Influence of pH on Ca²⁺ current and its control of electrical and Ca²⁺ signaling in ventricular myocytes

Modulation of L-type Ca(2+) current (I(Ca,L)) by H(+) ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of I(Ca,L) was investigated (whole cell voltage clamp) while measuring intracellular Ca(2+) (Ca(2+)(i)) or pH(i) (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pH(o) 6.5 and pH(i) 6.7) had opposite effects on I(Ca,L) gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pH(o), this decreased I(Ca,L), whereas at low pH(i), it increased I(Ca,L) at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca(2+)(i) was buffered with BAPTA, the stimulatory effect of low pH(i) was even more marked, with essentially no inhibition. We conclude that extracellular H(+) ions inhibit whereas intracellular H(+) ions can stimulate I(Ca,L). Low pH(i) and pH(o) effects on I(Ca,L) were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H(+) ion screening of fixed negative charge at the sarcolemma, with additional channel block by H(+)(o) and Ca(2+)(i). Action potential duration (APD) was also strongly H(+) sensitive, being shortened by low pH(o), but lengthened by low pH(i), caused mainly by H(+)-induced changes in late Ca(2+) entry through the L-type Ca(2+) channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca(2+) signaling. We conclude that the pH(i)-versus-pH(o) control of I(Ca,L) will exert a major influence on electrical and Ca(2+)-dependent signaling during acid-base disturbances in the heart.     

Ca2+-binding Motif of βγ-Crystallins

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca²⁺ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca²⁺-binding sites. βγ-Crystallins make a separate class of Ca²⁺-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca²⁺ binding to βγ-crystallins in mediating biological processes are yet to be elucidated.     

Molecular topography of the Ca2+ — ATPase of sarcoplasmic reticulum

Summary The Ca²⁺ binding site region of the Ca²⁺ — ATPase of skeletal muscle sarcoplasmic reticulum was labeled with several fluorescent analogs of dicyclohexylcarbodiimide. As has been shown by Chadwick and Thomas [1, 2], in the absence of Ca²⁺ in the medium, labeling with the naphthyl carbodiimide results in the inhibition of enzyme activity. Further, Ca²⁺ occupancy of the high affinity sites of the enzyme protects against incorporation into the site(s). The fluorescent carbodiimide has been used to determine the depth of the site of label incorporation relative to the aqueous-bilayer interfaces by quenching studies using spin-labeled fatty acid derivatives. The series of quenchers used have their spin-label moiety located at different positions along the fatty acid chain. It was found that after suitable correction for differences in partitioning of the various derivatives, the order of quenching efficiency was 16 - > 12- > 10- > 7- > 5-NS, indicating that the naphthyl moiety is near the center of the bilayer. In contrast, quenching with the aqueous-restricted I^– indicated that the label is accessible from the external milieu, likewise for a presumed aqueous quencher, acrylamide. The aqueous quenchers accessibilities were altered upon Ca²⁺ binding to the ATPase. Quenching of the intrinsic fluorescence with the x-NS derivatives indicates that the ATPase tryptophan residues are primarily localized at the aqueous-membrane interfaces, with the order of quenching being 5- > 7- > 10- > 12- > 16-NS. The trp residue(s) which changes its fluorescence upon Ca²⁺ binding is shown to be near the membrane surface.     

New mechanisms of pulmonary arterial hypertension: role of Ca²⁺ signaling

Pulmonary arterial hypertension (PAH) is a severe and progressive disease that usually culminates in right heart failure and death if left untreated. Although there have been substantial improvements in our understanding and significant advances in the management of this disease, there is a grim prognosis for patients in the advanced stages of PAH. A major cause of PAH is increased pulmonary vascular resistance, which results from sustained vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness. In addition to other signal transduction pathways, Ca(2+) signaling in pulmonary artery smooth muscle cells (PASMCs) plays a central role in the development and progression of PAH because of its involvement in both vasoconstriction, through its pivotal effect of PASMC contraction, and vascular remodeling, through its stimulatory effect on PASMC proliferation. Altered expression, function, and regulation of ion channels and transporters in PASMCs contribute to an increased cytosolic Ca(2+) concentration and enhanced Ca(2+) signaling in patients with PAH. This review will focus on the potential pathogenic role of Ca(2+) mobilization, regulation, and signaling in the development and progression of PAH.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Is Ca2+ antagonists binding protein from cytosolic fraction the precursor of alpha 1-subunit of Ca2+ channel?

