expression of multiple plasma membrane ca2+-atpases in rat pancreatic islet cells

Research

Expression of multipleplasma membrane Ca2+-ATPases inrat pancreatic islet cells

A. Kamagate, 1 A. Herchuelz, 1 A. Bollen, 2 F. Van Eylen 1

1 Laboratory of Pharmacology, Brussels Free University School of Medicine, Brussels, Belgium 2Laboratory of Applied Genetics, Brussels Free University – Faculty of Science, Institut de Biologie et de Médecine Moléculaires, Gosselies, Belgium

Summary When stimulated by glucose, the pancreatic β-cell displays large oscillations of intracellular free Ca2+ con-centration ([Ca2+]i). To control [Ca2+]i, the β-cell must be equipped with potent mechanisms for Ca2+ extrusion. We stud-ied the expression of the plasma membrane Ca2+-ATPases (PMCA) in three insulin secreting preparations (a pureβ-cell preparation, RINm5F cells and pancreatic islet cells), using reverse-transcribed PCR, RNase protection assayand Western blotting. The four main isoforms, PMCA1, PMCA2, PMCA3 and PMCA4 were expressed in the threepreparations. Six alternative splice mRNA variants, characterized at splice sites A, B and C were detected in the threepreparations (rPMCA1xb, 2yb, 2wb, 3za, 3zc, 4xb), plus two additional variants in pancreatic islet cells (PMCA4za,1xkb). The latter variant corresponded to a novel variant of rat PMCA1 gene lacking the exon coding for the 10th trans-membrane segment, at splice site B. At the mRNA and protein level, five variants predominated (1xb, 2wb, 3za, 3zc,4xb), whilst one additional isoform (4za), predominated at the protein level only. This provides the first evidence for thepresence of PMCA2 and PMCA3 isoforms at the protein level in non-neuronal tissue. Hence, the pancreatic β-cell isequipped with multiple PMCA isoforms with possible differential regulation, providing a full range of PMCAs for [Ca2+]i

regulation. © 2000 Harcourt Publishers Ltd

Cell Calcium (2000) 27 (4), 231–246© 2000 Harcourt Publishers Ltd

DOI: 10.1054/ceca.2000.0116, available online at http://www.idealibrary.com on

INTRODUCTION

For years, insulin secretion from the pancreatic β-cell wasknown to be a Ca2+-dependent process; a rise in cytosolicfree Ca2+ concentration ([Ca2+]i) is now recognized as play-ing an essential role in this process [1–3]. To allow such arole, [Ca2+]i must be tightly controlled. In both excitableand non-excitable cells, Ca2+ can be actively extruded bytwo processes: the plasma membrane Ca2+-ATPase andthe Na/Ca exchanger, that play a major role in Ca2+ home-ostasis. Classically, it is considered that while Na/Caexchange has a low affinity but a high capacity for Ca2+,the Ca2+-ATPase has a high affinity but low capacity for

Received 10 January 2000Revised 23 March 2000Accepted 23 March 2000

Correspondence to: A. Herchuelz, Laboratoire de Pharmacodynamie et deThérapeutique, Université Libre de Bruxelles, Faculté de Médecine, Routede Lennik, 808 – Bâtiment GE, B-1070 Bruxelles, Belgium. Tel.: +32 2 55562 75; fax +32 2 555 63 70; e-mail: [email protected]

the divalent cation [4]. Therefore, Na/Ca exchange takescare of large intracellular Ca2+ loads, while the Ca2+-ATPase performs the fine tuning of intracellular Ca2+ levelaround basal [Ca2+]i, namely 0.1 to 0.2 µM [4,5].

The plasma membrane Ca2+-ATPase (PMCA) belongs tothe P-type family of transport ATPases which form a phos-phorylated intermediate during the reaction cycle [6]. ThePMCA has been recently cloned from rat brain [7–8] andtestis [9]. PMCA presents ten transmembrane domains aspredicted from hydropathy plots. About 80% of the pumpmass protrudes into the cytoplasm, with very short loopsconnecting the putative transmembrane domains on theexternal side [7]. Four different genes corresponding tofour isoforms PMCA1, PMCA2, PMCA3, and PMCA4 havebeen found. Diversity among the ATPases is generated byalternative splicing of the primary transcripts that mayinvolve three different sites termed A, B and C [10–12]. Afourth splice site named D [7] was initially suggested butwas proven to be a cloning artefact [13].

Alternative splice site A is located in between theputative transmembrane helices 2 and 3, upstream a

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232 A Kamagate, A Herchuelz, A Bollen, F Van Eylen

regulatory binding site for acidic phospholipids [14].Alternative splicing at site C, located on the intracytoplas-mic COOH-terminus, involves a calmodulin (CaM)-bind-ing site, that may affect the affinity of the pump for CaMas well as the autoinhibitory activity exerted by this CaM-binding domain [15]. The splicing at site B leads to theloss of the 10th transmembrane domain causing a reor-ganization of the pump topology leading to the elimina-tion of the 9th transmembrane domain [11,16].Alternative splicing at sites A and C has been observedfor all four isoforms except that PMCA1 is never splicedout at site A [10]. Alternative splicing at site B has beendescribed only for human isoforms 1 and 4 [11–12]. Inaddition to the functional differences, the PMCA isoformsshow tissue-specific expression [8,13,17–18]. While geneproducts 1 and 4 are transcribed in a majority of tissues,PMCA2 and PMCA3 mRNAs are expressed in a relativetissue-specific way, i.e. predominantly in brain and heart,and in brain and skeletal muscle respectively. Recently,the study of PMCA isoforms at the protein level hasprovided direct evidence of the regulation of calciumpump using the alternate splicing options [19].

In pancreatic β-cells, one splice variant of PMCA1,PMCA2 and PMCA4 has already been identified focusingon the CaM-binding region (site C). An additional splicevariant of PMCA4 was also shown to be expressed inpancreatic islet (non-β) cells [20]. However, alternativesplicing at the other sites, A and B, has not yet beenexamined in insulin producing cells.

The aim of the present study was to further character-ize PMCA isoforms in insulin releasing cells. Usingreverse transcription (RT) PCR, ribonuclease protectionassay (RPA) and Western blotting we have investigatedthe transcription pattern and expression level of PMCAisoforms and splice variants at sites A, B, and C. Our datashow that PMCA1, PMCA2, PMCA3 and PMCA4 areexpressed in the three insulin-releasing preparations. Sixalternative splice mRNA variants, characterized at thethree splice sites A, B and C were detected in the threepreparations, plus two additional variants in pancreaticislets. One of the latter variants corresponded to a novelvariant of rat PMCA1 gene lacking the exon at splice siteB. All splice variants, except the latter were expressed to asignificant level in pancreatic islet cells. At the proteinlevel, islet cells expressed substantial amounts of onePMCA2 and two PMCA3 splice variants that up to nowwere observed only in neuronal tissue.

These various PMCAs, by being both of the spliced-inor of the spliced-out type of the four isoforms, could dis-play differential autoinhibitory and regulatory behaviouras well as differential functional importance that wouldhelp the β-cell to maintain appropriate Ca2+ homoeosta-sis as required for optimal cellular activity and insulinsecretion.

