Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15–21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni²⁺ was found to be the strongest stimulator, while Zn²⁺ was found to inhibit CaN phosphatase activity. Mn²⁺ was a relatively less effective stimulator compared to Ni²⁺. Fe²⁺ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe²⁺ slightly inhibited CaN activity from bovine brain. The residues at 97–201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407–456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457–480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.     

Effects of lanthanum and calcium on photoelectron transport activity and the related protein complexes in chloroplast of cucumber leaves

The effects of lanthanum and calcium ions on electron transport, dichlorephenol indophenol (DCIP) photoreduction, and oxygen evolution activities in chloroplast from cucumber (Cucumis satives L.) were determined. The lanthanum inhibited the whole electron chain-transport activity of chloroplast. DCIP photoreduction and oxygen evolution activities of the photosystem I (PSII) also decrease after treatment with La³⁺. But the diminished activities of PSII and chloroplast caused by La³⁺ could be reversed by Ca²⁺ and even became higher than the control level. The concentration analysis of related protein complexes to photoelectron transport in chloroplast included that La³⁺ induced the concentration of chlorophyll protein complexes increasing but caused some nonchlorophyll protein complexes to decompose partially. This increasing effect of La³⁺ on chlorophyll protein complexes results in the improvement of chlorophyll content, which will improve the absorption of photoelectron and energy transport in the process of photosynthesis.     

Lanthanum Induces Extracellular Signal-regulated Kinase Phosphorylation through Different Mechanisms in HeLa Cells and NIH 3T3 Cells

Lanthanum ion (La³⁺) was generally regarded as calcium antagonist and was used as calcium channel blocker. However, its potential biological effects on cells were poorly understood. In the present work, it was found that La³⁺ could induce rapid extracellular signal-regulated kinase (ERK) phosphorylation in both HeLa cells and NIH 3T3 cells, but different mechanisms were involved. At a concentration of 30μM or higher, La³⁺ enters the cells and activates ERK through a mechanism involving calmodulin activation inside the cells, which is similar to the action of intracellular Ca²⁺. However, at lower concentration, free La³⁺ promoted ERK phosphorylation in NIH 3T3 cells outside the cells through an unknown La³⁺ sensing mechanism, while Ca²⁺ exerted much weaker effect. The present results suggested that the biological effects of La³⁺ on cells maybe involve mechanisms beyond calcium antagonist.     

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K⁺ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca²⁺/calmodulin (CaM)–dependent phosphatase, in modulating K⁺ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca²⁺ concentration ([Ca²⁺] _i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca²⁺] _i , we speculated that inhibiting CaN enhances Ca²⁺-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca²⁺/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6 μM) and CaMKII (0.15 U/ml) to the bath at 10^?6 M Ca²⁺ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K⁺ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.     

Computational design of calmodulin mutants with up to 900-fold increase in binding specificity

Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca²⁺ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca²⁺/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca²⁺ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

Evidence of metal binding activities of pentadecapeptide from Panax ginseng

A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg²⁺. Mobility was also affected in the presence of La³⁺, Mn²⁺, Ca²⁺ and Zn²⁺ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca²⁺-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg²⁺ and Mn²⁺ ions.     

Metal cations as synaptosomal calcium blockers in studies with Fura-2

Metal ions are often used to block calcium channels in various tissues, including synaptosomes. In the present study, Fura-2 was used to determine the effectiveness of various metal ions as calcium channel blockers in rat brain synaptosomes in vitro. In buffer solutions, La³⁺ and Cd²⁺ increased the Fura-2 fluorescence in a manner similar to Ca²⁺. Ni²⁺ and Mn²⁺ appeared to be fluorescence quenching cations, and Sr²⁺ and Co²⁺ had little effect on the fluorescence of Fura-2. In suspensions of synaptosomes under resting conditions, Cd²⁺, Ni²⁺ and Mn²⁺ were found to be not suitable for use in synaptosome studies. On the other hand, La³⁺ and Co²⁺ had little effect on the Fura-2 fluorescence of resting synaptosomes, and under depolarizing conditions, La³⁺ and Co²⁺ decreased the Fura-2 fluorescence. These resuls, therefore, suggest that La³⁺ and Co²⁺ may be suitable as calcium channel blockers in synaptosome studies.     

Calmodulin mediates differential sensitivity of CaMKII and calcineurin to local Ca2+ in cardiac myocytes

Calmodulin (CaM) mediates Ca-dependent regulation of numerous pathways in the heart, including CaM-dependent kinase (CaMKII) and calcineurin (CaN), yet the local Ca²⁺ signals responsible for their selective activation are unclear. To assess when and where CaM, CaMKII, and CaN may be activated in the cardiac myocyte, we integrated new mechanistic computational models of CaM, CaMKII, and CaN with the Shannon-Bers model of excitation-contraction coupling in the rabbit ventricular myocyte. These models are validated with independent in vitro data. In the intact myocyte, model simulations predict that CaM is highly activated in the dyadic cleft during each beat, but not appreciably in the cytosol. CaMKII-δ_C was almost insensitive to cytosolic Ca due to relatively low CaM affinity. Dyadic cleft CaMKII exhibits dynamic frequency-dependent responses to Ca, yet autophosphorylates only when local phosphatases are suppressed. In contrast, dyadic cleft CaN in beating myocytes is predicted to be constitutively active, whereas the extremely high affinity of CaN for CaM allows gradual integration of small cytosolic CaM signals. Reversing CaM affinities for CaMKII and CaN also reverses their characteristic local responses. Deactivation of both CaMKII and CaN seems dominated by Ca dissociation from the complex (versus Ca-CaM dissociation from the target). In summary, the different affinities of CaM for CaMKII and CaN determine their sensitivity to local Ca signals in cardiac myocytes.     

