Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

A Potent and Specific CD38 Inhibitor Ameliorates Age-Related Metabolic Dysfunction by Reversing Tissue NAD+ Decline.

Aging is characterized by the development of metabolic dysfunction and frailty. Recent studies show that a reduction in nicotinamide adenine dinucleotide (NAD+) is a key factor for the development of age-associated metabolic decline. We recently demonstrated that the NADase CD38 has a central role in age-related NAD+ decline. Here we show that a highly potent and specific thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, reverses age-related NAD+ decline and improves several physiological and metabolic parameters of aging, including glucose tolerance, muscle function, exercise capacity, and cardiac function in mouse models of natural and accelerated aging. The physiological effects of 78c depend on tissue NAD+ levels and were reversed by inhibition of NAD+ synthesis. 78c increased NAD+ levels, resulting in activation of pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs. Furthermore, in animals treated with 78c we observed inhibition of pathways that negatively affect health span, such as mTOR-S6K and ERK, and attenuation of telomere-associated DNA damage, a marker of cellular aging. Together, our results detail a novel pharmacological strategy for prevention and/or reversal of age-related NAD+ decline and subsequent metabolic dysfunction.

Plasma membrane Ca²âº-ATPases can shape the pattern of Ca²âº transients induced by store-operated Ca²âº entry.

Calcium (Ca(2+)) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca(2+) is regulated by multiple proteins, including the plasma membrane Ca(2+)-ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca(2+) out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca(2+) transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca(2+) transients elicited by conditions that trigger store-operated Ca(2+) entry (SOCE) and that blocked Ca(2+) uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced long-lasting Ca(2+) oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundance-sensitive clearance of SOCE-mediated Ca(2+) transients, whereas PMCA4a reduced cytosolic Ca(2+), resulting in the establishment of a higher than baseline cytosolic Ca(2+) concentration. Mathematical modeling showed that slow activation was critical to the sustained oscillation induced by the "slow" PMCA4b pump. The modeling and experimental results indicated that the distinct properties of PMCA isoforms differentially regulate the pattern of SOCE-mediated Ca(2+) transients, which would thus affect the activation of downstream signaling pathways.

Novel TRAF1-ALK fusion identified by deep RNA sequencing of anaplastic large cell lymphoma.

Chromosomal translocations leading to expression of abnormal fusion proteins play a major role in the pathogenesis of various hematologic malignancies. The recent development of high-throughput, "deep" sequencing has allowed discovery of novel translocations leading to a rapid increase in understanding these diseases. Translocations involving the anaplastic lymphoma kinase (ALK) gene leading to ALK fusion proteins originally were discovered in anaplastic large cell lymphomas (ALCLs). Among ALCLs, NPM1-ALK fusions are most common and lead to nuclear localization of the fusion protein. Here, we present a 50-year-old male with ALCL demonstrating cytoplasmic ALK immunoreactivity only, suggesting the presence of a non-NPM1 fusion partner. We performed deep RNA sequencing of tumor tissue from this patient and identified a novel transcript fusing Exon 6 of TRAF1 to Exon 20 of ALK. The TRAF1-ALK fusion transcript was confirmed at the mRNA level by Sanger sequencing and the fusion protein was visualized by Western blot. The discovery of this TRAF1-ALK fusion expands the diversity of known ALK fusion partners and highlights the power of deep sequencing for fusion transcript discovery. © 2013 Wiley Periodicals, Inc.

Alternative pathways for association and dissociation of the calmodulin-binding domain of plasma membrane Ca(2+)-ATPase isoform 4b (PMCA4b).

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

Diving Into the Lipid Bilayer to Investigate the Transmembrane Organization and Conformational State Transitions of P-type Ion ATPases.

Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca(2+) pump (SERCA) revealing different conformations, and recently, crystal structures of the H(+)-ATPase and the Na(+)/K(+)-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca(2+) signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na(+)/K(+)-ATPase, and other P-type ATPases.

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells.

The "w" splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.

Validation of fractal-like kinetic models by time-resolved binding kinetics of dansylamide and carbonic anhydrase in crowded media.