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MATERIALS AND METHODS

Cell preparations

Pancreatic islets were isolated by the collagenase tech-nique from the pancreas of wistar female rats [21]. Themethod used to isolate pancreatic islet cells has beendescribed elsewhere [22]. Clonal insulin-producingRINm5F cells were grown in RPMI medium supple-mented with 10% foetal calf serum, 2 mM L-glutamine,100 U/ml penicillin and 100 µg/ml streptomycin (GibcoBRL, Merelbeke, Belgium). RINm5F cells were scrapedand pelleted for direct RNA extraction. Ninety-nine percent pure β-cells were obtained using flow cytometry ofdispersed islet cells labelled with the Ca2+-sensitive fluo-rochrome fluo-3 [23]. For immunoblot use, islets wereisolated in Hanks buffer containing protease inhibitors [1mM PMSF, 1 mM Benzamidine (Sigma-Aldrich SA), 1 mMEDTA, 2 mM DTT, 1 mM Pefabloc and 10 µM Leupeptin(Boehringer Mannheim)].

Total RNA preparation

Pancreatic islet cells, purified β-cells and RINm5F cellswere stored 1 h at 4°C in ‘RNAlater’ (Ambion), a RNA sta-bilization solution, according to the protocol provided bythe manufacturer, and then pelleted at 3000 × g and 4°Cfor 15 min. Total RNA was isolated using the ‘RNA now’method (Biogentex, Seabrook, TX, USA). The resultingRNA was dissolved in DEPC-treated water and stored at–70°C. The concentration of RNA was determined byabsorbance at 260 nm (1A260 unit = 40 µg/ml).

Design of polymerase chain reaction primers

Using specific primer pairs flanking either site A alone orsites B and C (Table 1), PMCA isoforms 1, 2, 3, and 4 wereamplified to detect the different splice variant-specificmRNAs. All primers were based on rat PMCA cDNAsequences (rPMCA1, rPMCA2 [7], rPMCA3 [8] andrPMCA4 [9]).

Reverse transcription and polymerase chain reaction

RNA (2 µg) was heated 10 min at 70°C, to denaturate theRNA, and reverse transcribed for 50 min at 42°C, using200 units of Superscript II RT (Gibco BRL), with 25 µg/mlof oligo(dT) primer and 2.5 µg/ml random primer(Promega, Leiden, The Netherlands), and triphosphatenucleosides (0.5 mM each) (Boehringer Mannheim,Brussels, Belgium) in 20 µl reaction volume, as recom-mended by the manufacturer. RNA complementary tocDNA was removed using 2 units of E. coli RNase H(Boehringer Mannheim) for 20 min at 37°C. The mediumwas then diluted with 30 µl of 16 mM EDTA and the

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Ca2+-ATPase isoforms in pancreatic β-cells 233

Table 1 Position and sequence of PCR primers used for determination of alternative splicing at site A (panel A), and atsites B and C (panel B)

A)

Gene Direction Sequence Starting Primerbase number

PMCA 1 Forward 5′-CTTACCTTACTTGGAGCTG-3′ 1125 1Reverse 5′-GTTGTTATCCTTCATCATTTTCTT-3′ 1629 2

PMCA 2 Forward 5′-CTGTGGGTGTCAACTCTCAA-3′ 1376 3Reverse 5′-GTGAGCTTGCCCTGAAGCA-3′ 1589 4

PMCA 3 Forward 5′-CATGTCATGGAAGGTTCTGG-3′ 1491 5Reverse 4′-GTTATTGTCCTTCATCATTTTCTT-3′ 2012 6

PMCA 4 Forward 5′-GTGACTGCTGTGGGAATCAA-3′ 978 7Reverse 5′-GTTGTTGTCCTTCATCATTTTCTT-3′ 1502 8

B)


PMCA 1 Forward 5′-ATCTTCTGCACAATTGTCTTAG-3′ 3286 9Reverse 5′-GAGCTACGAATGCATTCACC-3′ 3798 10

PMCA 2 Forward 5′-CATCTTCTGCACCATCGTTC-3′ 3507 11Reverse 5′-AGCCATGAAGTTATGGATGGA-3′ 3924 12

PMCA 3 Forward 5′-ATCTTCTGTACCATTGTCCTG-3′ 3666 13Reverse 5′-GAGCTACGGAATGCTTTCAC-3′ 4178 14

PMCA 4 Forward 5′-TCTGCTCTGTTGTTTAGGCA-3′ 3157 15Reverse 5′-ATGAAATACTTTGACCACTCTG-3′ 3675 16

All primers were based on rat PMCA cDNA sequences: rPMCA1 and rPMCA27; rPMCA38 and rPMCA49. The 5′ positionof the primer sequence on the cDNA is indicated. For the sake of clarity, the primers were numbered 1–16 andreference is made to these numbers in the text.

reaction was terminated by heating the medium up to70°C for 15 min; 3 µl of single strand cDNA was amplifiedby polymerase chain reaction (PCR) in a 50 µl volumeusing ‘Pwo DNA polymerase’ Kit (Boehringer Mannheim),30 pmol of each primer and 0.5 unit of ‘Pwo DNA poly-merase’. The amplification was conducted in a thermalcycler (GeneAmp PCR system 2400; Perkin Elmer,Zaventem, Belgium) under the following conditions: ini-tial denaturation at 94°C for 2 min; 10 cycles of 94°C 30s, 58°C 30 s, 72°C 1 min; 25 cycles of 94°C 30 s, 58°C 30s, 72°C 1 min increased by 20 s at each cycle; and then72°C 7 min (final extension). After 35 cycles, a 10 µlaliquot was subjected to electrophoresis in 1% (w/v)agarose gel containing 0.5 µg/ml ethidium bromide in 1X TBE buffer. RNA without reverse transcriptase wereused in every PCR reaction for each set of primers as con-trol, to check possible amplification of contaminant. Allexperiments were repeated at least three times.

Cloning and sequencing of PCR products

PCR products were subcloned into the PCR-Blunt plas-mid vector (Invitrogen, Leek, The Netherlands) accordingto the manufacturer’s protocol. Plasmid DNA was pre-pared from recombinant colonies identified by blue-white colour selection. DNA sequencing of selectedclones was determined using cycle sequencing with


AmpliTaq® DNA polymerase, FS (Perkin-Elmer). Allsequencing products were separated on 5% (v/v) LongRanger gels (FMC® Bioproducts, Rockland, USA) contain-ing urea in the 1 X TBE Buffer, using 373-DNA sequenc-ing system (Perkin Elmer).

Templates for RNA probes and targets synthesis

We generated transcription templates by PCR withoutcloning. The procedure required standard PCR reagentsand two genes specific oligonucleotide primers (Table2A), at least one of which including an additional stretchof 19–20 bp nucleotides corresponding to the T7 phagepromoter sequence. Templates for antisense RNA probeswere synthesized using reverse primer including phagepromoter followed by first 18–20 bp of antisense strandand other forward primer, which was gene-specific withno additional bases. We used primer pairs 18–19, 22–23,26–27, 30–31 for PMCA1–4 respectively. To produce tar-get RNA template, we used T7 phage promoter sequencefollowed by the first 18–20 bp of the sense strandsequence and the other reverse primer which is gene-specific with no additional bases. We used primer pairs17–20, 21–24, 25–28, 29–32 for PMCA1–4 respectively.