Calcium Site Specificity : Early Ca2+-related Tight Junction Events

The molecular mechanisms by which Ca²⁺ and metal ions interact with the binding sites that modulate the tight junctions (TJs) have not been fully described. Metal ions were used as probes of these sites in the frog urinary bladder. Basolateral Ca²⁺ withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca²⁺ is readmitted, and is followed by a complete recovery of the TJ seal. Mg²⁺ and Ba²⁺ were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg²⁺ causes a reversible concentration-dependent inhibition of the Ca²⁺-induced TJ recovery. The effects of extracellular Ca²⁺ manipulation on the TJs apparently is not mediated by changes of cytosolic Ca²⁺ concentration. The transition elements, Mn²⁺ and Cd²⁺, act as Ca²⁺ agonists. In the absence of Ca²⁺, they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca²⁺ withdrawal. In addition, Mn²⁺ promotes an almost complete recovery of the TJ seal. Cd²⁺, in spite of stabilizing the TJs in the closed state and halting TJ opening, does not promote TJ recovery, an effect that apparently results from a superimposed toxic effect that is markedly attenuated by the presence of Ca²⁺. The interruption of TJ opening caused by Ca²⁺, Cd²⁺, or Mn²⁺, and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La³⁺ (2 μM) to the basolateral Ca²⁺-containing solution causes an increase of TJ permeability that fully reverses when La³⁺ is removed. This effect of La³⁺, observed in the presence of Ca²⁺ (1 mM), indicates a high La³⁺ affinity for the Ca²⁺-binding sites. This ability of La³⁺ to open TJs in the presence of Ca²⁺ is a relevant aspect that must be considered when using La³⁺ in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives.     

Calmodulin modulates insect odorant receptor function

Insect odorant receptors (ORs) are heteromeric complexes of an odor-specific receptor protein (OrX) and a ubiquitous co-receptor protein (Orco). The ORs operate as non-selective cation channels, also conducting Ca²⁺ ions. The Orco protein contains a conserved putative calmodulin (CaM)-binding motif indicating a role of CaM in its function. Using Ca²⁺ imaging to monitor OR activity we investigated the effect of CaM inhibition on the function of OR proteins. Ca²⁺ responses elicited in Drosophila olfactory sensory neurons by stimulation with the synthetic OR agonist VUAA1 were reduced and prolonged by CaM inhibition with the potent antagonist W7 but not with the weak antagonist W5. A similar effect was observed for Orco proteins heterologously expressed in CHO cells when CaM was inhibited with W7, trifluoperazine or chlorpromazine, or upon overexpression of CaM-EF-hand mutants. With the Orco CaM mutant bearing a point mutation in the putative CaM site (K339N) the Ca²⁺ responses were akin to those obtained for wild type Orco in the presence of W7. There was no uniform effect of W7 on Ca²⁺ responses in CHO cells expressing complete ORs (Or22a/Orco, Or47a/Orco, Or33a/Orco, Or56a/Orco). For Or33a and Or47a we observed no significant effect of W7, while it caused a reduced response in cells expressing Or22a and a shortened response for Or56a.     

Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution

Ca²⁺ activates SK Ca²⁺-activated K⁺ channels through the protein Ca²⁺ sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca²⁺ regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca²⁺ concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca²⁺, SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca²⁺, 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca²⁺, the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca²⁺ and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca²⁺ or with CaM in molar excess. In low Ca²⁺ both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca²⁺. These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.     

Addition and reaction products of triethyl thiophosphate and metal perchlorates at just over ambient and elevated temperatures

Triethyl thiophosphate (tetp) invariably forms adducts with various metal perchlorates (M=Mg²⁺, Al³⁺, Cr³⁺, Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺) at 35–40°C in ethanol-triethyl orthoformate (teof). Only certain of these adducts, which involve S-bonded tetp in the thione form for soft or borderline metal ions and O-bonded tetp in the thiol tautomeric form for hard metal ions, could be isolated in solid form, owing to their tendency to decompose yielding diethylthiophosphato (detp) metal complexes and ethyl perchlorate, at temperatures ranging between ambient and 80–90°C, depending on the metal ion. Several well-defined detp and detpperchlorato metal complexes were obtained by heating solutions of mixtures of tetp and metal perchlorates in ethanol-teof at 80–90°C, and characterized. In most cases, linear polymeric or dimeric complexes involving double or triple bridges of O,S-bonded bidentate detp between adjacent metal ions were isolated. However, in a number of occasions, heavily hydrated monomeric species, containing terminal S-bonded detp were obtained.     

