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E Garcia-Martin

Publications and source records attributed to E Garcia-Martin.

8 recordsLinked to original sources
PubMed1998

The calcium pump of the plasma membrane: membrane targeting, calcium binding sites, tissue-specific isoform expression.

The two Ca2+ pumps of higher eucaryotes are strictly targeted to different membrane systems: the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). Chimeric constructs of the two pumps expressed in COS-7 cells have revealed a strong signal for endoplasmic reticulum retention in the N-terminal cytosolic portion of the SERCA pump: the signal is contained in a stretch of 28 amino acids that follows the N-terminus. A second, but masked, endoplasmic reticulum retention signal is contained in a cytosolic C-terminal sequence immediately preceding the calmodulin-binding domain of the Ca2+ pump. Selective mutations on the SERCA pump have led to the conclusion that 5 conserved residue membrane domains (TM)4, 5, and 6 form the Ca2+ channel through the pump protein. A comparative sequence inspection has failed to reveal any of these residues in TM5 of the PMCA pump. Mutation of the conserved residue in TM4 and of two in TM6 abolished the ability of the pump to form the Ca(2+)-dependent phosphoenzyme. However, one of the mutations (N979, TM6) also caused retention of the PMCA pump in the reticulum, suggesting structural alterations. Of the four basic isoforms of the pump, two (1, 4) are ubiquitously expressed, two (2, 3) are essentially brain specific. Isoform 2 has the highest calmodulin affinity. Primary cultures of cerebellar granule cells from newborn rats did not express isoforms 2 and 3 at plating time. Incubation of the cells in depolarizing concentrations of KCl, which promote Ca2+ influx, promoted the expression of isoforms 2 and 3, and of a brain specific spliced variant of isoform 1. Incubation of the cells in L-type Ca2+ channel blockers abolished the upregulation of the pump genes.

Amino Acid Sequence↗
PubMed1997

Hypothalamic hypophyseal inhibitory factor (HHIF) increases intrasynaptosomal free calcium concentration.

We have isolated from bovine hypothalamic and pituitary tissues a sodium pump inhibitor that is structurally different from ouabain. By mass spectrometric analysis, this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from that of ouabain. It has been previously shown that this factor inhibits the Ca2+, Mg(2+)-ATPase of the plasma membrane of synaptosomes. Because Ca2+ plays a major role in cellular excitability, we carried out a systematic study of the effects of this inhibitor on the Ca2+ transport processes across the plasma membrane of synaptosomes: We measured ATP-dependent calcium uptake, Na(+)-Ca2+ exchange, and passive permeability using 45Ca2+ and Millipore filtration, chlortetracycline fluorescence, and light-scattering, respectively. This factor inhibits the Na+, K(+)-ATPase activity of the synaptosomal plasma membrane vesicles in the same range of concentrations that produced an increase of intrasynaptosomal free calcium, with nearly the same K0.5 value. In addition, in this concentration range, this factor stimulated 10- to 11-fold the passive flux of Ca2+ and 2.5- to 3-fold the Ca2+ influx via the Na(+)-Ca2+ exchange in these membranes with respect to control values. Measurements of fluorescence anisotropy showed that in this concentration range, the inhibitor did not significantly change the order parameter (fluidity) of these membranes. These results suggest that besides its known inhibition of the sodium pump, this factor could play a role in the control of Ca2+ homeostasis by direct modulation of transport systems implicated in the control of intracellular calcium.

Animals↗
PubMed1996

The plasma membrane calcium pump: recent developments and future perspectives.

The Ca2+ pump of the plasma membrane (PMCA) is regulated by a number of agents. The most important is calmodulin (CaM), which binds to a domain located in the C-terminal portion of the pump, removing it from an autoinhibitory site next to the active site. The CaM-binding domain is preceded by an acidic sequence which contains a hidden signal for endoplasmic reticulum (ER) retention. Chimeras of the PMCA and endoplasmic reticulum (SERCA) pumps have revealed the presence of a strong signal for ER retention in the first 45 residues of the SERCA pump. Four gene products of the PMCA pump are known: two of them (1 and 4) are ubiquitously expressed, two (2 and 3) are specific for nerve cells and may be induced by their activation. Mutagenesis work has identified four residues in three of the transmembrane domains of the pump which may be components of the trans-protein Ca2+ path. The mutation of two of these residues alters the membrane targeting of the pump.

Amino Acid Sequence↗
PubMed1993

Properties of the purified hypothalamic pituitary NA/K-ATPase inhibitor.

Previously we described the isolation and final purification of an endogenous sodium-pump inhibitor from the CNS, mainly from bovine hypothalamus and pituitary. The purification protocol consisted of lipophilic chromatography, followed by lipid extraction, and semipreparative and analytical reverse-phase high-pressure liquid chromatography. The bioassays used were in vitro Na+/K(+)-ATPase inhibition, and 3H-ouabain displacement from its specific binding site in the enzyme structure, as well as inhibition of 86Rb uptake from human red blood cells. We have obtained, from both tissues, a low-molecular-weight, nonpeptidic, nonlipidic substance that elutes as a single peak highly pure according to criteria of coincidence of its spectra properties. When rechromatographed in two different chromatographic systems, the same homogeneous peak is obtained suggesting complete purity. This pure substance can be isolated from other bovine tissues as well as from human plasma and human placenta. It shows a very distinctive fluorescence spectrum and it behaves as a potent inhibitor of the Ca2+ pump of synaptosomal plasma membrane.

Animals↗
PubMed1990

Modulation of the Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membrane by the local anesthetics dibucaine and lidocaine.

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.

Adenosine Triphosphate↗
PubMed1986

Local anesthetics inhibit the Ca2+, Mg2+-ATPase activity of rat brain synaptosomes.

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.

Anesthetics, Local↗