Amplified DNA was sequenced using AmpliTaq® DNApolymerase, FS (Perkin-Elmer) directly with no furtherpurification after PCR reaction. Typically, 90 µl of each

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Table 2 Position and sequence of PCR primers used for RNase protection assay (panel A) and quantitative PCR (panel B)

A)


PMCA 1 Forward 5′-TAATACGACTCAC TATAGGGTGTGGTGTTAGTGACGG-3′ 775 175′-GTGTGTGGTGTTAGTGACGG-3′ 18

Reverse 5′-TAATACGACTCAC TATAGGGTACCTGAAAGAAGCAAGGG-3′ 1036 195′-GTACCTGAAAGAAGCAAGGG-3′ 20

PMCA 2 Forward 5′-TAATACGACTCAC TATAGGGAAGGAGACATATGGGGAC-3′ 708 215′-AAGGAGACATATGGGGAC-3′ 22

Reverse 5′-TAATACGACTCAC TATAGGGTTCACCTTCATCTTCTGC-3′ 973 235′-TTCACCTTCATCTTCTGC-3′ 24

PMCA 3 Forward 5′-TAATACGACTCAC TATAGGGCACAGCCTTCAATGACTG-3′ 1205 255′-CACAGCCTTCAATGACTG-3′ 26

Reverse 5′-TAATACGACTCAC TATAGGGCCTTCCATGACATGAGTC-3′ 1504 275′-CCTTCCATGACATGAGTG-3′ 28

PMCA 4 Forward 5′-TAATACGACTCAC TATAGGGAGAAGGTCTGTCTGGGAAC-3 356 295′-AGAAGGTCTGTCTGGGAAC-3′ 30

Reverse 5′-TAATACGACTCAC TATAGGGACCACAATGATCACCGAG-3′ 655 315′-ACCACAATGATCACCGAG-3′ 32

B)


PMCA 1 Forward 5′-ATCTTCTGCACAATTGTCTTAG-3′ 3286 33Reverse 5′-GAGCTACGAATGCATTCACC-3′ 3798 34

PMCA 2 Forward 5′-GAAGAGGAAGAGAAGAAAGAC-3′ 1432 35Reverse 5′-TCTTGTCATCTGCATCAC-3′ 1537 36

PMCA 3 Forward 5′-TGGGAAGGATGAGATGAC-3′ 3872 37Reverse 5′-GAGCTACGGAATGCTTTCAC-3′ 4178 38

PMCA 4 Forward 5′-AAGAGGAGATCAGCAAGG-3′ 3364 39Reverse 5′-TAGAAGCCAACGAAGGAC-3′ 3811 40

All primers were based on rat PMCA cDNA sequences: rPMCA1 and rPMCA27; rPMCA38 and rPMCA49. The 5′ position of the primersequence on the cDNA is indicated. For the sake of clarity, the primers were numbered 17–40 and reference is made to these numbers inthe text. T7 phage promoter sequence is written in bold.

PCR reaction was purified with High Pure PCR ProductPurification Kit (Boehringer Mannheim) and concen-trated by isopropanol precipitation for RNA synthesis.

Linearized pTRIPLEscript DNA (Ambion) in TE buffercontaining a 250 bp fragment of mouse β-actin cDNA inantisense orientation relative to tandem SP6, T7, and T3promoters was used as control template for probe syn-thesis. The mouse β-actin probe contained 11 single-basemismatches compared to the same region in the rat β-actin mRNA. In spite of these differences, mouse probewill be protected by rat β-actin mRNA to give mostly full-length 250 base-protected fragments.

Synthesis of RNA probes and targets for plasmamembrane Ca 2+-ATPase isoforms and RNA probe for β-actin.

RNA probes were transcribed with T7 RNA polymerasefrom template sequences using MAXIscripts™ kits(Ambion), according to manufacturer’s protocol.Reactions contained 20 nmol each ATP, CTP, GTP, 12nmol unlabelled UTP, 8 nmol biotin-16-UTP (Boehringer

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Mannheim), 0.2 µg PCR template or 0.5–1 µg plasmidtemplate, 40 units T7 RNA polymerase and 20 unitsribonuclease inhibitor in a final volume of 20 µl.Reactions were carried out at 37°C for 2 h. Following syn-thesis of RNA, DNA templates were destroyed by addi-tion of 1 µl of RNase-free DNase I (Ambion) andincubated at 37°C for 15 min. Samples were thenextracted by gel purification on denaturing polyacry-lamide gel (5%) and RNA was eluted from gel usingProbe Elution Buffer (Ambion).

RNA targets were prepared by the same procedure,except that we used only unlabelled NTP, and reactionscontained 20 nmol each ATP, CTP, GTP and UTP.Synthetic RNA targets for PMCA1–4 protecting a 300 ntfragment of the antisense probe were used to calibratethe RNase protection assays.

Ribonuclease protection assays

Ribonuclease protection assays (RPAs) were performedusing RPA II™ Kit® (Ambion) as described by the manufac-turer’s protocol. RNA standard tubes were prepared with



increasing amounts of in vitro synthesized sense-strandRNA combined with 4-fold molar excess of probe overthe RNA target. RNA preparation to be examined and twocontrol tubes containing a yeast RNA equivalent to thehighest amount of sample RNA were combined witheither 160 pg PMCAs or β-actin, or PMCAs and β-actinRNA probe per 10 µg total RNA, lyophilized and dis-solved in 20 µl hybridization buffer. RNA was denaturedby incubation at 95°C for 3 min and hybridized by incu-bation at 45°C overnight. Following overnight incuba-tion, unhybridized RNA of all experimental tubes, andone tube of each pair of yeast control tubes weredigested by addition of 200 µl of a mix containing RNasedigestion buffer, 0.25 units/ml RNase A and 10 units/mlRNase T1 (Ambion). The remaining tubes contained 200µl of RNase digestion buffer without RNase. All tubeswere incubated at 37°C for 30 min. Digestion was termi-nated by addition of 300 µl RNase inactivation/precipita-tion mixture (Ambion) and transferred to a –20°C freezerfor at least 15 min in the presence of carrier yeast RNA(20 µg). Purified RNA was run on denaturing polyacry-lamide gels (5% acrylamide/8 M urea; 16 cm wide × 20cm long, 0.75 mm thick, with 20 wells) at 250 volts for 3h and transferred to a positively-charged nylon mem-brane (Ambion) by electroblotting. Nucleic acids werecrosslinked to the membrane and submitted to the detec-tion procedure for visualization of the non-isotopic probe(Ambion’s BrightStar BioDetect Kit). Bands were visual-ized by autoradiography of membrane using HyperfilmECL (Amersham). Biotinylated RNA Century-Plus markerand pUC 19 DNA-Sau 3A I digested DNA size standard(Ambion) were used for RNA bands molecular weightdetermination. The RNA bands were quantified by scan-ning densitometry.

Quantitative comparison of PCR products

To determine the transcription pattern of the Ca2+-ATPasesplice variants, quantitative reverse-transcribed PCR(RT–PCR) method was carried out as described above. Weused primer pairs 33–34, 35–36, 37–38 and 39–40 forPMCA1–4 respectively (Table 2B). After amplification, thesamples were analyzed on 1.2% agarose gel stained withethidium bromide and the cDNA bands were quantifiedby scanning densitometry.

