Plasma Membrane Calcium Pump Research Articles

Differential effects of G- and F-actin on the plasma membrane calcium pump activity.

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641-1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca(2+) extrusion through the membrane. Our results provide further evidence of the activation-inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-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.

On Ca2+ signalling research

As a highly versatile intracellular signal, calcium (Ca) regulates many different cellular processes in both animal and plant systems. Disruption of Ca homeostasis contributes to several human diseases. Owing to the importance of Ca signalling, its research is now an active field in life science. There are numerous Ca signalling systems, consisting of a diverse array of signalling units that deliver Ca signals with different spatial and temporal properties [1,2], playing roles in ubiquitous biological processes including gene regulation, fuel generation, substance transport, hormone and neurotransmitter secretion, cell motility and muscle contraction [3]. Consequently, exquisite homeostasis of Ca cycling is the key for health of humans, the disruption of which is related to many human diseases such as heart failure, neuron-degeneration, and diabetes [46]. Many remarkable achievements have greatly enhanced our understanding of Ca signaling, including those from Chinese scientists [710]. The 17th International Symposium on Ca-binding Proteins and Ca Function in Health and Disease was held in Beijing, China, on July 16–20, 2011 [11], accompany which, a special issue of Science China Life Sciences was published for transducing Ca signals to effectors. The first part focused on the mechanisms in maintaining a low cytosolic level of Ca, with two articles reviewing the properties of the plasma membrane calcium ATPases (PMCA) in ejecting Ca into the extracellular space. First, Carafoli [12] reviewed the role of the plasma membrane calcium pump, PMCA2, in the hearing process. As an important plasma membrane Ca ATPases, PMCA2 plays an important role in maintaining intracellular Ca homeostasis in the inner ear. It is involved in the maintenance of intracellular free calcium levels and the dysfunction of which leads to deafness. Recent studies show that another important isoform of PMCA, PMCA4, has a number of roles for heart functioning [13,14]. Cartwright et al. [15] then reviewed the roles of plasma membrane calcium pumps related to cardiovascular disease, focusing on PMCA4, including its function in heart and vasculature, linking PMCA4 to contractile function, cardiac hypertrophy, cardiac rhythm, blood pressure control and hypertension. Undoubtedly, further understanding of the roles of PMCAs will facilitate the development of new treatment strategies for related diseases. As a versatile intracellular messenger, Ca performs roles in many different ways to regulate cellular processes [16]. The second part of this special issue focuses on mechanisms for spatiotemporally-specific increases in cytosolic Ca. First, Lee [17] reviewed studies on the fraternal twin messengers for calcium signaling, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Since the initial finding by Lee and his colleagues that cADPR and NAADP could liberate stored Ca, they have been shown to be active in mobilizing Ca in a wide range of cells spanning three biological kingdoms. He summarized the cADPR/NAADP/CD38signalling pathway and also described the structure and function of its components. Kimlicka and van Petegem [18] then reviewed the structural biology aspect of ryanodine receptors (RyRs) which are high-conductance ion channels

A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes.

Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid–protein interaction

The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.

Inverse problems in neuronal calcium signaling

Calcium is the most important of the brain's second messengers. Thanks to engineered fluorescent indicators and caged compounds we have an excellent qualitative picture of its regulation and impact.With the advent of new scanning technology that permits one to observe the calcium signal throughout a highly branched neuron the potential exists for functional, single cell, quantitative calcium imaging. To help realize that potential we analyze a sequence of four inverse problems that infer the parameters of the cytosolic calcium buffers and plasma membrane calcium pumps and channels from the light shed by fluorescent indicators following specific stimulus protocols. Our analyses lead in each case to practical algorithms that we illustrate and test on synthetic data.

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2

Local Ca2+ signaling requires proper targeting of the Ca2+ signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca2+ pump (PMCA) are responsible for Ca2+ extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca2+ signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.