The binding of Ca2+ antagonists to soluble proteins obtained by ammonium sulphate precipitation from cytosol fraction of rabbit skeletal muscles was studied. The KD values for 3H D-888 and 3H PN 200-110 binding to soluble proteins were 21.3 +/- 3.1 nmol.l-1 and 28.8 +/- 8.9 nmol.l-1 respectively. Photoaffinity labelling of the soluble proteins with the arylazide 1,4-dihydropyridine probe 3H azidopine resulted in labelling of the 85-95 K protein band as determined by SDS polyacrylamide gel electrophoresis. Partial purification of prelabelled soluble sample by gel filtration on Sephadex G-150 gave a more precise molecular weight of 90 +/- 2.5K. Polyclonal antibodies prepared against Ca2+ channel complex from rabbit muscle T-tubules inhibited the 3H PN 200-110 binding. Our results suggest that the soluble protein with Mr = 90K +/- 2.5K may be a precursor of the large subunit of the membrane bound L-type Ca2+ channel in rabbit skeletal muscle.     

Transsynaptic control of presynaptic Ca²⁺ influx achieves homeostatic potentiation of neurotransmitter release

Given the complexity of the nervous system and its capacity for change, it is remarkable that robust, reproducible neural function and animal behavior can be achieved. It is now apparent that homeostatic signaling systems have evolved to stabilize neural function. At the neuromuscular junction (NMJ) of organisms ranging from Drosophila to human, inhibition of postsynaptic neurotransmitter receptor function causes a homeostatic increase in presynaptic release that precisely restores postsynaptic excitation. Here we address what occurs within the presynaptic terminal to achieve homeostatic potentiation of release at the Drosophila NMJ. By imaging presynaptic Ca(2+) transients evoked by single action potentials, we reveal a retrograde, transsynaptic modulation of presynaptic Ca(2+) influx that is sufficient to account for the rapid induction and sustained expression of the homeostatic change in vesicle release. We show that the homeostatic increase in Ca(2+) influx and release is blocked by a point mutation in the presynaptic CaV2.1 channel, demonstrating that the modulation of presynaptic Ca(2+) influx through this channel is causally required for homeostatic potentiation of release. Together with additional analyses, we establish that retrograde, transsynaptic modulation of presynaptic Ca(2+) influx through CaV2.1 channels is a key factor underlying the homeostatic regulation of neurotransmitter release.     

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney α-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification

Summary NADH inhibition of bovine kidney -ketoglutarate dehydrogenase complex was compared at 10 m free Ca²⁺ or in the absence of Ca²⁺ (i.e., < 1.0 nM free Ca²⁺). In the presence of Ca^2–, NADH inhibition was appreciably decreased for a wide range of NADH : NAD⁺ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 m free Ca²⁺ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca²⁺ on the S_0.5 for -ketoglutarate which was decreased by Ca²⁺ with a half-maximal effect at a similar concentration. The effect of Ca²⁺ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the -ketoglutarate dehydrogenase complex is required to elicit the effect of Ca²⁺ on NADH inhibition.At a fixed -ketoglutarate concentration (50 m), removal of Ca² reduced the activity of the -ketoglutarate dehydrogenase complex by 8,5-fold (due to an increase in S_0.5 for -ketoglutarate) and, in the presence of different NADH : NAD⁺ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxP_i) or a combination of the phosphate potential and NADH :NAD⁺ ratio are also described. The possibility that the level of intramitochondrial free Ca²⁺ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.     

Automated region of interest analysis of dynamic Ca²⁺ signals in image sequences

Ca(2+) signals are commonly measured using fluorescent Ca(2+) indicators and microscopy techniques, but manual analysis of Ca(2+) measurements is time consuming and subject to bias. Automated region of interest (ROI) detection algorithms have been employed for identification of Ca(2+) signals in one-dimensional line scan images, but currently there is no process to integrate acquisition and analysis of ROIs within two-dimensional time lapse image sequences. Therefore we devised a novel algorithm for rapid ROI identification and measurement based on the analysis of best-fit ellipses assigned to signals within noise-filtered image sequences. This algorithm was implemented as a plugin for ImageJ software (National Institutes of Health, Bethesda, MD). We evaluated the ability of our algorithm to detect synthetic Gaussian signal pulses embedded in background noise. The algorithm placed ROIs very near to the center of a range of signal pulses, resulting in mean signal amplitude measurements of 99.06 ± 4.11% of true amplitude values. As a practical application, we evaluated both agonist-induced Ca(2+) responses in cultured endothelial cell monolayers, and subtle basal endothelial Ca(2+) dynamics in opened artery preparations. Our algorithm enabled comprehensive measurement of individual and localized cellular responses within cultured cell monolayers. It also accurately identified characteristic Ca(2+) transients, or Ca(2+) pulsars, within the endothelium of intact mouse mesenteric arteries and revealed the distribution of this basal Ca(2+) signal modality to be non-Gaussian with respect to amplitude, duration, and spatial spread. We propose that large-scale statistical evaluations made possible by our algorithm will lead to a more efficient and complete characterization of physiologic Ca(2+)-dependent signaling.     