Membrane preparation from islets of Langerhans

The fresh islets were washed twice in Hanks buffer con-taining proteinase inhibitors (see islets isolation proce-dure) and homogenized in a glass homogenizer inice-cold 5 mM Tris-HCl buffer (pH 8.0) containing pro-teinases inhibitors and incubated for 1 h at 4°C. Thehomogenate was centrifuged at 900 × g for 10 min to


remove nuclei and the final pellet (0.5–1 mg/ml protein)was resuspended in 20 mM MOPS buffer (pH 7.4) con-taining proteinases inhibitors. Membrane preparationswere aliquoted, frozen immediately in liquid nitrogenand stored at –70°C. Protein concentration was deter-mined using Bio Rad Dc Protein Assay (Bio-Rad) with BSAas standard.

Immunoprecipitation

Membrane samples (100 µg membrane proteins) wereused to perform immunoprecipitation using Immuno-precipitation Kit (Protein G) (Boehringer Mannheim). Theexperiment was carried out as described in protocolprovided by the manufacturer. The proteins wereimmunoprecipitated for 12–16 h at 4°C using four differ-ent specific rabbit antibodies directed against PMCA iso-forms 1, 2, 3, 4, at saturating concentration (dilution1:1000; SWant, Bellinzona, Switzerland).

Western blot analysis

Immunoprecipitated proteins were separated by elec-trophoresis on 1 mm-thick 7.5% SDS-polyacrylamide gel(SDS-PAGE) according to Laemmli [24]. Proteins weretransferred electrophoretically onto Hybond™ ECL™nitrocellulose membrane (Amersham) using an IMM-2Semi-Dry Blotting Device System (W.E.P. Company,California) with a continuous buffer system at 200 mA(10 V) for 2 h at room temperature. KaleidoscopePrestained Standards (Bio-Rad) were used for molecularmass determination of protein bands. Nitrocellulosemembranes (Amersham) were analyzed using BMChemiluminescence Western Blotting Kit (Mouse/Rabbit) (Boehringer Mannheim) and experiment wasdone as described in the protocol provided by the manu-facturer. Membrane proteins were incubated for 12–16 hat 4°C with four different rabbit-specific antibodiesdirected against PMCA isoforms 1, 2, 3, 4 diluted 1:1000in blocking solution. The bound antibodies weredetected with a POD-labelled secondary antibody (40mU/ml) and visualized by autoradiography of membraneusing Hyperfilm ECL (Amersham).

Statistics

The results are expressed as mean ± SEM.

RESULTS

To identify the alternatively spliced variants of rPMCA1,rPMCA2, rPMCA3 and rPMCA4 at sites A, B and C ininsulin-producing cells, reverse-transcribed PCR tech-nique was carried out using primers flanking the

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Fig. 1 Scheme summarizing described splice variants at site A(panel A), site B (panel B) and site C (panel C). The scheme alsoshows the genomic structure as deduced from data on the ratPMCA genes [for detailed references, see text]. Splice-out variantsat site B for PMCA2 and PMCA3 have not yet been described. APMCA3g splice variant has been described in human18, that, to ourunderstanding, corresponds to the splice variant PMCA3f found inrat13. Likewise, a PMCA4g has been described32, that wouldcorrespond to PMCA4xka or 4zka.

expected splice sites, in rat pancreatic β-cells, RINm5Fcells, and rat islets of Langerhans. The identity of thePCR products was determined by subcloning andsequencing at least two independent clones from distinctPCR amplifications (determined sequences not shown).

Site A

The described splice variants at site A and the genomicstructure of PMCA genes are illustrated in Figure 1. InPMCA2 and PMCA3, a 42-bp exon may be alternativelyspliced [25–27]. In PMCA4, a corresponding 36-bp exonmay also undergo alternative splicing [18,28], while inPMCA1, a corresponding 39-bp exon exists which isapparently never spliced out [18,28,29].

In the case of PMCA1, PCR amplification, using primerpair 1–2 (Table 1A) led to the identification of a single

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504-bp fragment containing the 39-bp fragment in thethree cellular preparations (Figs 2A–B, data of RINm5Fcells not shown). It corresponds to the isoform initiallydetected in rats and humans [7,30]. Hence, according tothe classification used by Stauffer et al. [1993], and ini-tially used by Adamo and Penniston [1992] to designatethe variants of isoform 2, insulin-producing cells areequipped with PMCA1x, like the other tissues studied sofar [18, 26].

For PMCA2 gene, previous results from PCR analysis ofcDNA libraries, as well as data on the genomic structure atsplice site A, revealed, in addition to the 42-bp exon, a sup-plemental 93-bp insertion, encoded by two distinct exonsof 33 and 60 bp, which could be included into the maturemRNA in four different combinations [26–27]. In the caseof PMCA2, PCR amplification, using primers 3–4 (Table1A), yielded only two bands of 306 and 348 bp in the threecellular preparations (Figs 2A–B, data of RINm5F cells notshown). These PCR products correspond to PMCAsincluding at site A the 93-bp insertion (33- and 60-bpexons; PMCA2y) or the three exons of 33, 60 and 42 bp(PMCA2w), the latter being the most abundant. These twosplice variants have been found to be transcribed in theheart, whilst PMCA2w was found also in stomach, liver,kidney, skeletal muscle, lung and uterus [18,26,27].

For PMCA3, cDNA amplification by PCR, using primers5–6 (Table 1A), yielded a single fragment of 521 bp in thethree insulin-secreting preparations (Figs 2A–B, data ofRINm5F cells not shown), representing the mRNA specieswithout the exon of 42 bp at site A (PMCA3z). This splicevariant corresponds to that detected by Burk and Shull(1992) in rat brain and by Stauffer et al. (1993) in cerebralcortex [18,25]. Stauffer et al. did not look for its presencein the heart but in a further work, it was found neither inthe foetal nor in the adult heart [31]. A minor fragment ofabout 600 bp was also found in pancreatic islets, that didnot correspond to a PMCA sequence.

PCR at site A of PMCA4, using primers 7–8 (Table 1A),yielded only one band of 524-bp in pancreatic β-cells andRINm5F cells, and two bands of 488 and 524 bp in ratislets of Langerhans (Figs 2A–B, data of RINm5F cells notshown). Sequence analysis showed that the 488-bp frag-ment represented the mRNA species without the exon of36 bp (PMCA4z) at site A, whereas the 524-bp fragmentcontained the exon of 36 bp (PMCA4x). Alternatively,spliced variants corresponding to PMCA4x have beenfound previously in most tissues while PMCA4z has beendetected in heart and testis only [18,28]. The presence ofboth PMCA4x and PMCA4z in the heart was recentlyconfirmed [31].

The sequences obtained (data not shown) were identi-cal to published sequences of rat gene for each PMCAisoform and subtype. Thus, data obtained indicate that β-cells, RINm5F cells, and islets of Langerhans express at


site A PMCA 1x, 2y, 2w, 3z, 4x, whereas islets ofLangerhans express PMCA4z in addition.


