Plasma Membrane Calcium Pump Isoform Research Articles

PMCA2 pump mutations and hereditary deafness.

Hair cells of the inner ear detect sound stimuli, inertial or gravitational forces by deflection of their apical stereocilia. A small number of stereociliary cation-selective mechanotransduction (MET) channels admit K+ and Ca2+ ions into the cytoplasm promoting hair cell membrane depolarization and, consequently, neurotransmitter release at the cell basolateral pole. Ca2+ influx into the stereocilia compartment is counteracted by the unusual w/a splicing variant of plasma-membrane calcium-pump isoform 2 (PMCA2) which, unlike other PMCA2 variants, increases only marginally its activity in response to a rapid variation of the cytoplasmic free Ca2+ concentration ([Ca2+]c). Missense mutations of PMCA2w/a cause deafness and loss of balance in humans. Mouse models in which the pump is genetically ablated or mutated show hearing and balance impairment, which correlates with defects in homeostatic regulation of stereociliary [Ca2+]c, decreased sensitivity of mechanotransduction channels to hair bundle displacement and progressive degeneration of the organ of Corti. These results highlight a critical role played by the PMCA2w/a pump in the control of hair cell function and survival, and provide mechanistic insight into the etiology of deafness and vestibular disorders.

Enhancement of intrarenal plasma membrane calcium pump isoform 1 expression in chronic angiotensin II-infused mice.

Plasma membrane calcium pump isoform 1 (PMCA1) is encoded by ATPase plasma membrane Ca 2+ transporting 1 (ATP2B1), the most likely candidate gene responsible for hypertension. Although PMCA1 is highly expressed in the kidney, little is known about regulation of its renal expression in various pathological conditions in vivo. Our study was designed to elucidate regulation of renal PMCA1 expression in mice. We employed three mouse models for kidney disease. These were the unilateral ureteral obstruction (UUO), the remnant kidney using 5/6 nephrectomy, and chronic angiotensin II administration models. Mice were assessed for systolic blood pressure and renal injury in accordance with the damage induced in the specific model. Kidney PMCA1 mRNA levels were measured in all mice. The UUO model showed renal fibrosis but no changes in blood pressure or renal PMCA1 mRNA expression. Similarly, the 5/6 nephrectomy model exhibited declined renal function without changes in blood pressure or renal PMCA1 mRNA expression. In contrast, chronic angiotensin II administration increased albuminuria and blood pressure as well as significantly increasing renal PMCA1 mRNA and protein expression. These results suggest that renal PMCA1 has a role as one of the molecules involved in angiotensin II‐induced hypertension and kidney injury.

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.

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.

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.

E0037 Effects of enalapril and irbesartan on aorta remodelling and ion pumps in renovascular hypertensive rats

ObjectiveTo investigate the effects of single-drug or combination therapy of enalapril and irbesartan on aorta remodelling and its mechanisms.MethodsRenovascular hypertensive rats (RHD) induced by two-kidney one-clip method were treated with...