Abstract 9943: Prevention and Reversal of Hypertrophy Through a Novel Drug-Like Compound That Inhibits Local Calcium Oscillations in a Pmca4/calcineurin Microdomain

In pathological hypertrophy, the calcium/calmodulin dependent phosphatase calcineurin has been shown to transmit upstream calcium signals through dephosphorylation of the transcription factor NFAT which subsequently translocates to the nucleus and initiates a hypertrophic gene programme. We have previously shown that plasma membrane calcium ATPase isoform 4 (PMCA4) binds to calcineurin when overexpressed in HEK293 cells. We here investigated whether targeting PMCA4 by genetic or pharmacological strategies provides novel opportunities for the reduction of cardiac hypertrophy through modulation of calcineurin. Germline PMCA4 ablation resulted in the attenuation of pathological hypertrophy in response to pressure overload (Heart weight /tibia length ratio after transverse aortic constriction (TAC), PMCA4-/-: 6.74 ± 0.33 mg/mm vs WT: 8.34 ± 0.88 mg/mm, P10). Mechanistically, PMCA4 ablation led to a significant reduction in calcineurin at the sarcolemma as shown by Western blot and electron microscopy. We then developed a specific pharmacological inhibitor of PMCA4, which had not previously been available. Using a modified colorimetric ATPase assay we screened a library of medically optimized drug-like molecules and identified AP2 which has an IC50 of 150 nM for PMCA4. Injection of AP2 in mice (5 mg/kg body weight/day ip) significantly reduced cardiac hypertrophy following TAC for 2 weeks by 50% (n=10 each group, p<0.01). Moreover, AP2 reversed pre-established TAC-induced hypertrophy (HW/TL (mg/mm): sham, 5.7+0.3, TAC+vehicle, 9.1+0.7, TAC+AP2, 7.6+0.6, n=4-6 each group, P<0.05). Using a novel fluorescent Ca2+ sensor (GCaMP2) attached to the N-terminus of PMCA4 we found that AP2 reduced calcium oscillations in the microdomain around PMCA4 by 45% leading to reduced calcineurin activation. Furthermore, AP2 inhibited the NFAT pathway as shown by expression of the bona fide calcineurin target RCAN 1.4 and NFAT phosphorylation levels. Overall, our results demonstrated that specific inhibition of PMCA4 prevents and reverses pressure-overload hypertrophy, likely through regulation of local calcium oscillations around calcineurin making the plasma membrane calcium pump a potential target for the treatment of cardiac hypertrophy.

Plasma Membrane Calcium Pump (PMCA4)-Neuronal Nitric-oxide Synthase Complex Regulates Cardiac Contractility through Modulation of a Compartmentalized Cyclic Nucleotide Microdomain

Identification of the signaling pathways that regulate cyclic nucleotide microdomains is essential to our understanding of cardiac physiology and pathophysiology. Although there is growing evidence that the plasma membrane Ca(2+)/calmodulin-dependent ATPase 4 (PMCA4) is a regulator of neuronal nitric-oxide synthase, the physiological consequence of this regulation is unclear. We therefore tested the hypothesis that PMCA4 has a key structural role in tethering neuronal nitric-oxide synthase to a highly compartmentalized domain in the cardiac cell membrane. This structural role has functional consequences on cAMP and cGMP signaling in a PMCA4-governed microdomain, which ultimately regulates cardiac contractility. In vivo contractility and calcium amplitude were increased in PMCA4 knock-out animals (PMCA4(-/-)) with no change in diastolic relaxation or the rate of calcium decay, showing that PMCA4 has a function distinct from beat-to-beat calcium transport. Surprisingly, in PMCA4(-/-), over 36% of membrane-associated neuronal nitric-oxide synthase (nNOS) protein and activity was delocalized to the cytosol with no change in total nNOS protein, resulting in a significant decrease in microdomain cGMP, which in turn led to a significant elevation in local cAMP levels through a decrease in PDE2 activity (measured by FRET-based sensors). This resulted in increased L-type calcium channel activity and ryanodine receptor phosphorylation and hence increased contractility. In the heart, in addition to subsarcolemmal calcium transport, PMCA4 acts as a structural molecule that maintains the spatial and functional integrity of the nNOS signaling complex in a defined microdomain. This has profound consequences for the regulation of local cyclic nucleotide and hence cardiac β-adrenergic signaling.

Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells

Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca2+-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca2+-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca2+-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca2+ loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase Vmax values much higher than those ever measured by PMCA-mediated Ca2+ extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism.

High-dose glucocorticoids induce decreases calcium in hypothalamus neurons via plasma membrane Ca2+ pumps

In our previous studies, we occasionally found that high-dose glucocorticoids (GC) induced decrease in [Ca(2+)](i) in hypothalamus neurons. In previous articles, modulation of Ca(2+) channels by GC has been shown to contribute to the elementary regulation of several neuronal functions. However, little is known about the regulation of the Ca efflux pathways that counterbalance the Ca(2+) influx in neurons caused by high-dose GC. In this study, we demonstrate that a high-dose of GC (10 M dexamethasone) caused a 20% decrease in [Ca(2+)](i) within 2 s in cultured hypothalamic neurons; furthermore, we show that an antagonist of the GC receptor blocks this action. To ascertain the temporal sequence of relevant calcium transport mechanisms we selectively blocked the main calcium transporters, including sodium/calcium exchanger (NCX), plasma membrane calcium pumps (PMCA), and P-type Ca(2+)-ATPases of the sarcoplasmic reticulum (SERCA). The GC-induced [Ca(2+)](i) decrease disappeared completely when PMCA was blocked, but not when NCX and SERCA were blocked. These results suggest that high-dose GC (10(-6) M) rapidly decreases [Ca(2+)](i) by activating PMCA but not NCX or SERCA.

Gene expression pattern in PC12 cells with reduced PMCA2 or PMCA3 isoform: selective up-regulation of calmodulin and neuromodulin

Cellular calcium homeostasis is controlled predominantly by the plasma membrane calcium pump (PMCA). From four PMCA isoforms, PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are found in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in non-differentiated PC12 cells changed the cell morphology and triggered neuritogenesis. Using the microarrays, real-time PCR and immunodetection, we analyzed the effect of PMCA2 or PMCA3 reduction in PC12 cells on gene expression, with emphasis on calmodulin (CaM), neuromodulin (GAP43) and MAP kinases. In PMCA-suppressed lines total CaM increased, and the calm I and calm II genes appeared to be responsible for this effect. mRNA and protein levels of GAP43 were increased, however, the amount of phosphorylated form was lower than in control cells. Localization of CaM/GAP43 and CaM/pGAP43 differed between control and PMCA-reduced cells. In both PMCA-modified lines, amounts of ERK1/2 increased. While pERK1 decreased, the pERK2 level was similar in all examined lines. PMCA suppression did not change the p38 amount, but the p-p38 diminished. JNK2 protein decreased in both PMCA-reduced cells without changes in pJNK level. Microarray analysis revealed distinct expression patterns of certain genes involved in the regulation of cell cycle, proliferation, migration, differentiation, apoptosis and cell signaling. Suppression of neuron-specific PMCA isoforms affected the phenotype of PC12 cells enabling adaptation to the sustained increase in cytosolic Ca(2+) concentration. This is the first report showing function of PMCA2 and PMCA3 isoforms in the regulation of signaling pathways in PC12 cells.

