Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin

Filoteo, Adelaida G.; Enyedi, Ágnes; Verma, Anil Kumar; Elwess, Nancy L.; Penniston, John T.

doi:10.1074/jbc.275.6.4323

Cited by 16 publications

(20 citation statements)

References 28 publications

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“…Unfortunately, the information presently available on functional differences among isoforms is fragmentary at best. The problem is further exacerbated by the existence, next to the four basic gene products of the pump, of numerous alternatively spliced variants, which also display tissue-specific ex- pression and which in some cases may be even more abundantly expressed than the unspliced versions (12,13). Although the most extensively studied splice variants involve splice site C in the C-terminal cytosolic unit, splice variants involving site A, located immediately downstream of the domain that mediates the regulatory action of acidic phospholipids in the Nterminal half of the pump would also be however potentially interesting.…”

Section: Discussionmentioning

confidence: 99%

“…A C-terminally spliced variant of PMCA3, termed 3f (or 3CVI), in which a short insert leads to the loss of most of the C-terminal portion downstream of the calmodulin-binding domain (11) has also been analyzed. This isoform, which has been detected in skeletal muscle and to a lesser extent in brain, has very weak calmodulin affinity (12,13). Unfortunately, these studies were performed on isolated pump domains or on membrane preparations from overexpressing mammalian or insect cells in which possible physiological pump regulators may have become lost.…”

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confidence: 99%

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A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

Brini

Coletto

Pierobon

et al. 2003

Journal of Biological Chemistry

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The four basic isoforms of the plasma membrane Ca 2؉ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII (3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca 2؉ in the native intracellular environment. The level of [Ca 2؉ ] in the endoplasmic reticulum and the height of the [Ca 2؉ ] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca 2؉ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

Brini

Coletto

Pierobon

et al. 2003

Journal of Biological Chemistry

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“…Modeled concentration of each species as a function of time for dissociation of C28W(1b) from CaM-AF488. Sequences of the CaM binding domains for different isoforms of PMCA (37,38). The first 18 residues (underlined) are identical for all isoforms.…”

Section: Discussionmentioning

confidence: 99%

“…However, differences in the ten Cterminal residues of these isoforms are sufficient to dramatically alter their CaM binding affinities (36,37). For instance, a recent study reported dissociation constants of CaM binding to C28W peptides from isoforms 3f and 2b that were nearly two orders of magnitude apart (38), and these differences must originate in the sequence differences in the C-terminal portions of the respective CaM binding regions. Thus, the differences in the kinetics reported for isoforms 1b and 4b must be due to the amino-acid sequence differences (only three of them, shown in lower case in Figure 6) in the C-terminal 10 residues of the CaM binding domains of isoforms 1b and 4b.…”

Section: Relationship To Isoform 4bmentioning

confidence: 99%

Mechanism of Calmodulin Recognition of the Binding Domain of Isoform 1b of the Plasma Membrane Ca²⁺-ATPase: Kinetic Pathway and Effects of Methionine Oxidation

Slaughter

Urbauer

et al. 2007

Biochemistry

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Calmodulin (CaM) binds to a domain near the C-terminus of the plasma-membrane Ca 2+ -ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca 2+ -CaM with a 28-residue peptide (C28W(1b)) corresponding to the CaM binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or on the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism where an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the Nterminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the lower effectiveness of oxidized CaM in activating the Ca 2+ pump.Calmodulin is a cellular Ca 2+ sensor that interacts with numerous target enzymes in response to changes in intracellular Ca 2+ concentrations. The geometries of complexes between CaM and target domains are diverse, as CaM and the target may form not only compact structures, but also more extended bound conformations (1). It is well established that the C-terminal domain of CaM binds Ca 2+ with about an order of magnitude higher affinity than the N-terminal domain (2,3), and it also binds more tightly than the N-terminal domain to some targets (4,5).One important CaM target is the plasma membrane Ca 2+ -ATPase (PMCA), a membrane Ca 2+ pump critical for regulation of intracellular Ca 2+ levels (6,7). Several researchers have speculated that an intermediate in the CaM-mediated activation of PMCA corresponds to a * Corresponding Author E-mail: ckjohnson@ku.edu Telephone (785) (5), shows the C-terminal domain of CaM bound to the peptide while the N-terminal domain is free (pdb 1cff (4)). Other experiments show that a peptide with just four more residues (C24W) binds in a final structure where both terminal domains of CaM interact with the target (9,10). Squier and co-workers showed that CaM C (res...