012 Differential roles of the plasma membrane calcium pump isoforms 1 and 4 in modulating cardiac contractility

Heart failure is a syndrome currently affecting almost one million people in the UK. Abnormal calcium handling is one the characteristic features of heart failure. The plasma membrane calcium pump (PMCA) is one of the calcium transporters in the cardiomyocytes. Two isoforms of PMCA, PMCA1 and 4, are expressed in the myocardium. Our group has shown previously that PMCA4 modulates the beta-adrenergic response in the heart through its interaction with nNOS. Currently, in order to elucidate the physiological importance of both PMCA isoforms in the modulation of cardiac contractility we generated mice carrying a genetic deletion of either PMCA1 or PMCA4. Given that PMCA4 is a calcium extrusion pump it was unexpected that in vivo basal contractility was enhanced in PMCA4 KO mice (dP/dtmax in 8049±628 vs 6604±296 mmHg/s in KO and WT respectively, p 2+ transients in cardiomyocytes from PMCA4 KO mice showed an increase in amplitude (298.8±24.3 nM calcium vs 492.2±28.2 nM calcium in WT and KO respectively; p 2+ decay. Again, this phenotype was imitated by nNOS specific inhibition in WT adult CMC. Although, there is no difference in total nNOS protein expression between PMCA4 KO and WT, the nNOS localisation and activity at the sarcolemmal membrane was decreased by 52% in PMCA4 KO. Intracellular cardiac cGMP levels in PMCA4 KO were markedly decreased (619±16.7 fmol/mg protein in KO vs 713.4±24.2 fmol/mg protein in WT) suggesting that the phenotype was likely through nNOS modulation. Since the global deletion of PMCA1 is embryonic lethal, we generated PMCA1 cardiac-specific knockout mice (PMCA1cko) using Cre/LoxP technology. PMCA1cko showed a reduction in the rate of relaxation (logistic τ; 6.9±0.34 vs 5.8±0.29 ms in PMCA1cko and PMCA1flox/flox control respectively; p 2+ decay (τ, 0.201±0.009 vs 0.164±0.006 ms in PMCA1cko and PMCA1flox/flox respectively; p 2+ transient amplitude remained unchanged. The nNOS protein expression, localisation and activity in PMCA1cko mice were similar to those in PMCA1flox/flox. In conclusion, our results suggest differential rather than redundant roles of the cardiac PMCAs. PMCA4 regulates cardiac signalling through modulation of membrane nNOS activity, while PMCA1 modulates fine tuning of diastolic calcium in the excitation-contraction coupling. The use of non-isoform-specific inhibitors in previous work was unable to detect these differential roles as the effects of PMCA1 and 4 inhibition cancel each other out, at least in part.

The Plasma Membrane Ca2+ Pump Isoform 4a Differs from Isoform 4b in the Mechanism of Calmodulin Binding and Activation Kinetics: IMPLICATIONS FOR Ca2+ SIGNALING

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., Török, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.

Differential modulation of myocardial beta-adrenergic signalling by plasma membrane calcium pump isoforms

Differential modulation of myocardial beta-adrenergic signalling by plasma membrane calcium pump isoforms

A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines

The plasma membrane Ca 2+ ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca 2+ plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca 2+ regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.

Expression and cellular distribution pattern of plasma membrane calcium pump isoforms in rat pancreatic islets.

This work is aimed at identifying the presence and cellular distribution pattern of plasma membrane calcium pump (PMCA) isoforms in normal rat pancreatic islet. Microsomal fractions of isolated islets and exocrine tissue were analyzed to detect different PMCA isoforms. The cellular distribution pattern of these PMCAs in the islets was also studied in fixed pancreas sections incubated with antibodies against PMCAs and insulin. Antibody 5F10, which reacts with all PMCA variants, showed multiple bands in the blots in the 127-134 kDa region, indicating the presence of several isoforms. Microsomes also reacted positively with specific antibodies for individual PMCA isoforms, generating a band of the expected size. Antibody 5F10 immunocytochemically labeled the plasma cell membrane of both b- and non-b-cells, but predominantly the former. All islet cells were also labeled with antibodies against isoforms 1 and 4, while the antibody reacting with isoform 3 labeled exclusively b-cells. A few b- and non-b-cells were positively labeled with the antibody reacting with PMCA b variant. Negative results were obtained with the antibody against isoform 2. Further studies, together with previous reports on the modulatory effect of insulin secretagogues and blockers upon PMCA activity, may provide evidence of the importance of this particular PMCA expression for islet function under normal and pathological conditions.

Expression and cellular distribution pattern of plasma membrane calcium pump isoforms and spliced variants in rat pancreatic islets

Expression and cellular distribution pattern of plasma membrane calcium pump isoforms and spliced variants in rat pancreatic islets

Plasma Membrane Calcium Pump Isoform 1 Gene Expression Is Repressed by Corticosterone and Stress in Rat Hippocampus