The plasma membrane calcium pump in the hearing process: physiology and pathology

Mammalian cells express four different plasma membrane Ca(2+) ATPases. Two of them (PMCA1 and PMCA4) are expressed ubiquitously, and are considered housekeeping isoforms. Two (PMCA2 and PMCA4) have tissue restricted distribution. They are abundantly expressed in the brain and in nervous tissue-derived cell types. The primary transcripts of all PMCAs undergo alternative splicing, generating a large number of additional isoforms. Splicing occurs at site A, in the N-terminal moiety of the pump, and at site C, within the C-terminal calmodulin binding domain: The pumps are canonical targets of calmodulin stimulation. The site C insertion leads to a truncation of the pump about 50 residues short of the original C-terminal. One of the pumps (PMCA2) has special properties: It displays high activity even in the absence of the natural activator calmodulin, and has a particularly complex pattern of alternative splicing at both sites A and C. A variant of the PMCA2 pump containing an insert at site A and truncated C-terminally is the resident isoform of the pump in the stereocilia of hair cells of the inner ear. It exports Ca(2+) to the endolymph that bathes the stereocilia less efficiently than the full length, non-inserted PMCA2 pump. The proper functioning of hair cells demands the precise maintenance of the Ca(2+) balance between hair cells and the endolymph. Disturbances in the balance affect the process of mechano-electrical transduction, which depends on the ability of the stereociliar bundle to deflect in response to sound waves. The tip links that organize the bundle are formed by the Ca(2+) binding protein cadherin 23 and by protocadherin 15: Disturbances of the Ca(2+) binding by cadherin 23 and/or of the ability of the PMCA2 variant of the stereocilia to export Ca(2+) to the endolymph generate hearing loss phenotypes. Such phenotypes have now been described in mice and humans. In some cases they are linked to mutations of both cadherin 23 and the PMCA2 pump, but in other cases they may be generated by mutations of particular severity in only one of the two proteins. The PMCA2 defect that leads to deafness has now been analyzed molecularly: It affects the long range, unstimulated ability of PMCA2 to export Ca(2+).

Ca2+ signalling in cardiovascular disease: the role of the plasma membrane calcium pumps

The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca(2+) from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca(2+) signalling, depending on the cell type. In the cardiovascular system, Ca(2+) is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca(2+) homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.

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.

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 Ca2+ 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 Ca2+ 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. Keywords: Ca2+-ATPase, hydrophobic photo-labeling, lipid-protein interaction, membrane protein, Na+/K+-ATPase, PMCA, P-type ATPase, photo-labeling group TPD, 8-(5'-azido-O-hexanoylsalicylami-do)octanoic acid (AS86), Ca2+-ATPase activity, SERCA

Ion Channels, Action Potentials and Ca 2+ Handling in Human Pancreatic Beta-Cells. a Computational Approach

In this study, we examined the ionic mechanisms mediating depolarization-induced spike activity in human pancreatic beta-cells. We formulated a Hodgkin-Huxley-type ionic model for the action potential (AP) in these cells based on experimental voltage- and current-clamp results from our laboratory and literature. The model contains the equations for the currents: inward L-, P/Q and T-type Ca2+, a “rapid” delayed rectifier K+, the voltage-gated and Ca2+-activated K+ (BK-type), a voltage-independent Ca2+-activated K+ (SK-type), an ATP-sensitive K+, a plasma membrane calcium pump, a voltage-gated Na+ and a Na+ background. Ionic model is coupled to an equation describing intracellular Ca2+ homeostasis. The model simulates the behavior of human beta-cell APs under a wide range of experimental conditions, including changes in the period and amplitude of AP in response to glucose challenge and changes in islet electrical activity due to K+, Na+ and Ca+ channel blockade. The model was used to study the role of specific ionic currents in human pancreatic beta-cell firing. Particularly, modeling supports the importance of constitutively active tetrodotoxin-sensitive Na+ and voltage-gated Ca2+-activated K+ channels (BK-type) in maintaining spontaneous spikes. This model provides acceptable fits to voltage-clamp, action potential and Ca2+ concentration data and can be used to seek biophysically based explanations of the electrophysiological activity and Ca2+ influx in human beta-cells for in silico analysis of physiological conditions and channel modulator actions.

