Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis.Fil: Dalghi, Marianela Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Fernández, Marisa Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Malchiodi, Emilio Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Strehler, Emanuel E.. Mayo Clinic College of Medicine; Estados UnidosFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

“Fast” plasma membrane calcium pump PMCA2a concentrates in GABAergic terminals in the adult rat brain

The plasma membrane Ca2+-ATPases (PMCA) represent the major high-affinity Ca2+ extrusion system in the brain. PMCAs comprise four isoforms and over 20 splice variants. Their different functional properties may permit different PMCA splice variants to accommodate different kinds of local [Ca2+] transients, but for a specific PMCA to play a unique role in local Ca2+ handling it must be targeted to the appropriate subcellular compartment. We used immunohistochemistry to study the spatial distribution of PMCA2a–one of the two major carboxyl-terminal splice variants of PMCA2–in the adult rat brain, testing whether this isoform, with especially high basal activity, is targeted to specific subcellular compartments. In striking contrast to the widespread distribution of PMCA2 as a whole, we found that PMCA2a is largely restricted to parvalbumin-positive inhibitory presynaptic terminals throughout the brain. The only major exception to this targeting pattern was in the cerebellar cortex, where PMCA2a also concentrates postsynaptically, in the spines of Purkinje cells. We propose that the fast Ca2+ activation kinetics and high Vmax of PMCA2a make this pump especially suited for rapid clearance of presynaptic Ca2+ in fast-spiking inhibitory nerve terminals, which face severe transient calcium loads

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.Fil: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Pignataro, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Strehler, Emanuel E.. Mayo Clinic College of Medicine; Estados UnidosFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Changes in the expression of plasma membrane calcium extrusion systems during the maturation of hippocampal neurons

Spatial and temporal control of intracellular calcium signaling is essential for neuronal development and function. The termination of local Ca2+ signaling and the maintenance of basal Ca2+ levels require specific extrusion systems in the plasma membrane. In rat hippocampal neurons developing in vitro, transcripts for all isoforms of the plasma membrane Ca2+ pump (PMCA) and the Na/Ca2+ exchanger (NCX), and the major non-photoreceptor Na+/Ca2+,K+ exchangers (NCKX) were strongly upregulated during the second week in culture. Upregulation of PMCA1, 3, and 4 mRNA coincided with a splice shift from the ubiquitous b-type to the neuron-specific a-type with altered calmodulin regulation. Expression of all PMCA isoforms increased over five-fold during the first two weeks. PMCA immunoreactivity was initially concentrated in the soma and growth cones of developing hippocampal neurons. As the cells matured, PMCAs concentrated in the dendritic membrane and often co-localized with actin-rich dendritic spines in mature neurons. In the developing rat hippocampal CA1 region, immunohistochemistry confirmed the upregulation of all PMCAs and showed that by the end of the second postnatal week, PMCAs 1, 2 and 3 were concentrated in the neuropil, with less intense staining of cell bodies in the pyramidal layer. PMCA4 staining was restricted to a few cells showing intense labeling of the cell periphery and neurites. These results establish that all major Ca2+ extrusion systems are strongly upregulated in hippocampal neurons during the first two weeks of postnatal development. The overall increase in Ca2+ extrusion systems is accompanied by changes in the expression and cellular localization of different isoforms of the Ca2+ pumps and exchangers. The accumulation of PMCAs in dendrites and dendritic spines coincides with the functional maturation in these neurons, suggesting the importance of the proper spatial organization of Ca2+ extrusion systems for synaptic function and development

Cellular and subcellular localization of the neuron-specific plasma membrane calcium ATPase PMCA1a in the rat brain

Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca2+ extrusion from the cell, the plasma membrane calcium-dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a “housekeeping” function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites and spines. Thus, rather than serving a general “housekeeping” function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca2+ transients

Emanuel Strehler’s work on calcium pumps and calcium signaling

Cells are equipped with mechanisms to control tightly the influx, efflux and resting level of free calcium (Ca2+). Inappropriate Ca2+ signaling and abnormal Ca2+ levels are involved in many clinical disorders including heart disease, Alzheimer’s disease and stroke. Ca2+ also plays a major role in cell growth, differentiation and motility; disturbances in these processes underlie cell transformation and the progression of cancer. Accordingly, research in the Strehler laboratory is focused on a better understanding of the molecular “toolkit” needed to ensure proper Ca2+ homeostasis in the cell, as well as on the mechanisms of localized Ca2+ signaling. A long-term focus has been on the plasma membrane calcium pumps (PMCAs), which are linked to multiple disorders including hearing loss, neurodegeneration, and heart disease. Our work over the past 20 years or more has revealed a surprising complexity of PMCA isoforms with different functional characteristics, regulation, and cellular localization. Emerging evidence shows how specific PMCAs contribute not only to setting basal intracellular Ca2+ levels, but also to local Ca2+ signaling and vectorial Ca2+ transport. A second major research area revolves around the calcium sensor protein calmodulin and an enigmatic calmodulin-like protein (CALML3) that is linked to epithelial differentiation. One of the cellular targets of CALML3 is the unconventional motor protein myosin-10, which raises new questions about the role of CALML3 and myosin-10 in cell adhesion and migration in normal cell differentiation and cancer

Natural products : biochemical-chemical characterization, and evidence for therapeutic potential

This Special Issue follows from a previous Issue entitled “Mechanism-Based Development of Natural Products for Human Health” arising from an inaugural conference on Natural Products Development held at Whistler Mountain, Canada from September 21-22, 2012. The timeliness of this issue reflects the continued growth in Natural Product research and the success of the First Issue, as judged from feedback we received and citations received. We were also encouraged by the interest in natural products research in recent years, as exemplified by the recent publication on Traditional Medicine in the prestigious journal Science