Synchronization of stochastic Ca²(+) release units creates a rhythmic Ca²(+) clock in cardiac pacemaker cells

In sinoatrial node cells of the heart, beating rate is controlled, in part, by local Ca2(+) releases (LCRs) from the sarcoplasmic reticulum, which couple to the action potential via electrogenic Na(+)/Ca2(+) exchange. We observed persisting, roughly periodic LCRs in depolarized rabbit sinoatrial node cells (SANCs). The features of these LCRs were reproduced by a numerical model consisting of a two-dimensional array of stochastic, diffusively coupled Ca2(+) release units (CRUs) with fixed refractory period. Because previous experimental studies showed that β-adrenergic receptor stimulation increases the rate of Ca2(+) release through each CRU (dubbed I(spark)), we explored the link between LCRs and I(spark) in our model. Increasing the CRU release current I(spark) facilitated Ca2(+)-induced-Ca2(+) release and local recruitment of neighboring CRUs to fire more synchronously. This resulted in a progression in simulated LCR size (from sparks to wavelets to global waves), LCR rhythmicity, and decrease of LCR period that parallels the changes observed experimentally with β-adrenergic receptor stimulation. The transition in LCR characteristics was steeply nonlinear over a narrow range of I(spark), resembling a phase transition. We conclude that the (partial) periodicity and rate regulation of the "Calcium clock" in SANCs are emergent properties of the diffusive coupling of an ensemble of interacting stochastic CRUs. The variation in LCR period and size with I(spark) is sufficient to account for β-adrenergic regulation of SANC beating rate.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Effects of amyloid-β peptides on voltage-gated L-type Ca(V)1.2 and Ca(V)1.3 Ca(2+) channels

Overload of intracellular Ca²⁺ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities in intracellular Ca²⁺ homeostasis systems. L-type Ca²⁺ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β (Aβ) peptides. However, most studies of L-type Ca²⁺ channels in Aβ-related mechanisms have been limited to Ca_V1.2, and surprisingly little is known about the involvement of Ca_V1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of Ca_V1.3 after Aβ_25–35 exposure for 24 h and compared them with the expression patterns of Ca_V1.2. The expression levels of Ca_V1.3 were not significantly changed by Aβ_25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of Ca_V1.3 were significantly increased by Aβ_25–35, but not by Aβ_35–25. We next found that acute treatment with Aβ_25–35 increased Ca_V1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β₃ subunits of Ca²⁺ channels instead of Ca_V1.2 or Ca_V1.3 α₁ subunits. These results show that Aβ_25–35 chronically or acutely upregulates Ca_V1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that Ca_V1.3 has a potential role along with Ca_V1.2 in the pathogenesis of Alzheimer’s disease.     

Are Ca2+ channels targets of praziquantel action?

Praziquantel is the current drug of choice for the control of schistosomiasis. It is highly effective against all species of schistosomes and shows minimal adverse effects. Though introduced for the treatment of schistosomiasis more than 20 years ago, the mode of action of praziquantel remains to be elucidated. This review will focus on advances in defining the molecular target of praziquantel action, with particular emphasis on recent work indicating an important role for voltage-gated calcium channels.     

Stimulation of the extracellular Ca²⁺-sensing receptor by denatonium

The extracellular Ca(2+)-sensing receptor (CASR) is a promiscuous G-protein-coupled receptor closely related to the taste receptors T1R1-T1R3. Here we analyzed the possibility that apart from being stimulated by external Ca(2+) and amino acids, the substances effective as tastants, CASR might serve as a receptor for other sapid compounds. CASR was heterologously expressed in HEK-293 cells, and their responsivity to a variety of bitter and sweet substances was examined. Among them, solely denatonium was found to stimulate Ca(2+) signaling in CASR-positive HEK-293 cells. Apparently, these Ca(2+) responses were specific, as those were inhibited by the CASR antagonist NSP-4123. Altogether, our findings indicate that denatonium stimulates CASR by shifting a dose-response curve for the principal CASR agonist Ca(2+) to lower concentrations.     

