Plasma Membrane Ca&lt;sup&gt;2+&lt;/sup&gt; Pump: Recent Developments

Penniston, John T.; Enyedi, Ágnes

doi:10.1159/000154718

Cited by 34 publications

(8 citation statements)

References 22 publications

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“…On the other hand, the slope corresponding to the whole pump is consistent with the presence of between 8 and 10 transmembrane segments, in agreement with the current models for the calcium pump (Penniston & Enyedi, 1994;Stokes et al, 1994;Mdler et al, 1996). However, in this particular case, one should bear in mind that differential exposure to the lipid phase and/or the presence of unusually reactive side chains could bias the interpretation of this piece of data.…”

Section: The First Hydrophobic Cluster (Hci) Includes Two Distinct Trsupporting

confidence: 89%

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The membrane topology of the amino‐terminal domain of the red cell calcium pump

et al. 1997

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A systematic study of the membrane-associated regions in the plasma membrane Ca2+ pump of erythrocytes has been performed by hydrophobic photolabeling. Purified Ca2+ pump was labeled with 3-(trifluoromethyl)-3-(rn- [ '251]iodophenyl)-diazirine ([ '251]TID), a generic photoactivatable hydrophobic probe. These results were compared with the enzyme labeled with a strictly membrane-bound probe, [3H]bis-phosphatidylethanolamine (trifluoromethyl) phenyldiazirine. A significant light-dependent labeling of an M, 135,000-140,000 peptide, corresponding to the full Ca2+ pump, was observed with both probes. After proteolysis of the pump labeled with each probe and isolation of fragments by SDS-PAGE, a common pattern of labeled peptides was observed. Similarly, labeling of the Ca2+ pump with ['251]TID, either in isolated red blood cell membranes or after the enzyme was purified, yields a similar pattern of labeled peptides. Taken together, these results validate the use of either probe to study the lipid interface of the membrane-embedded region of this protein, and sustain the notion that the conformation of the pump is maintained throughout the procedures of solubilization, affinity purification, and reconstitution into proteoliposomes. In this work, we put special emphasis on a detailed analysis of the N-terminal domain of the CaZ+ pump. A labeled peptide of M, 40,000 belonging to this region was purified and further digested with V8 protease. The specific incorporation of [1251]TID to proteolytic fragments pertaining to the amino-terminal region indicates the existence of two transmembrane stretches in this domain. A theoretical analysis based on the amino acid sequence 1-322 predicts two segments with high probability of membrane insertion, in agreement with the experimental data. Each segment shows a periodicity pattern of hydrophobicity and variability compatible with a-helical structure. These results strongly suggest the existence of a transmembrane helical hairpin motif near the N-terminus of the Ca2+ pump.Keywords: calcium pump; detection of the outside face of the helices; hydrophobic photolabeling; membrane proteins; secondary structure prediction for transmembrane segments; transmembrane topologyThe erythrocyte CaZ+ pump is a monomeric integral membrane protein that actively transports Ca2+ from the cytoplasm to the external milieu. The main portion of the pump faces the cytoplasm.

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Section: The First Hydrophobic Cluster (Hci) Includes Two Distinct Trsupporting

confidence: 89%

“…By contrast, it is believed that short external loops connect 8-10 putative transmembrane segments (Penniston & Enyedi, 1994;Stokes et al, 1994;Lutsenko & Kaplan, 1995;Moller et al, 1996).…”

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The membrane topology of the amino‐terminal domain of the red cell calcium pump

et al. 1997

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“…PMCA1 and -4 may thus represent housekeeping isoforms, while PMCA2 and -3 may be required for specialized functions in a limited number of tissues. Diversity among the ATPases is also generated by alternative splicing of the primary transcripts that may involve two major sites, termed A and C (for review see 19,39,40). In a previous work, we identified the PMCA transcripts expressed in the ␤-cell and characterized them at alternative splicing sites A and C (20).…”

Section: Discussionmentioning

confidence: 99%

Plasma Membrane Ca2+-ATPase Overexpression Reduces Ca2+ Oscillations and Increases Insulin Release Induced by Glucose in Insulin-Secreting BRIN-BD11 Cells

Kamagaté¹,

Herchuelz²,

Eylen³

2002

Diabetes

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“…To date, at least four different genes have been found which encode for PMCA (2). Additional variability is obtained by alternate splices occurring at two sites in the pump (3)(4)(5)(6)(7).…”