Modulation of the Activity of an Integral Membrane Protein by Phospholipids in Mixed Micelles

Although the importance of lipid-protein interactions in determining the biological function of integral membrane proteins is well recognized, the underlying molecular mechanisms remain unclear. In this work we explore the modulation of the enzymatic activity of an integral membrane protein by phospholipids, using the Plasma Membrane Calcium Pump (PMCA) reconstituted in mixed detergent C12E10-phospholipid micelles as model system. Increasing amounts of phospholipids were added to purified PMCA preparations reconstituted in detergent micelles producing a reversible increase in activity reaching a maximum value. No major structural changes occur during this process neither in the protein as assessed by far UV circular dichroism (CD) and tryptophan fluorescence nor in the micellar system as determined by fluorescence spectroscopy and fluorescence correlation spectroscopy (FCS). In addition the relative affinities of phospholipids for the PMCA transmembrane region were evaluated by a FRET method using a pyrene labelled PC as fluorescent probe. These results were analyzed in terms of a macroscopic model that includes the affinities of the phospholipids covering the PMCA transmembrane region and a transduction parameter that correlates the composition of the boundary monolayer with the enzyme activity. The model predictions show good agreement with the experimental data, linking amphiphile/protein interactions with enzymatic activity.

PMCA Differential Exposure of Hydrophobic Domains after Calmodulin and Phosphatidic Acid Activation

The exposure of plasma membrane calcium pump to surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable reagent [125I]TID-PC/16 into the membrane regions of this pump. In the absence of activators, Ca2+ increases the incorporation of [125I]TID-PC/16. On the contrary, in the presence of Ca2+ and either calmodulin or phosphatidic acid the incorporation of the labeled phospholipids is decreased. Proteolysis of PMCA with V8 protease results in 3 main fragments: fragment N (TM 1 and 2), fragment M (TM 3 and 4) and fragment C (TM 5 to 10). In the presence of Ca2+, CaM decreased the level of incorporation of [125I]TID-PC/16 to fragments M and C, while phosphatidic acid decreased the incorporation of [125I]TID-PC/16 to fragments N and M, suggesting that the conformational changes induced by calmodulin or phosphatidic acid extend to the transmembrane domains. The result also indicates differences between the active conformations produced by calmodulin and acidic phospholipids. To verify this, we also measured FRET between PMCA labeled with eosin isothiocianate at the ATP binding site and Rho-PE included in PMCA-containing micelles. CaM decreased the efficiency of the energy transfer between these two probes while PA did not. The result indicates that activation by CaM increases the distance between the ATP binding site and the membrane, but acidic phospholipids do not. Moreover, the access of two proteases to their sites of cleavage was different in the presence of calmodulin or phosphatidic acid. The results indicate structural differences between the PMCA conformations induced by these activators.

Changes in gravity rapidly alter the magnitude and direction of a cellular calcium current

In single-celled spores of the fern Ceratopteris richardii, gravity directs polarity of development and induces a directional, trans-cellular calcium (Ca(2+)) current. To clarify how gravity polarizes this electrophysiological process, we measured the kinetics of the cellular response to changes in the gravity vector, which we initially estimated using the self-referencing calcium microsensor. In order to generate more precise and detailed data, we developed a silicon microfabricated sensor array which facilitated a lab-on-a-chip approach to simultaneously measure calcium currents from multiple cells in real time. These experiments revealed that the direction of the gravity-dependent polar calcium current is reversed in less than 25 s when the cells are inverted, and that changes in the magnitude of the calcium current parallel rapidly changing g-forces during parabolic flight on the NASA C-9 aircraft. The data also revealed a hysteresis in the response of cells in the transition from 2g to micro-g in comparison to cells in the micro-g to 2-g transition, a result consistent with a role for mechanosensitive ion channels in the gravity response. The calcium current is suppressed by either nifedipine (calcium-channel blocker) or eosin yellow (plasma membrane calcium pump inhibitor). Nifedipine disrupts gravity-directed cell polarity, but not spore germination. These results indicate that gravity perception in single plant cells may be mediated by mechanosensitive calcium channels, an idea consistent with some previously proposed models of plant gravity perception.