Glucocorticoids (GCs) are critical to learning and memory, in large part because of their actions in the hippocampus. Chronic high levels of GCs have profound effects on hippocampal structure and function and can even result in irreversible neurodegeneration. Hippocampal GC actions are mediated by intracellular receptors that modulate the transcription of specific target genes. In a screen for genes repressed by GCs in rat hippocampus, we identified plasma membrane calcium pump isoform 1 (PMCA1), a plasma membrane calcium ATPase. In Northern blots, PMCA1 was repressed approximately 33% after a high, but not a low dose of the GC, corticosterone (B), suggesting glucocorticoid (but not mineralocorticoid) receptor-mediated repression. Furthermore, in situ hybridization demonstrated that B significantly downregulated PMCA1 mRNA in all brain regions examined. Repression of PMCA1 was also observed in cultured hippocampal neurons, but only when the cells were in the differentiated state. Stress also repressed PMCA1 expression in hippocampus of adrenal-intact animals, and a clear inverse correlation between B level and PMCA1 mRNA could be discerned. However, other non-B-dependent factors appeared to be involved in the response of PMCA1 to stress because, unlike exogenous B, cold stress did not repress PMCA1 in brain regions other than hippocampus. Moreover, in the presence of constant B (B-replaced, adrenalectomized animals), cold stress led to increased hippocampal PMCA1 expression. These observations suggest that repression of PMCA1 represents one molecular mechanism by which corticosteroids regulate Ca(2+) homeostasis and hence influence neuronal activity. Moreover, other stress-related neurohumoral factors appear to counter the repressive effects of B. Defects in the balance between GC-mediated and non-GC-mediated effects on PMCA1 expression may have adverse effects on neuronal function and ultimately result in irreversible neuronal damage.

Selective decrease of mRNAs encoding plasma membrane calcium pump isoforms 2 and 3 in rat kidney

Although the existence of multiple isoforms of plasma membrane calcium ATPase (PMCA) is now well documented, their biological functions are not yet known. In this study, we set out to investigate the potential role of PMCA isoforms, previously identified in renal cortical tissue, in tubular reabsorption of calcium (Ca2+). With use of reverse transcription-polymerase chain reaction analysis, we determined levels of mRNAs encoding isoforms of PMCA1 through PMCA4 in renal cortex, liver, and brain of rats with hypercalciuria induced by feeding with a low-phosphate diet (LPD) as compared with Ca2+-retaining rats that were fed a high-phosphate diet (HPD). We observed that in hypercalciuric LPD-fed rats, the mRNAs encoding isoforms PMCA2b and PMCA3(a + c) are significantly lower (Delta approximately-50%) than in HPD-fed hypocalciuric rats, whereas no changes in mRNAs encoding isoforms PMCA1b and PMCA4 were observed, and mRNA encoding calbindin 28 kDa was increased. On the other hand, the content of mRNAs encoding PMCA2b and PMCA3(a + c) in liver and brain, respectively, was not changed. These findings are evidence that expression of PMCA isoforms in the kidney can be selectively modulated in response to pathophysiologic stimuli. The association of a decrease in mRNA encoding PMCA2b and PMCA3(a + c) with hypercalciuria suggests that the two PMCA isoforms may be operant in tubular reabsorption of Ca2+ and its regulation.

Plasma Membrane Ca2+ Pump in Rat Brain: PATTERNS OF ALTERNATIVE SPLICES SEEN BY ISOFORM-SPECIFIC ANTIBODIES

The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.

Co-ordinated regulation of the plasma membrane calcium pump and the sarco(endo)plasmic reticular calcium pump gene expression by Ca 2+