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“…A cell that utilizes PMCA to respond rapidly to a Ca 2ϩ spike requires an isoform that has a high basal activity, such as isoform 3f in heart muscle (2), and/or one that is activated rapidly by calmodulin, such as isoform 2a in inner ear hair cells. In contrast, a cell that requires a sustained Ca 2ϩ signal may utilize isoforms with low basal activity and slow activation by Ca 2ϩ -calmodulin, such as isoform 4b in Jurkat T lymphocytes (3).…”

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confidence: 99%

Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

Penheiter¹,

Caride²,

Enyedi³

et al. 2002

Journal of Biological Chemistry

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Tryptophan 1093 resides in the 28-residue calmodulinbinding/autoinhibitory domain of the plasma membrane Ca 2؉ pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was ϳ2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K 0.5 of 9 nM for wild type to 144 nM (assayed at 700 nM free Ca 2؉ ). Pre-steady state measurements of the rate of activation by Ca 2؉ -calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the halftime of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca 2؉ -calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulinactivated pump.The plasma membrane Ca 2ϩ ATPase or pump (ATP2B1-4, referred to here as PMCA) 1 and the Na ϩ /Ca 2ϩ exchanger are the main mechanisms for removing Ca 2ϩ from the cell. Ca 2ϩ removal is required to maintain a steady Ca 2ϩ disequilibrium of ϳ10 5 between the cytoplasm and the extracellular milieu and to create the Ca 2ϩ spikes, waves, and oscillations that are the most ubiquitous signaling systems in the cell. Regulation of PMCA occurs via allosteric activation by Ca 2ϩ -calmodulin. Before activation, PMCA is inhibited by an intramolecular interaction between the inhibitory domain at the C terminus and the catalytic core of the molecule. The inhibitory domain contains the calmodulin-binding region of the molecule. A rise in intracellular Ca 2ϩ results in 4 Ca 2ϩ binding to calmodulin; the Ca 2ϩ -calmodulin complex then binds to the inhibitory domain, activating the pump. Activation results in an increase in the affinity of the pump for Ca 2ϩ at its transport site as well as an increase in its maximal turnover.PMCA in mammals is encoded by four genes with additional diversity provided by alternate splicing at two or more sites (for a recent review of PMCA genes, splicing variants, and nomenclature, see Strehler and Zacharias (1)). PMCA isoforms differ among themselves in three ways: 1) basal activities in the absence of calmodulin, 2) rates of activation by Ca 2ϩ -calmodulin, and 3) rates of inactivation by calmodulin dissociation. These differences are caused by differences in the interactions between the inhibitory domain, calmodulin, and the catalytic core. The present study investigates the effect of...

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Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin

Cited by 16 publications

References 28 publications

A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

Mechanism of Calmodulin Recognition of the Binding Domain of Isoform 1b of the Plasma Membrane Ca²⁺-ATPase: Kinetic Pathway and Effects of Methionine Oxidation

Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

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Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin

Cited by 16 publications

References 28 publications

A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

A Comparative Functional Analysis of Plasma Membrane Ca2+ Pump Isoforms in Intact Cells

Mechanism of Calmodulin Recognition of the Binding Domain of Isoform 1b of the Plasma Membrane Ca2+-ATPase: Kinetic Pathway and Effects of Methionine Oxidation

Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

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Mechanism of Calmodulin Recognition of the Binding Domain of Isoform 1b of the Plasma Membrane Ca²⁺-ATPase: Kinetic Pathway and Effects of Methionine Oxidation