two Lys residues of an ATPase protein fragment, while SERCA3 does not have a similar sequence nor is labeled by I-125, and 2) a sequence similarity between the PLN binding site located less than 50 amino acid downstream of the active ATPase site (forming E-P SERCA1, expressed in fast-twitch skeletal muscle which is devoid of PLN) and the SERCA2 PLN-binding site, thus indicating that the differential regulation by PLN is a result of tissue-specific PLN expression. The amino acid sequence around the PLN binding region in SERCA isoforms is shown in Figure 6.

Construction of a series of chimeras of SERCA2 and SERCA3, subjected to assay for determining Ca uptake rates, provided additional insights of the PLN and SERCA molecular interactions. Co-expression of PLN inhibits SERCA2 by shifting the Ca-dependence curve to higher Ca²⁺ concentrations, but it has no effect on SERCA3. Chimera CH2, mostly derived from SERCA2, does not react to co-expressed PLN, similar to the sequence data. However, Chimera CH9, mostly derived from SERCA3 with a PLN-binding sequence derived from SERCA2, shows no reaction to PLN co-expression, which differs from sequence data. Further study revealed that a secondary interaction site in the ATPase protein within the SR membrane is required for PLN to fully inhibit the Ca uptake rate.

An electrochemical milieu that allows PLN to bind to the PLN-binding domain of SERCA2 is established by the interaction between the cluster of charged residues (K-D-D-K plus PV in SERCA2) and charged residues in the PLN domain IA. Incorporation of phosphate into Ser16/Thr17 in PLN perturbs the charged interactions by introducing negative charges and results in dissociation of PLN from SERCA, which allows augmentation of ATPase activity.

The mutation of transmembrane residues of PLN provided insights into the molecular interactions between the TM regions of PLN and SERCA2. Studies showed functional polarization, i.e., gain of function residues located in one region and loss of function residues located in another. This functional polarization is important in establishing the transmembrane interaction of PLN and SERCA2, since gain of function mutants of PLN were about 75% monomeric and 25% pentameric, whereas the wild-type PLN, the loss of function mutants and the no change mutants were about 75% pentameric and 25% monomeric. In other words, gain of function mutants with about a 3-fold increase in their inhibitory function accompanied the 3- to 4-fold enhancement of monomer formation. The results indicated that a direct consequence of monomer formation may be gain of function mutations. In terms of loss of function mutants, the pentameter stability remained unchanged compared to wild-type PLN, suggesting that the interacting surface for SERCA2 lies on one face of the helix. Thus, PLN monomers are thought to represent the functional form, while the PLN pentameters provide a reservoir for the active monomer. This leads to the notion that the key determinant of inhibitory function must be the concentration of the PLN monomer/SERCA2 complex formation.

PLN was shown to be directly associated with the M6 segment of SERCA2. Three of 10 transmembrane segments of SERCA (M4, M5, M6) were shown to form a pore for two Ca²⁺ ions, providing further evidence that PLN can alter the affinity of SERCA2 for Ca²⁺ by a direct protein-protein interaction.

Two SR proteins, among others, Ca²⁺-release channel/ryanodine receptor (RyR) and SERCA2 plus PLN, play major roles in controlling the rise and fall of intracellular Ca²⁺ to induce contraction-relaxation cycles. Myofibrillar contraction is initiated by Ca²⁺-induced Ca²⁺-release by the RyR stored in the lumen of SR, when voltage-sensitive Ca²⁺ channels on the sarcolemma open to allow Ca²⁺ entry into the cell. Relaxation is caused by the uptake of Ca²⁺ through SERCA and PLN. Under pathologic conditions, when cells are overloaded with Ca²⁺, the removal of Ca²⁺ by the Na⁺/Ca²⁺ exchanger is activated to supplement the SERCA2 activity.

Work by other investigators shows the consequences of PLN/SERCA2 transgenic mutations in vivo. This works has shown 1) that the ablation of PLN in a mouse model of dilated cardiomyopathy rescues the phenotype that resembles human heart failure; and 2) that superinhibition of SERCA2 by PLN mutants causes cardiac hypertrophy and failure. Work by Tada and colleagues aims to show that mutation of the PLN-binding sequence in SERCA2 prevents PLN from forming the active complex, so that Ca²⁺ uptake is augmented to attenuate pressure overload-induced cardiac hypertrophy.

PLN ablation has been shown by other investigators to rescue macroscopic and ultrastructural defects of the MLP knockout mice myocardium. In this setting, Ca signaling defects seen in dilated cardiomyopathy are rescued when PLN is ablated and restored to the normal pattern compared to the effects in wild-type mice. Cardiac contractility is activated by inhibition of the interaction between SERCA2 and Val⁴⁹ to ALA mutation of PLN, a loss of inhibitory function mutation. The data indicate the possibility that PLN ablation and mutation rescue the "Ca cycling defect" in dilated cardiomyopathy.

Tada and colleagues showed that a mutant SERCA2 transgene, constructed such that positively-charged K³⁹⁷ and K⁴⁰⁰ are mutated to negatively charged glutamic acids, had a 10-fold greater affinity for Ca²⁺ in the transgenic mice, compared to the wild-type and non-transgenic mice. Further, in the mutant SERCA transgenic mice, pressure overload hypertrophy is attenuated and prevented.