Study on the toxic mechanism of La3+ to Escherichia coli

The toxic mechanism of La³⁺ to Escherichia coli is investigated by detecting the concentration change of La³⁺ in E. coli cells growing in La³⁺-containing medium. Stimulatory action and inhibitory effect of La³⁺ in different concentrations can be attributed to the permeability alteration of the cell. Low concentration of La³⁺ increases the nutrition absorbability of the cells from the cultures as a result of increased cell permeability, and high concentration of La³⁺ causes the accumulation of La³⁺ in cells, resulting in the toxic effects on the E. coli cells.     

Both N- and C-lobes of calmodulin are required for Ca-dependent regulations of CaV1.2 Ca channels

We investigated the concentration- and Ca²⁺-dependent effects of CaM mutants, CaM₁₂ and CaM₃₄, in which Ca²⁺-binding to its N- and C-lobes was eliminated, respectively, on the Ca_V1.2 Ca²⁺ channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM₁₂ and CaM₃₄ (0.7-10 μM) applied with 3 mM ATP produced channel activity after “rundown”. Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca²⁺], suggesting that both functional lobes of CaM were necessary for the Ca²⁺-dependent shift. However, channel activity induced by the CaM mutants showed Ca²⁺-dependent decrease, implying a Ca²⁺ sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca²⁺-dependent regulations of Ca_V1.2 Ca²⁺ channels.     

钙调神经磷酸酶B亚基及钙调素的EPR研究

根据顺磁离子Mn~(2+)的取代特性,用EPR方法研究了钙调神经磷酸酶B亚基与其4个Ca~(2+)的结合位点,以及它们亲和力的细微差别。并同时进行了钙调素的对比研究。实验和Scatchard作图表明,B亚基有4个Ca~(2+)结合位点,2个高亲和力结合位点,其解离常数为4×10~(-6)mol/L;2个低亲和力结合位点,解离常数为9×10~(-5)mol/L。钙调素也有2个Ca~(2+)高亲和力结合位点,其解离常数为8×10~(-6)mol/L,2个低亲和力结合位点,解离常数为7×10~(-5)mol/L。钙调神经磷酸酶B亚基和钙调素Mn~(2+)结合位点的EPR研究对B亚基和钙调素在共同调节钙调神经磷酸酶中的作用提供了有用的信息。     

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro

Since its initial discovery as Ca²⁺/calmodulin (CaM)-dependent serine/threonine protein phosphatase, calcineurin (CaN) has been extensively studied in many mammalian tissues. CaN has been shown to be involved in various biological and Ca²⁺-dependent signal transduction pathways. Over the last decade, our laboratory has been interested and has carried out numerous experiments on this specific protein phosphatase. While, a lot of research has been performed studying CaN’s involvement in ischemia, the immune system, and various mammalian tissues, not much is known about the potential role of CaN in various eye diseases. This review focuses on the studies that have been carried out in our laboratory on CaN, and specifically CaN’s involvement in the eye. We demonstrated that CaN is localized in various eye tissues (cornea, iris, ciliary body, vitreous body, retina, choroid, sclera, and optic nerve) and that both its protein expression and activity were observed in high amounts in the retina, optic nerve and cornea. Recently, we have cloned and characterized the CaN A and B subunits in the bovine retina. These initial findings suggest that CaN may play a potential role in visual transduction and various ocular diseases, including cancer.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

La<Superscript>3+</Superscript> uptake and its effect on the cytoskeleton in root protoplasts of<Emphasis Type="Italic"> Zea mays</Emphasis> L.

La³⁺ ions are known to antagonize Ca²⁺ and are used as a Ca²⁺ channel blocker but little is known on the direct effects of La³⁺. Micromolar La³⁺ concentrations promoted root growth while higher concentrations were inhibitory. The uptake of La³⁺ in maize root protoplasts revealed a membrane binding component (0.14 and 0.44 pmol min^–1 protoplast^–1 for 100 and 1,000 M La³⁺) followed by a slower concentration and time-dependent uptake. Uptake was reduced by Ca²⁺, but had no substantial effect on other ions. La³⁺ shifted microtubule organization from random to parallel but caused aggregation of microfilaments. Our data suggest that La³⁺ is taken up into plant cells and affects growth via stabilization of the cytoskeleton.     

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca²⁺-pump (PMCA) in the membrane region upon interaction with Ca²⁺, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [¹²⁵I]TID-PC/16, measuring the shift of conformation E₂ to the auto-inhibited conformation E₁I and to the activated E₁A state, titrating the effect of Ca²⁺ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca²⁺ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca²⁺ transport but does not modify the affinity for Ca²⁺ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E₁I to the activated E₁A conformation and thus modulating the transport of Ca²⁺. This is reflected in the different apparent constants for Ca²⁺ in the absence of CaM (calculated by Ca²⁺-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca²⁺ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca²⁺ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca²⁺ binding and activation.