Pruified β Cells Pruified β Cells

M C 1 C 2 C 3 C 4 C 1 C 2 C 3 C 4 M

bp

1636

1018

517396344298220

bp

1636

1018

517396344298220

M C 1 C 2 C 3 C 4 C 1 C 2 C 3 C 4 M

Islets of Langerhans Islets of Langerhans

A C

DB

bp

1636

1018

517396344298220

bp

1636

1018

517396344298220

Fig. 2RT-PCR amplifications at site A and both sites B and C of PMCA1, PMCA2, PMCA3 and PMCA4 in purified β-cells (panels A andC) and pancreatic islet cells (panels B and D). PCR amplifications of cDNA at site A from rat purified β-cells (A) and pancreatic islets (B)were performed using primer pairs 1–2, 3–4, 5–6 and 7–8 (lanes 1, 2, 3 and 4 respectively). PCR amplification of cDNA at sites B and Cfrom rat purified β-cells (C) and pancreatic islets cells (D) were performed using primer pairs 9–10, 11–12, 13–14 and 15–16 (lanes 1, 2, 3and 4 respectively). The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. C: control RT-PCR (RNA without reverse transcriptase), M: marker (the molecular weights of the markers are indicated on the left or the right of thefigure).

Sites B/C

The possible splice variants at sites B and C with thegenomic structure of PMCA genes are illustrated inFigure 1. For site B, a human PMCA1 and PMCA4 cDNAwere identified in which the splicing process leads to theinclusion or exclusion of a 108-bp exon [11,12].Alternative splicing at site C has been demonstrated forall four isoforms [for a review, see ref. 10].

At site C, the mRNAs derived from the PMCA1 gene have six known spliced forms designatedPMCA1a–PMCA1f [7,13,30,32]. The alternative splicing atsite C leads to the inclusion or the removal of a singleexon of 154 bp containing three internal donor sites fromwhich inserts corresponding to 87-, 114- or 152-bp origi-nate. For PMCA1, PCR amplification, using primer pair9–10 (Table 1B), yielded a 358-bp fragment in all threepreparations (Figs 2C–D, data of RINm5F cells notshown). Sequencing revealed the absence of 154-bp exonat site C in PCR products. Thus, splice variant PMCA1b


with the 108 bp at site B is present in all three prepara-tions. Sequencing also revealed two clones (amongseven), originating from islet cells, that contained a 250-bp fragment, characterized by the absence of 154-bpexon at site C and the absence of the 108-bp exon at siteB, providing an additional 1b splice variant lacking the108-bp fragment in islet cells. In fact, in Figures 2C–D, a3 µl aliquot of the initial PMCA1 PCR reaction mixturewas reamplified giving rise to two PCR fragments of 250and 358 bp. In islet cells, splicing at site B occurred atposition 3325–3432 of rPMCA1 (7) (Genbank,AF076783). The presence of 1b splice variant in insulin-secreting cells was predicted from previous cDNA analy-sis experiments in pancreatic β-cells, RINm5F cells andislets of Langerhans [20]. Previous data on rat mRNA iso-forms [13] indicated that PMCA1b mRNA was detected insignificant levels in all tissues.

The alternative splicing at site C of PMCA2 mRNA ischaracterized by two exons (172 and 55 bp). The 172-bpexon or the two exons (227-bp) can be either included orexcised [13,18,26]. PMCA2 cDNA amplification, usingprimer pair 11–12 (Table 1B), yielded one band (417 bp)in pancreatic β-cells, RINm5F cells and islets of

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Langerhans (Figs 2C–D, data of RINm5F cells not shown).The 417-bp product lacking the 227 bp was analogous tothe 358-bp product found in PMCA1 (containing the108-bp exon at site B), providing the splice variantPMCA2b. This splice variant corresponds to that initiallyidentified by Shull and Greeb (1988) and to that previ-ously described in pancreatic β-cells, RINm5F cells andislets of Langerhans [7,20]. According to published data,PMCA2b corresponds to the most abundant formexpressed in rat brain, uterus, liver and kidney [13] andwas detected in significant levels in most tissues [18,26].

Site C of PMCA3 gene contains two exons of 68 and154 bp, that can be inserted or excluded. Internal donorsites in the exon of 154 bp can also be used, producingsplice variants analogous to those of PMCA1 gene[8,13,18,25]. A 154-bp splice variant followed by a 88-bpsequence, being an extension of the genomic DNA imme-diately downstream of the potential splice site at position154, has also been described [13]. PCR amplification ofPMCA3 cDNA, using primer pair 13–14 (Table 1B),resulted in two fragments of 512 and 445 bp in the threepreparations (Figs 2C–D, data of RINm5F cells notshown). Both isoforms presented the 108-bp exon at siteB. The predominant isoform (646 bp) contained the 154-bp exon at site C (PMCA3a), whereas the second con-tained a partial 87-bp exon at this site (PMCA3c). Frompublished data on PMCA3 mRNA, most of the PCR prod-ucts that have been identified lacked the 68-bp exon,and were directly analogous to PMCA1a, b, c, and d[13,18,25]. The most abundant splice variant in brain andin most other tissues is PMCA3a, whereas PMCA3c ispresent in low levels in kidney and testis, and in traceamounts in several other tissues [13]. A minor fragmentof about 390 bp was also found in RINm5F cells and pan-creatic islets, that did not correspond to a PMCAsequence.

For PMCA4, alternatively spliced variants at site C,result from the insertion or the exclusion of a 175-bp(175 bp in rat, 178 bp in human) exon that correspondsto the 154-bp exon of PMCA1 gene [9,13,17,33]. An inter-nal donor site, located 108 bp from the beginning ofexon C, may also be used [31]. cDNA amplification byPCR, using primer pair 15–16 (Table 1B), yielded onefragment of 343 bp in pancreatic β-cells and RINm5Fcells and two fragments of 343 and 518 bp in islets ofLangerhans (Figs 2C–D, data of RINm5F cells not shown).The two different PCR fragments contained the 108-bpexon at site B. The longest fragment contained the 175-bp exon at site C, providing the splice variant PMCA4a,whereas in the shorter, the 175-bp exon was excluded,providing the splice variant PMCA4b. These isoformscorrespond to those previously detected [20] in pancre-atic β-cells, RINm5F cells, and islets of Langerhans. PCRanalysis of the splicing pattern at site C demonstrated the

Cell Calcium (2000) 27(4), 231–246

presence of PMCA4a and PMCA4b mRNAs in most tis-sues [13]. A minor fragment of about 500-bp was alsofound in pancreatic islets, that did not correspond to aPMCA sequence.

The sequences obtained (data not shown) were identi-cal to published sequences of rat gene for each PMCAisoform and splice variants. Thus, for both sites B and C,data obtained indicate that β-cells, RINm5F, and islets ofLangerhans express PMCA 1b, 2b, 3a, 3c, and 4b, allwithout splicing at site B, with two supplemental iso-forms PMCA1b (with splicing at site B) and PMCA4a(without splicing at site B) in islets of Langerhans.

Characterization of RNA probes for PMCA1–4 and β-actin

RNA probes for PMCA1–4 and β-actin were designed tobe complementary to 300 nt and 250 nt of PMCA1–4and β-actin mRNA respectively. Calibration of RNase pro-tection assay using PMCA2 probe and target was carriedout as described in MATERIALS AND METHODS, usingvarying amounts of probes (200–1000 pg) and PMCA2target mRNA (50–250 pg) (Fig. 3A). A band of 300 bp wasobtained on the autoradiogram of the gel analysis ofRNase protection assay. Using densitometry scan to ana-lyze the autoradiogram, the RNase protection assayexhibited linearity within the range of 0–200 pg using a300 nt PMCA2 target (Fig. 3B). Owing to the weak con-centration of PMCA mRNA in islet cells and in order toperform quantification with more precision, PMCA1–4standard curves were made using amounts of corre-sponding target mRNAs between 0 and 36 pg. For bestestimation of PMCA1–4 and β-actin mRNA, 20 and 10 µgof cell RNA respectively, was needed.