Prevalence of Ca²⁺-ATPase-mediated carbonate dissolution among cyanobacterial euendoliths

Recent physiological work has shown that the filamentous euendolithic cyanobacterium Mastigocoleus testarum (strain BC008) is able to bore into solid carbonates using Ca²⁺-ATPases to take up Ca²⁺ from the medium at the excavation front, promoting dissolution of CaCO₃ there. It is not known, however, if this is a widespread mechanism or, rather, a unique capability of this model strain. To test this, we undertook a survey of multispecies euendolithic microbial assemblages infesting natural carbonate substrates in marine coastal waters of the Caribbean, Mediterranean, South Pacific, and Sea of Cortez. Microscopic examination revealed the presence of complex assemblages of euendoliths, encompassing 3 out of the 5 major cyanobacterial orders. 16S rRNA gene clone libraries detected even greater diversity, particularly among the thin-filamentous forms, and allowed us to categorize the endoliths in our samples into 8 distinct phylogenetic clades. Using real-time Ca²⁺ imaging under a confocal laser scanning microscope, we could show that all communities displayed light-dependent formation of Ca²⁺-supersaturated zones in and around boreholes, a staple of actively boring phototrophs. In 3 out of 4 samples, boring activity was sensitive to at least one of two inhibitors of Ca²⁺-ATPase transporters (thapsigargin or tert-butylhydroquinone), indicating that the Ca²⁺-ATPase mechanism is widespread among cyanobacterial euendoliths but perhaps not universal. Function-community structure correlations point to one particular clade of baeocyte-forming euendoliths as the potential exception.     

The effect of OPA1 on mitochondrial Ca²⁺ signaling

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca²⁺ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca²⁺ to the transporters in the crista membrane and thus would enhance Ca²⁺ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca²⁺ signals evoked with K⁺ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca²⁺] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca²⁺ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca²⁺ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na⁺/Ca²⁺ and Ca²⁺/H⁺ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca²⁺ metabolism.     

Revisiting the ionic mechanisms of early afterdepolarizations in cardiomyocytes: predominant by Ca waves or Ca currents?

Early afterdepolarizations (EADs) have been implicated in severe cardiac arrhythmias and sudden cardiac deaths. However, the mechanism(s) for EAD genesis, especially regarding the relative contribution of Ca(2+) wave (CaW) vs. L-type Ca current (I(Ca,L)), still remains controversial. In the present study, we simultaneously recorded action potentials (APs) and intracellular Ca(2+) images in isolated rabbit ventricular myocytes and systematically compared the properties of EADs in the following two pharmacological models: 1) hydrogen peroxide (H(2)O(2); 200 μM); and 2) isoproterenol (100 nM) and BayK 8644 (50 nM) (Iso + BayK). We assessed the rate dependency of EADs, the temporal relationship between EADs and corresponding CaWs, the distribution of EADs over voltage, and the effects of blockers of I(Ca,L), Na/Ca exchangers, and ryanodine receptors. The most convincing evidence came from the AP-clamp experiment, in which the cell membrane clamp was switched from current clamp to voltage clamp using a normal AP waveform without EAD; CaWs disappeared in the H(2)O(2) model, but persisted in the Iso + BayK model. We postulate that, although CaWs and reactivation of I(Ca,L) may act synergistically in either case, reactivation of I(Ca,L) plays a predominant role in EAD genesis under oxidative stress (H(2)O(2) model), while spontaneous CaWs are a predominant cause for EADs under Ca(2+) overload condition (Iso + BayK model).     

Quantitative measurement of Ca²(+) in the sarcoplasmic reticulum lumen of mammalian skeletal muscle

Skeletal muscle stores Ca²⁺ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²⁺ in the SR ([Ca²⁺]_SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²⁺]_SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²⁺]_{SR, free} is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²⁺]_{SR, free} to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²⁺ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²⁺ and for future studies of the role of SR Ca²⁺ in healthy and diseased mammalian muscle.     

Impairment of the activity of the plasma membrane Ca²⁺-ATPase in Alzheimer's disease

AD (Alzheimer's disease) is an age-associated neurodegenerative disorder where the accumulation of neurotoxic Aβ (amyloid β-peptide) in senile plaques is a typical feature. Recent studies point out a relationship between Aβ neurotoxicity and Ca2+ dyshomoeostasis, but the molecular mechanisms involved are still under discussion. The PMCAs (plasma membrane Ca2+-ATPases) are a multi-isoform family of proteins highly expressed in brain that is implicated in the maintenance of low intraneural Ca2+ concentration. Therefore the malfunction of this pump may also be responsible for Ca2+ homoeostasis failure in AD. We have found that the Ca2+-dependence of PMCA activity is affected in human brains diagnosed with AD, being related to the enrichment of Aβ. The peptide produces an inhibitory effect on the activity of PMCA which is isoform-specific, with the greatest inhibition of PMCA4. Besides, cholesterol blocked the inhibitory effect of Aβ, which is consistent with the lack of any Aβ effect on PMCA4 found in cholesterol-enriched lipid rafts isolated from pig brain. These observations suggest that PMCAs are a functional component of the machinery that leads to Ca2+ dysregulation in AD and propose cholesterol enrichment in rafts as a protector of the Aβ-mediated inhibition on PMCA.