Kinetic studies of biochemical reactions are typically carried out in a dilute solution that rarely contains anything more than reactants, products, and buffers. In such studies, mass-action-based kinetic models are used to analyze the progress curves. However, intracellular compartments are crowded by macromolecules. Therefore, we investigated the adequacy of the proposed generalizations of the mass-action model, which are meant to describe reactions in crowded media. To validate these models, we measured time-resolved kinetics for dansylamide binding to carbonic anhydrase in solutions crowded with polyethylene glycol and Ficoll. The measured progress curves clearly show the effects of crowding. The fractal-like model proposed by Savageau was used to fit these curves. In this model, the association rate coefficient k(a) allometrically depends on concentrations of reactants. We also considered the fractal kinetic model proposed by Schnell and Turner, in which k(a) depends on time according to a Zipf-Mandelbrot distribution, and some generalizations of these models. We found that the generalization of the mass-action model, in which association and dissociation rate coefficients are concentration-dependent, represents the preferred model. Other models based on time-dependent rate coefficients were inadequate or not preferred by model selection criteria.

Plasma membrane calcium pump (PMCA) differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation.

The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.

Switch of PMCA4 splice variants in bovine epididymis results in altered isoform expression during functional sperm maturation.

Ca(2+) and Ca(2+)-dependent signals are essential for sperm maturation and fertilization. In mouse sperm the plasma membrane Ca(2+)-ATPase (PMCA) isoform 4 plays a crucial role in Ca(2+) transport. The two major splice variants of PMCA4 are PMCA4a and PMCA4b. PMCA4a differs from PMCA4b in the mechanism of calmodulin binding and activation. PMCA4a shows a much higher basal activity and is more effective than PMCA4b in returning Ca(2+) to resting levels. Knock-out mice carrying a PMCA4-null mutation are infertile because their sperm cannot achieve a hyperactivated state of motility. As sperm reach functional maturity during their transit through the epididymis, the expression of PMCA4a and 4b was assessed in bull testis and epididymis. Quantitative PCR revealed that PMCA4b is the major splice variant in testis, caput, and corpus epididymidis. In contrast, PMCA4a is the major splice variant in cauda epididymidis, whereas sperm are transcriptionally silent. Immunohistochemical staining using a new antibody against bovine PMCA4a located the PMCA4a to the apical membrane of the epithelium of cauda epididymidis, whereas testis, caput, and corpus epididymidis were negative. Western blotting of testis, epididymis, and sperm isolated from caput and cauda epididymidis showed a much higher level of PMCA4a in cauda epididymidis and sperm from cauda epididymidis compared with testis membranes and sperm from caput epididymidis. These findings suggest that PMCA4a is transferred to bovine sperm membranes in cauda epididymidis. This isoform switch may facilitate a higher calcium turnover in sperm necessary to traverse the female genital tract.

Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10.

Myo10 is an unconventional myosin with important functions in filopodial motility, cell migration, and cell adhesion. The neck region of Myo10 contains three IQ motifs that bind calmodulin (CaM) or the tissue-restricted calmodulin-like protein (CLP) as light chains. However, little is known about the mechanism of light chain binding to the IQ motifs in Myo10. Binding of CaM and CLP to each IQ motif was assessed by nondenaturing gel electrophoresis and by stopped-flow experiments using fluorescence-labeled CaM and CLP. Although the binding kinetics are different in each case, there are similarities in the mechanism of binding of CaM and CLP to IQ1 and IQ2: for both IQ motifs Ca(2+) increased the binding affinity, mainly by increasing the rate of the forward steps. The general kinetic mechanism comprises a two-step process, which in some cases may involve the binding of a second IQ motif with lower affinity. For IQ3, however, the kinetics of CaM binding is very different from that of CLP. In both cases, binding in the absence of Ca(2+) is poor, and addition of Ca(2+) decreases the K(d) to below 10 nM. However, while the CaM binding kinetics are complex and best fitted by a multistep model, binding of CLP is fitted by a relatively simple two-step model. The results show that, in keeping with growing structural evidence, complexes between CaM or CaM-like myosin light chains and IQ motifs are highly diverse and depend on the specific sequence of the particular IQ motif as well as the light chain.

A new conformation in sarcoplasmic reticulum calcium pump and plasma membrane Ca2+ pumps revealed by a photoactivatable phospholipidic probe.

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca(2+) pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2'-[(125)I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([(125)I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [(125)I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca(2+). This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca(2+) and calmodulin decreases by approximately the same amount. However, incubation with Ca(2+) alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [(125)I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E(1) conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca(2+) + C28 for SERCA and Ca(2+) alone for PMCA), and one in which the enzyme is fully active (Ca(2+) for SERCA and Ca(2+)-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.

Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations.

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.

Interaction with the IQ3 motif of myosin-10 is required for calmodulin-like protein-dependent filopodial extension.

Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility.

The plasma membrane Ca2+ pump isoform 4a differs from isoform 4b in the mechanism of calmodulin binding and activation kinetics: implications for Ca2+ signaling.

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., Török, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.

Plasma membrane calcium pump activity is affected by the membrane protein concentration: evidence for the involvement of the actin cytoskeleton.

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca(2+) from the cell. Specific Ca(2+)-ATPase activity of erythrocyte membranes increased steeply up to 1.5-5 times when the membrane protein concentration decreased from 50 microg/ml to 1 microg/ml. The activation by dilution was also observed for ATP-dependent Ca(2+) uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca(2+) or Ca(2+)-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.

Plasma membrane Ca2+ ATPases as dynamic regulators of cellular calcium handling.

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.

Modification of human hearing loss by plasma-membrane calcium pump PMCA2.

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.

Kinetic analysis of the calmodulin-binding region of the plasma membrane calcium pump isoform 4b.

The sequence L(1086)RRGQILWFRGLNRIQTQIKVVKAFHSS(1113) (peptide C28) is responsible for calmodulin binding to PMCA4b. In this work, peptides following the above sequence were progressively shortened either at the N-terminus (C28NDelta3, C28NDelta5, or C28NDelta6) or at the C-terminus (C20, C22, C23, and C25). Competitive inhibition of PMCA activity was used to measure apparent dissociation constants of the complexes between calmodulin and C28 or progressively shortened peptides. Additionally, equilibrium titrations were used to measure the apparent dissociation constants of the various peptides with TA-calmodulin by changes in TA-calmodulin fluorescence and Trp fluorescence of the peptides. At the N-terminus, deletion of five residues did not change calmodulin affinity, but deletion of six residues resulted in a 5-fold decrease in affinity. There were no major differences in the time course of TA-CaM binding, but C28NDelta6 exhibited a different time course of Trp fluorescence change. At the C-terminus, deletion of five residues (C23) or more resulted in a net increase in fluorescence of TA-CaM upon binding, while longer peptides (C25 and C28) produced both a transient increase and a net decrease in the fluorescence of TA-CaM. Global regression analysis revealed that binding of TA-CaM to the C23 peptide could be fit by a two-step model, while longer peptides required three-step models for adequate fitting. TA-calmodulin dissociated rapidly from C23, C22, and C20, resulting in a marked increase in apparent K(d). Thus, the sequence I(1091)LWFRGLNRIQTQIKVVKAF(1110) (C25NDelta5) is required to reproduce the calmodulin-binding properties of C28. When F(1110) was replaced by A, the TA-calmodulin association and dissociation kinetics resembled C23 kinetics, but changing V(1107) to A produced a smaller effect, suggesting that F(1110), rather than V(1107), is the main anchor for the N-terminal lobe of calmodulin in PMCA4b.

A model for the activation of plasma membrane calcium pump isoform 4b by calmodulin.

Overexpression of the plasma membrane calcium pump (PMCA) isoform 4b by means of the baculovirus system enabled us, for the first time, to study the kinetics of calmodulin binding to this pump. This was done by stopped-flow fluorescence measurements using 2-chloro-(amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin (TA-calmodulin). Upon mixing with PMCA, the fluorescence of TA-calmodulin changed along a biphasic curve: a rapid and small increase in fluorescence was followed by a slow and large decrease that lasted about 100 s. The experiment was done at several PMCA concentrations. Global fitting nonlinear regression analysis of these results led to a model in which PMCA is present in two forms: a closed conformation and an open conformation. Calmodulin reacts with both conformations but reacts faster and with higher affinity for the open conformation. Measurements of the ATPase activity of PMCA under similar conditions revealed that the open form has higher ATPase activity than the closed one. Contrasting with the reaction with the whole pump, TA-calmodulin reacted rapidly (in about 2 s) with a calmodulin-binding peptide made after the sequence of the calmodulin-binding domain of PMCA (C28). Results of TA-calmodulin binding to C28 are explained by a simpler model, in which only an open conformation exists.