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confidence: 99%

“…The isoforms whose mRNA contains a spliced-in exon are called "a," while those isoforms lacking the additional exon are called "b." 2 The a variants of the isoforms have a less basic calmodulin-binding domain as well as a different carboxyl terminus than the b variants. When synthetic peptides corresponding to representative a and b forms of the calmodulinbinding domain were compared, the b form of the peptide showed a 10-fold higher affinity for calmodulin than the a form of the peptide (12).…”

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confidence: 99%

Plasma Membrane Ca2+ Pump Isoforms 2a and 2b Are Unusually Responsive to Calmodulin and Ca2+

Elwess¹,

Filoteo²,

Enyedi³

et al. 1997

Journal of Biological Chemistry

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118

103

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The full-length a and b variants of the rat plasma membrane calcium pump, isoform 2 (rPMCA2a and rPMCA2b), were constructed and expressed in COS-7 cells. To characterize these isoforms, calcium transport was determined in a microsomal fraction. Both rPMCA2a and rPMCA2b had a much higher affinity for calmodulin than the corresponding forms of hPMCA4, and rPMCA2b had the highest affinity among the isoforms that have been tested so far. When analyzed at a relatively high calmodulin concentration, rPMCA2b and, to a lesser extent, rPMCA2a showed higher apparent calcium affinity; i.e. they were more active at lower Ca 2؉ concentrations than hPMCA4b. This indicates that these two variants of rat isoform 2 will tend to maintain a lower free cytosolic Ca 2؉ level in cells where they are expressed. Both variants also showed a higher level of basal activity (in the complete absence of calmodulin) than hPMCA4b, a property which would reinforce their ability to maintain a low free cytosolic Ca 2؉ concentration. Experiments designed to determine the source of the higher apparent Ca 2؉ affinity of rPMCA2b showed that it came from the properties of the carboxyl terminus, rather than from any difference in the catalytic core.The plasma membrane Ca 2ϩ pump plays a key role in controlling the intracellular Ca 2ϩ concentration. This P-type ATPase is regulated by calmodulin and is responsible for the ATP powered removal of Ca 2ϩ from eukaryotic cells (1). The plasma membrane Ca 2ϩ pump (PMCA) 1 has a low level of activity in the absence of calmodulin. Calmodulin binds to an autoinhibitory domain (the C domain), and increases both the maximum velocity of the pump and the apparent Ca 2ϩ affinity.To date, at least four different genes have been found which encode for PMCA (2). Additional variability is obtained by alternate splices occurring at two sites in the pump (3-7). In each of the four genes, the alternative splice sites (8) are located in the middle of the cytosolic loop between transmembrane domains 2 and 3 (splice site A) (9) and downstream of the last transmembrane domain, in the middle of the calmodulinbinding domain (splice site C) (9 -11). The first 18 amino acids of the calmodulin-binding domain are conserved for all PMCA isoforms, but the presence of the alternative RNA splice site in the middle of this region (at splice site C) changes the remainder of the calmodulin-binding domain as well as the carboxyl terminus (10). The isoforms whose mRNA contains a spliced-in exon are called "a," while those isoforms lacking the additional exon are called "b." 2 The a variants of the isoforms have a less basic calmodulin-binding domain as well as a different carboxyl terminus than the b variants. When synthetic peptides corresponding to representative a and b forms of the calmodulinbinding domain were compared, the b form of the peptide showed a 10-fold higher affinity for calmodulin than the a form of the peptide (12). Additionally, full-length isoforms hPMCA4a and hPMCA4b were overexpressed in COS-1 cells and the calmodulin-res...

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Plasma Membrane Ca<sup>2+</sup> Pump: Recent Developments

Cited by 34 publications

References 22 publications

The membrane topology of the amino‐terminal domain of the red cell calcium pump

The membrane topology of the amino‐terminal domain of the red cell calcium pump

Plasma Membrane Ca2+-ATPase Overexpression Reduces Ca2+ Oscillations and Increases Insulin Release Induced by Glucose in Insulin-Secreting BRIN-BD11 Cells

Plasma Membrane Ca2+ Pump Isoforms 2a and 2b Are Unusually Responsive to Calmodulin and Ca2+

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