Quantification of PMCAs mRNA in islets of Langerhansby RPA using external β-actin as positive control

Quantification of absolute amounts of PMCA1–4 and β-actin mRNAs in islets of Langerhans was carried out usingRNase protection assay. In such assays, under condition inwhich a biotin-labelled antisense probe is in large excessover the complementary target sequence, the amount ofprobe protected from digestion is proportional to theamount of target sequences. By using varying amounts ofa synthetic PMCA1–4 sense RNA (300 nt each) as target,standard curves were constructed relating target RNA toPMCA1–4 antisense probe protected RNA. Parallel assayswere performed using RNA from rat islet cells and a β-actin probe in a simple reaction run as positive control(Fig. 4). Each reaction contained two control tubes withyeast RNA as described in MATERIALS AND METHODS.Recovery of RNA using RNase protection assay has beenshown to range from 70 to 95% [34]. The amount of target



1 2 3 4 5 6 7

300 bp

A

B

Fig. 3 Calibration of RNase protection assay using PMCA2 probeand target. RNase protection assay was carried out using varyingamounts of PMCA2 target mRNA (0–250 pg) with PMCA2 probe inmolar excess. (A) Autoradiogram of gel analysis. The position ofthe 300 nt protected PMCA2 fragment is indicated. Lanes 1 and 2represent two positive control tubes containing the same amount oflabeled probe used for experimental tubes plus yeast RNAequivalent to highest amount of sample RNA, without or withRNase, respectively. Lanes 3, 4, 5, 6, 7 represent RNaseprotection assay carried out using 50, 100, 150, 200 and 250 pg oftarget. Autoradiogram was subjected to densitometric analysis. (B)Plot of densitometric units associated with the 300 nt protectedfragment as a function of the amount (pg) of the target mRNAadded.

assayed directly was used to deduce the amount of nativemRNA in islet cell RNA preparations. Protected fragmentlength was 300 and 250 nt for PMCAs and β-actin respec-tively, and the value obtained was corrected for differencesin length between protected fragment and message lengthfor PMCA1–4 (average value for message length was 4070,7139, 5050, 3996 nt for PMCA1–4 respectively) and β-actin (3335 nt). We have estimated islet cell total RNA tobe 66.93 ± 7.64 ng/islet, and the amount and number ofcopies of PMCAs and β-actin in 1 µg of total RNA was


corrected by the amount of total RNA in one islet. Figure 4show representative autoradiograms of gel analysis ofRNase protection assay for PMCA1–4. The amounts ofPMCA1–4 mRNA obtained are shown in Figure 5B. Whilethe number of copies of PMCA2, PMCA3 and PMCA4(related to β-actin) were rather abundant and comparable(25–30%), the number of PMCA1 copies was lower (16%).

Quantification of PMCAs mRNA in islets of Langerhansby RPA using internal β-actin as positive control

Quantification of absolute amounts of PMCA1–4 and β-actin mRNAs in islets of Langerhans was also carried outusing RNase protection assay. In such assays, we used asingle RNA sample and two different biotin-labeled antisense probes directed against the individual PMCAisoforms and β-actin, in large excess over the comple-mentary target sequence (Fig. 5A). The amount of β-actinprotected from digestion was used as correction factor tocompare the amount of each PMCA protected fromdigestion. The ratio PMCA/β-actin was used to comparethe proportion of each PMCA mRNA in islet cells (Fig.5B), and the ratio of PMCA/β-actin are shown in Figure5B. The results obtained were comparable to thoseobtained using external β-actin.

Quantitative analysis by RT-PCR of isoform subtypes ofCa2+-ATPase present in islets of Langerhans

To determine the respective proportion of the PMCA1, 2, 3,4 splice variants (PMCA1xb and 1xb variant lacking exonof 108 bp at site B; PMCA2yb and wb; PMCA3za and zc;PMCA4za and xb) in rat pancreatic islets, the quantitativeRT–PCR was performed using primer pairs 33–34, 35–36,37–38, 39–40 for PMCA1–4 respectively (Table 2B).RT–PCR amplification yielded the following bands of 358,250, 241, 199, 306, 238, 448, 273 bp for PMCA1xb,PMCA1xb variant lacking exon of 108 bp at site B,PMCA2wb, PMCA2yb, PMCA3za, PMCA3zc, PMCA4zaand PMCA4xb respectively. Several precautions must betaken to insure that the amount of the amplified fragmentis quantitatively related to the amount of template. Indeed,after a certain number of cycles, PCR reaches a plateau,depending on different individual factors. Therefore, thenumber of cycles corresponding to the exponential phaseof the PCR amplification was first determined. PCR amplifi-cation was carried out using two specific primers flankingthe putative splicing areas of PMCA1 (splicing sites B andC), PMCA2 (splicing site A), PMCA3 (splicing site C) andPMCA4 (splicing site C) cDNA and focusing on cycles28–40, 30–38, 28–36, and 28–36 respectively. As shown inFigure 6, the linear part of the amplification processdiffered from one isoform to the other. For PMCA1, the

Cell Calcium (2000) 27(4), 231–246


MW1 1 2 3 4 5 6 7 8 MW2 MW1 1 2 3 4 5 6 7 8 MW2

MW1 1 2 3 4 5 6 7 8 MW2MW1 1 2 3 4 5 6 7 8 MW2

bp

750500400

300

200

bp

750500400

300

200

bp

750500400300

200

bp

750

500400

300

200

bp

955

585

341

258

bp

955

585

341

258

bp

955

585341

258

bp

955585

341

258

A C

DB

Fig. 4 Quantification of PMCA1 (panel A), PMCA2 (panel B), PMCA3 (panel C) and PMCA4 (panel D) mRNA steady-state levels in isletsof Langerhans. Representative autoradiograms of gel analysis of RNase protection assay of PMCA calibration standards (lanes 1–6) andRNA from islet cells (lanes 7–8). These figures show the position of the 300 nt fragment protected by PMCA probe (lanes 1–7) and the 250nt fragment protected by β-actin probe (lane 8). Lanes: (1) undigested probe mRNA, (2) digested probe mRNA, (3–6) standard curve carriedout with 5, 10, 15, 20 pg (A, C, D) or 4, 8, 16, 32 pg (B) of calibration target respectively, (7) PMCA probe plus 20 µg of islet cells RNA, (8)β-actin probe plus 10 µg of islet cell RNA. MW1: Biotinylated RNA Century Marker Plus size standard. MW2: Biotinylated pUC 19 DNA-Sau3A I-digested size standard.

band corresponding to the PMCA1xb variant lacking exonof 108 bp at site B could not be visualized during the expo-nential phase, and only appeared when PCR reached aplateau, so that the amount of this splice variant could notbe estimated. For PMCA2, 3 and 4, cycles 35, 34, and 32respectively, were chosen for further work.

By combining the results obtained by RNase protec-tion assay an RT-PCR, the number of RNA copies and therelative level of each mRNA splice variant could be deter-mined (Table 3). There were five predominant splice vari-ants: PMCA1xb (15%), PMCA2wb (22%), PMCA3za(18%), PMCA3zc (13%), PMCA4xb (23%) and two minorsplice variants: PMCA2yb (4%) and PMCA4za (5%). In aprevious work, all variants were found to be expressed inthe β-cell, except one, PMCA4za [15].

