Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.     

Intramolecular fluorescence resonance energy transfer between fused autofluorescent proteins reveals rearrangements of the N- and C-terminal segments of the plasma membrane Ca2+ pump involved in the activation

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca(2+) pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca(2+)-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45A. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca(2+)-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.     

The plasma membrane calcium pump displays memory of past calcium spikes. Differences between isoforms 2b and 4b

To understand how the plasma membrane Ca(2+) pump (PMCA) behaves under changing Ca(2+) concentrations, it is necessary to obtain information about the Ca(2+) dependence of the rate constants for calmodulin activation (k(act)) and for inactivation by calmodulin removal (k(inact)). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k(act) increased with Ca(2+) along a sigmoidal curve. At all Ca(2+) concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca(2+) concentration and also tested these simulations experimentally; PMCA was activated by 500 nm Ca(2+) and then exposed to 50 nm Ca(2+) for 10 to 150 s, and then Ca(2+) was increased again to 500 nm. During the second exposure to 500 nm Ca(2+), the activity reached steady state faster than during the first exposure at 500 nm Ca(2+). This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nm Ca(2+). The peptide inhibited the activity of PMCA2b when the exposure to 50 nm Ca(2+) was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca(2+). The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca(2+) more quickly in subsequent spikes after an initial activating spike.     

Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b

Tryptophan 1093 resides in the 28-residue calmodulin-binding/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 approximately 2-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 half-time 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 calmodulin-activated pump.     

Intramolecular interactions of the regulatory region with the catalytic core in the plasma membrane calcium pump

The access of three proteases to their sites of cleavage was used as a measure of regulatory interactions in the plasma membrane Ca2+ pump isoform 4b (PMCA4b). When the proteases could not cut at their sites in the C-terminal regulatory region, the interaction was judged to be tight. This was the case in the absence of Ca2+, when chymotrypsin and caspase cut PMCA only very slowly. Ca2+ accelerated the fragmentation, but the digestion remained incomplete. In the presence of Ca2+ plus calmodulin, the digestion became nearly complete in all cases, indicating a more flexible conformation of the carboxyl terminus in the fully activated state. The acceleration of proteolysis by Ca2+ or Ca2+ plus calmodulin occurred equally at the caspase site upstream of the calmodulin-binding domain and the chymotrypsin and calpain sites downstream of that domain. Replacing Trp1093 (a key residue within the calmodulin-binding domain) with alanine had a much more specific effect, because it exposed only proteolytic sites within the calmodulin-binding domain that had previously been shielded in the native protein. At these sites, both calpain and chymotrypsin cut the Trp1093 --> Ala mutant in the absence of calmodulin. These data indicate that, in the auto-inhibited conformation, the calmodulin-binding/auto-inhibitory sequence and the regions both upstream and downstream are in close contact with the catalytic core. Trp1093 plays an essential role not only in stabilizing the Ca2+-calmodulin/calmodulin-binding domain complex but also in the formation or stability of the inhibitory conformation of that domain when it interacts with the catalytic core of PMCA4b.     

Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A mutant with high affinity for Ca2+ resembling the acidic lipid-activated enzyme

The C-terminal segment of the loop between transmembrane helices 2 and 3 (A(L) region) of the plasma membrane Ca(2+) pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca(2+) pump isoform h4xb with deletions in the A(L) region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296-349 had full Ca(2+) transport activity, but deletions involving the segment of amino acids 350-356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V(max) of mutant d296-349 was similar to that of the recombinant wild type pump, but its K(0.5) for Ca(2+) was about 5-fold lower. The addition of calmodulin increased the V(max) and the apparent Ca(2+) affinity of both the wild type and d296-349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca(2+) and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296-349. Taken together these results suggest that (a) deletion of residues 296-349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the A(L) region; (b) the A(L) region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca(2+) affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the A(L) region is independent of the autoinhibition by the C-terminal calmodulin-binding region.     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

The plasma membrane Ca2+ pump contains a site that interacts with its calmodulin-binding domain.

A synthetic, 28-residue peptide derived from the calmodulin-binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the calmodulin-binding domain, has a molecular mass of 124 kDa and is fully active in the absence of calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the calmodulin-binding domain of the pump.     

Loss of autoinhibition of the plasma membrane Ca(2+) pump by substitution of aspartic 170 by asparagin. A ctivation of plasma membrane calcium ATPase 4 without disruption of the interaction between the catalytic core and the C-terminal regulatory domain

The plasma membrane calcium ATPase (PMCA) actively transports Ca(2+) from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca(2+)-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp(170), in the putative "A" domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca(2+). The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the C-terminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.     

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.     

Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4-6 associated calmodulins, is represented by the 130-kDa myosin I (or MI(130)) from rat liver. MI(130) translocates actin filaments in vitro in a Ca(2+)-regulated manner. A decrease in motility observed at higher Ca(2+) concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of rat myr-1; we refer to this truncated molecule here as MI(1IQ). Association of calmodulin to MI(1IQ) is Ca(2+)-insensitive. MI(1IQ) translocates actin filaments in vitro at a rate resembling MI(130), but unlike MI(130), does not exhibit sensitivity to 0.1-100 micrometer Ca(2+). In addition to demonstrating successful expression of a functional truncated mammalian myosin I in vitro, these results indicate that: 1) Ca(2+)-induced calmodulin dissociation from MI(130) in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca(2+) sensitivity of actin translocation exhibited by MI(130) involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.     

Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment.

Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the so-called transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca(2+)-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La(3+)-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 microM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.     

Calmodulin antagonizes amyloid-β peptides-mediated inhibition of brain plasma membrane Ca(2+)-ATPase

The synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in regulating intracellular Ca(2+) concentration in brain. We have recently found that PMCA is the only Ca(2+) pump in brain which is inhibited by amyloid-β peptide (Aβ), a neurotoxic peptide implicated in the pathology of Alzheimer's disease (AD) [1], but the mechanism of inhibition is lacking. In the present study we have characterized the inhibition of PMCA by Aβ. Results from kinetic assays indicate that Aβ aggregates are more potent inhibitors of PMCA activity than monomers. The inhibitory effect of Aβ could be blocked by pretreating the purified protein with Ca(2+)-calmodulin, the main endogenous activator of PMCA, and the activity of truncated PMCA lacking the calmodulin binding domain was not affected by Aβ. Dot-overlay experiments indicated a physical association of Aβ with PMCA and also with calmodulin. Thus, calmodulin could protect PMCA from inhibition by Aβ by burying exposed sites on PMCA, making them inaccessible to Aβ, and also by direct binding to the peptide. These results suggest a protective role of calmodulin against neuronal Ca(2+) dysregulation by PMCA inhibition induced by Aβ.     

A Ca2+-binding domain in RyR1 that interacts with the calmodulin binding site and modulates channel activity

A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.     

Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding

MI(1IQ) is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI(130)). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI(1IQ). Transient kinetics reveal that the affinity of MI(1IQ) for actin is 12 nm. The ATP-induced dissociation of actin-MI(1IQ) is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca(2+) sensitivity. The fast phase is eliminated by the addition of ADP, 10 micrometer being required for half-saturation of the effect in the presence of Ca(2+) and 3 micrometer ADP in the absence of Ca(2+). The slow phase shares the same rate constant as ADP release (8 and 3 s(-)(1) in the presence and absence of Ca(2+), respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI(130) and that the Ca(2+) sensitivity of the kinetics are in all likelihood due to Ca(2+) binding to the first IQ domain.     

Caloxin 1b3: a novel plasma membrane Ca(2+)-pump isoform 1 selective inhibitor that increases cytosolic Ca(2+) in endothelial cells

The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca(2+) pump(s) (PMCA) isoform 1. PMCA extrude Ca(2+) from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1-4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca(2+)-Mg(2+)-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant=17±2 μM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant=45±4 μM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca(2+) concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.     

Ganglioside GM2 modulates the erythrocyte Ca2+-ATPase through its binding to the calmodulin-binding domain and its 'receptor'

We have previously demonstrated that gangliosides were able to modulate the plasma membrane Ca2+-ATPase (PMCA) from porcine brain synaptosomes and porcine erythrocytes [Y. Zhao, X. Fan, F. Yang, X. Zhang, Arch. Biochem. Biophys. 427 (2004) 204-212 and J. Zhang, Y. Zhao, J. Duan, F. Yang, X. Zhang, Arch. Biochem. Biophys. 444 (2005) 1-6]. The results indicated that the PMCA from porcine erythrocytes responded to gangliosides was different from that from synaptosomes, suggesting that the effects of gangliosides on the PMCA are isoform specific. Most interestingly, GM2 activated the PMCA from porcine erythrocytes at lower concentrations, but inhibited it at higher concentrations. In the present study, we found that GD1b, GM1 and GM3 did not affect the calpain digested PMCA from porcine erythrocytes or the intact enzyme in the presence of calmodulin, while GM2 inhibited it. Moreover, a synthetic peptide of 17 amino acid residues corresponding to the 'receptor' of the calmodulin-binding domain of the enzyme interfered with the inhibition of the enzyme by GM2 in competition assays. Taken together, our results suggested that gangliosides GD1b, GM1, GM2 (lower concentrations) and GM3 stimulated the PMCA by the interaction with calmodulin-binding domain, while the interaction of GM2 with the 'receptor' of the calmodulin-binding domain of the enzyme led to the inhibition of the enzyme.