The plasma membrane calcium pump (PMCA) and sarco(endo)plasmic reticular calcium pump (SERCA) are important components of the Ca 2+ homeostasis system responsible for intracellular Ca 2+ signaling, yet the factors which govern their expression remain unclear. Recently, we have found that overexpression of PMCA by a gene transfection approach caused a down-regulation of the SERCA pump [Liu B.F., Xu X., Fridman R., Muallem S., Kuo T.H. Consequences of functional expression of the plasma membrane calcium pump isoform 1a. J Biol Chem 1996; 271: 5536–5544]. The results suggest an interdependence between PMCA and SERCA gene expression which has prompted us to investigate further on the mechanisms that regulate the expression of these Ca 2+ pump genes in various cultured cell lines. In the present study, we have analyzed the isoforms of the PMCA and SERCA in different cells and presented evidence in favor of a co-induction of the PMCA and SERCA gene expression by second messengers, such as protein kinase C, cAMP, and Ca 2+. Ectopic expression of PMCA or SERCA led to downregulation of the endogenous forms of both pumps. Changes in the level of mRNAs were paralleled by corresponding altered pump protein contents. The Ca 2+-mediated increase of gene expression is accompanied by increased transcription rates as indicated by nuclear run-on assay. The co-ordinated induction of the PMCA and SERCA gene expression by thapsigargin was not blocked by the cytosolic application of the Ca 2+ chelator BAPTA. We conclude that genes encoding components of the major Ca 2+, transport pathways, including pumps and IP 3 receptor channels, are regulatorally linked and this link is provided by the Ca 2+ load of the ER store. This study points to the importance of gene expression as an integral component in the regulation of cellular Ca 2+ homeostasis.

Plasma Membrane Calcium Pump Isoform 4a Has a Longer Calmodulin-Binding Domain Than 4b

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin-binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue calmodulin-binding region.

Plasma-membrane calcium-pump isoforms in human and rat liver.

Plasma-membrane Ca(2+)-pumping ATPases (PMCAs) extrude Ca2+ from the cytoplasm of all cells. Some previous studies of ATP-dependent Ca2+ transport by liver membranes suggested there exist specific properties of the hepatic PMCA, including regulation by hormones which affect calcium signalling. Multiple PMCA isoforms are now known to result from expression of four different genes (known as PMCA 1-4) and alternative RNA splicing at three possible sites (A, B and C). We investigated which isoforms are expressed in adult human and rat liver RNA using reverse-transcription polymerase chain reaction with mixed primers designed to amplify parts of all the known PMCA transcripts. In human liver, products were identified by sequencing from PMCA1, PMCA2 and PMCA4, but not from PMCA3 or from any new gene. In rat liver, by contrast, only PMCA1 and PMCA2 were detectable, although we confirmed that the primers were able to amplify from rat lung a new sequence which is part of rat PMCA4. Of the alternatively spliced variants, at site A in the PMCA2 sequences, all the exons were included in both adult and fetal human liver. In human liver, the exon at site B was excluded in some products from PMCA1 and PMCA4, and at site C, only PMCA1b and one form of PMCA4 were found. Blots of human liver RNA showed PMCA1 and PMCA4 were abundantly expressed, unlike PMCA2. On blots of rat liver RNA, PMCA1 was more abundant than PMCA2, and purified rat parenchymal cell RNA gave similar findings. In summary, no new hepatic PMCA isoforms have been demonstrated, but differences between the predominant human and rat isoforms may have consequences for Ca2+ signalling or the response to liver cell injury.

Use of the Polymerase Chain Reaction for the Detection of Alternatively Spliced mRNAs of Plasma Membrane Calcium Pump

Polymerase chain reaction [PCR, reverse transcriptase-PCR (RT-PCR)] has been used to amplify the mRNA subspecies of the plasma membrane calcium pump isoform 1 (PMCA1) in total RNA extracted from hamster tissues. Two primers were synthesized that encompass the site at which a 154-bp exon is included totally (PMCA1a), partially (PMCA1c and d), or completely excluded (PMCA1b) in the carboxy-terminal regulatory region. PCR amplification revealed two bands (PMCA1b and 1c) that are more abundant in various tissues, while Southern hybridization of the samples after PCR amplification has detected two additional mRNA variants corresponding to PMCA1a and 1d. The distribution of these mRNA variants are tissue specific and correlate well with the pump protein distribution patterns on immunoblot. Since these multiple bands on the immunoblot are not derived from proteolysis, it is suggested that they represent the PMCA1 isozymes encoded by these alternatively spliced mRNAs. To our knowledge, this is the first report to show all four alternatively spliced mRNAs that are simultaneously detected in one single RNA sample using PCR technique. Since these isozymes are different in their regulatory domain, their tissue-specific expression may be physiologically important.