Immunological detection of PMCA isoforms in islets ofLangerhans

Four different rabbit specific antibodies directed againstthe four different isoforms were used. Each of the four anti-

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bodies labelled 1 or 2 distinct bands having the expectedmolecular mass for each of the splice variants identified byPCR and RPA (Fig. 7), indicating that all of the identifiedisoforms are expressed at the protein level. The sensitivityof the antibodies, used in the present study, against PMCAs1, 3 and 4 has been shown to be equal, whilst the antibodydirected against PMCA2 gives a weaker signal [35].Therefore, because saturating amounts of the antibodieswere used for immunoprecipitation, our data indicate theexpression of substantial amounts of one splice variant ofPMCA1 (1xb) and PMCA2 (2wb), and of two splice variantsof PMCA3 (3za and 3zc) and PMCA4 (4za and 4xb).

DISCUSSION

The aim of the present study was to identify the variousPMCA isoforms expressed in the pancreatic β-cell and todetermine their expression at both the mRNA andprotein level.

The islet of Langerhans of the endocrine pancreas is anheterogeneous tissue containing different cellular types



MW1 1 2 3 4 5 6 7 8 9 10 11 MW2 bp

585

341

PMCA258β-actin

bp

500400

300

200

A

B

12

Fig. 5 Quantification of PMCA1–4 mRNA levels in islets of Langerhans using internal or external β-actin as control. (A) Representativeautoradiogram of gel analysis of RNase protection assay of PMCA1–4, using internal β-actin as a control. Position of the 300 nt fragmentprotected by PMCA1–4 mRNA, and 250 nt fragment protected by β-actin mRNA are indicated. Lane: (1, 4, 7, 10) undigested probe mRNA.Lanes: (2, 5, 8, 11) digested probe mRNA. Lanes: (3, 6, 9, 12) RNase protection assay carried out using 20 µg of islet cells RNA withPMCA1–4 probe mRNA and β-actin probe mRNA in molar excess, respectively. MW1: Biotinylated RNA Century Marker Plus size standard.MW2: Biotinylated pUC 19 DNA-Sau3A I-digested size standard. (B) Graph representing the mRNA levels of PMCA1–4 isoforms inpancreatic islet cells, using internal (closed columns) and external (open columns) β-actin as controls.

of which the insulin-producing β-cell only represents60–70% of the total population. The finding of the samesix isoforms in a 99% pure β-cell preparation, in theinsulinoma tumoural RINm5F cell line and in pancreaticislet cells, strongly argues for the presence of these sixisoforms in the pancreatic β-cell. It also suggests that,except in one case (see below), the isoforms found didnot correspond to very low-abundance mRNAs undulyamplified by RT–PCR. The finding of two supplementalisoforms only in islet cells goes along this line.


One interest of this work is the determination of theexpression pattern of the splice variants of the fourPMCAs at the three splicing sites A, B and C, simultane-ously. For instance, in the pancreatic β-cell, the splicevariants at only one site (C), have been previously identi-fied [20], and the determination of the expression patternof the splice variants of the PMCAs at the three splicingsites has only recently begun. Because multiple (2) splicevariants only occurred at one splice site at a time in thefour PMCAs of the β-cell and RINm5F cells, the present

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PMCA2

PM 1 2 3 4 5 PMbp

396298220

A

B D

PMCA3

PM 1 2 3 4 5 PM6

C

bp

506

298220154

PMCA4

PM 1 2 3 4 5 PM6

Ebp

506396298220

F

Fig. 6 Quantitative analysis of Ca2+-ATPase isoforms 2, 3 and 4 by reverse-transcribed PCR amplification in islet cells. (A) RT-PCRamplification of PMCA2 at cycles 30 to 38 (lanes 1–5). Bands at 241 and 199 bp represent PMCA2w and PMCA2y respectively. (B) Semi-logarithmic plots of the relative amplification of PMCA2w (filled circles) and PMCA2y (clear circles) at cycles 30 to 38. (C and E) Samepresentation as in A for PMCA3 and PMCA4, respectively, except that the number of cycles was 28 to 36. In C bands at 306 and 238 bprepresent PMCA3a PMCA3c respectively. In E, bands at 448 and 273 bp represent PMCA4a and PMCA4b respectively. (D and F) Samepresentation as in B. In D, PMCA3a (filled circles) and PMCA3c (open circles). In F, PMCA4a (open circles) and PMCA4b (filled circles) atcycles 28 to 36. RT–PCR was performed on cDNA from pancreatic islets. The PCR products were separated by agarose gelelectrophoresis and stained with ethidium bromide. PM: 1 kb DNA ladder (sizes in bases). The cDNA values were determined fromfluorescence of each PCR fragment by densitometry (arbitrary units). Data are means of three or four determinations. SEM when notpresented are smaller than symbols.

Table 3 Combination of RNase protection assay and RT-PCR for the determination of mRNA levels of Ca2+-ATPase splice variantspresent in islets of Langerhans

Islet cell mRNA level

PMCA1xb PMCA2wb PMCA2yb PMCA3za PMCA3zc PMCA4za PMCA4xb

Nb of copies/islet(x 105) 1.10 1.45 0.28 1.20 0.89 0.30 1.47Amount(fg/islet) 237 ± 9 550 ± 41 108 ± 8 323 ± 16 238 ± 12 63 ± 2 313 ± 11mRNA level (%)(PMCA/β-actin) 15.32 ± 1.04 21.69 ± 1.40 4.25 ± 0.28 18.08 ± 0.78 13.33 ± 0.57 4.60 ± 0.28 22.95 ± 1.41

Values represent the means ± SEM of three or four independent determinations.

study allows the exact determination of the various iso-forms present in β-cells and RINm5F cells, taking intoaccount the three splicing sites A, B and C. In islet cells,splicing of PMCA4 occurred at sites A and C simultane-

Cell Calcium (2000) 27(4), 231–246

ously, so that four splice variants could be generated. Foreach site, two bands of different abundance wereobserved, a small and a large one, the latter being ofcomparable size to that found in β-cells (4xb). Therefore,



Table 4 Summary of PMCA1, PMCA2, PMCA3, PMCA4 isoformsat the three sites A, B and C in insulin producing cells

Purified β-Cells RINm5F Cells Islets of Langerhans

rPMCA1xb rPMCA1xb rPMCA1xbrPMCA1xkb

rPMCA2yb rPMCA2yb rPMCA2ybrPMCA2wb rPMCA2wb rPMCA2wbrPMCA3za rPMCA3za rPMCA3zarPMCA3zc rPMCA3zc rPMCA3zcrPMCA4xb rPMCA4xb rPMCA4xb

rPMCA4za

All splice variants present the 108-bp exon at site B except 1 splicevariant of PMCA1. Its absence is designated by the letter ‘k’

1 2 3 4

kDa

200

135

81

Fig. 7 Immunological detection of plasma membrane Ca2+-ATPase isoforms in rat islets of Langerhans. 100 µg of membraneproteins were purified by immunoprecipitation using four differentrabbit antibodies directed against PMCA isoforms 1, 2, 3 and 4 andapplied in lanes 1–4 respectively. The gel was electroblotted ontonitrocellulose membrane which was then cut and incubated withcorresponding antibodies. The positions and sizes of themolecular-mass standards (MW) are indicated on the left.Antibodies directed against each of the four PMCAs label 1 or 2bands.

Fig. 8 Splice variants of the plasma membrane Ca2+-ATPasesfound in three insulin producing cell preparations. cDNAorganization of PMCAs is shown on top. Putative transmembranesegments 1–10 are indicated by black bars. Exons involved in thealternative splicing at sites A, B and C are represented by greyboxes. Flanking sequences that are conserved are represented bywhite boxes.

the abundant band corresponds to the splice variant 4xb,while the smaller would correspond to the combinationof the two other sequences identified at site A and C, pro-viding the splice variant 4za.

To designate splice variants at site A, the letters ‘x, z, y,w’ are used most frequently [26]. The splice variant withthe full 42-bp (or corresponding) insert is labelled ‘z’ andthe splice variant without the insert is labelled ‘x’. Thesplice variants labelled ‘y’ or ‘w’ contain extra exons tothe 42-bp insert. In the case of site C, the letters ‘b, a, c, d,e f, g’ are used [7,13,18,32,33]. The splice variant contain-ing the full exon is labelled ‘a’ and the splice variant with-out the insert is labelled ‘b’. Splice variants labelled ‘c’ to‘g’ contain partial or extra inserts. In the case of site B, nodefinite nomenclature has been proposed yet. Forinstance, in hPMCA4, a DNA fragment lacking the regioncorresponding to the 108-bp exon B and including a fulllength exon at site C was designated PMCA4 g [31]. Onthe other hand, a PMCA4 splice variant lacking both fulllength exons B and C [16] was designated PMCA4BICI,using two different letters (B and C) to characterize thetwo splice sites separately, in accordance with thenomenclature proposed by Carafoli (1994) [10]. In keep-ing with the logical of the nomenclature most frequentlyused to designate splice variants at sites A and C, and theproposal of Carafoli (1994) to use different (series of) let-ters to designate splice variants at different splice sites,we propose not to designate the fragment presenting the108-bp exon at site B and to designate ‘k’ the DNAfragment lacking the fragment. Therefore, we suggest the existence of rPMCA1xb, rPMCA2yb, rPMCA2wb,


rPMCA3za, rPMCA3zc and rPMCA4xb in rat pancreaticβ-cells and RINm5F cells, and of two supplemental iso-forms in rat islets, namely in non-β-cells of the islets ofLangerhans: rPMCA1xkb and rPMCA4za (Table 4 andFig. 8).

In previous work on alternative splice variants ofPMCAs at site C in insulin secreting cells, splice variants1b, 2b and 4b were found in pancreatic β-cells, and 4a inislets, namely in non β-cells of the islets of Langerhans

Cell Calcium (2000) 27(4), 231–246


[20]. Hence, with respect to site C, 2 additional splicevariants were identified in the present work: splice vari-ants 3a and 3c, providing PMCA3za and PMCA3zc (ascharacterized at the three sites).

Previous evidence of alternative splicing at site B hasbeen provided in human [11–12], but not yet in rat.Indeed, Keeton et al. [1993] have been unable to demon-strate alternative splicing at site B in rPMCA4 [13].Therefore, our PCR product demonstrates for the firsttime alternative splicing of rPMCA1 gene at site B.

The present study also shows that the identified splicevariants are expressed to a significant extent both at themRNA and protein level in pancreatic islet cells, with theexception of the newly identified variant PMCA1xkb.Alternative splicing at site B (exclusion of the 108-bpexon) leads to the deletion of the 10th, and apparentlyalso, of the 9th transmembrane domains of the ATPase[11,16]. The isoform of PMCA4 spliced out at site B wasfound to be inactive as an ATPase [16]. Hence, the pres-ence of splice variant PMCA1xkb in islet cells togetherwith its very low abundance at the RNA level points to itslow physiological importance and suggests that it couldresult from aberrations in splicing mechanisms, as previ-ously proposed [12,13,16]. Therefore, taking into accountthe estimated affinity of the antibodies for their respec-tive isoform (35), the most abundant splice variantsfound at the protein level in islet cells are PMCA1xb,2wb, 3za, 3zc, 4za and 4xb.

Possible functional consequences of alternativesplicing of PMCAs in pancreatic β-cells

The influence of splicing at site A, which is located nearthe phospholipid binding domain of the PMCA [14], hasbeen previously investigated in PMCA2, but no func-tional differences were observed between splice variants[36]. Whether the various splice variants generated bysplicing at site A differ or not in their regulation by acidicphospholipids, the splice variants expressed in the β-cellwere of the four types: 1x, 4x, 2y, 2w and 3z, providing afull range of A site spliced isoforms in this cell.

The calmodulin-binding domain is composed of a 28aa sequence rich in interspersed basic and hydrophobicresidues [37]. By binding to this region, CaM stimulatesthe pump by decreasing its Ca2+ affinity and increasingits maximum velocity. The CaM-binding domain alsoserves as an autoinhibitory region [37]. Alternate spliceof the PMCA genes at site C occurs in the middle of theregion coding the CaM-binding domain [8]. Hence, theinsertion of an exon at this place has been predicted,based on experiments with expressed C-terminal frag-ments of the PMCA1 pump, to alter the affinity of thepump for CaM [38]. Using synthetic peptides correspond-ing to this domain in ‘a’ and ‘b’ splice variants of PMCA4,

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Enyedi et al. (1991) showed that the peptide representingthe ‘a’ splice variant (insertion), has about a 10-fold loweraffinity for Ca2+-CaM and was also about 10-fold lesseffective as an inhibitor of the activated Ca2+ pump, thanthe ‘b’ splice variant (no insert) [39]. The study of theactivity of splice variants 4a and 4b, expressed in COS-cells [39], and of the properties of purified splice variants(4a and 4b) also showed a reduced calmodulin affinitywith higher activity in the absence of CaM, of ‘a’ com-pared to ‘b’ splice variants [16]. Four of the isoformsdetected in the β-cell were of the ‘b’ type, and two of the‘a’ or ‘c’ type. Therefore, the β-cell appears to be equippedwith four isoforms that would display ‘normal’ autoin-hibitory and regulatory behaviour, and with two supple-mental isoforms that, could display higher basal activity,a phenomenon that would help to transport Ca2+ at lowbasal [Ca2+]i or when insufficient calmodulin would beavailable to activate the pump [20]. In addition, their pre-sumed lower affinity to CaM could be interesting at high[Ca2+]i, such spliced-in isoforms constituting reservoirpumps coming into play when extrusion of high quanti-ties of Ca2+ is required, e.g. in excitable tissues [40].

Genes 1 and 4 are transcribed in most tissues, whilstgenes 2 and 3 are transcribed in specialized tissues.PMCA1 and PMCA4 may thus represent housekeepingisoforms whilst PMCA2 and PMCA3 may be required forspecialized functions in a limited number of tissues[13,17,25,32]. In fact, the housekeeping enzyme variantsare of the ‘b’ type (PMCA1b and PMCA4b), the other vari-ants showing a distinct cell type- and differentiation-spe-cific expression pattern [40]. For instance, 1a splice variantis found in myocytes and neuronal cells, whilst 1c variantis found in skeletal muscle and brain tissue [40].Undifferentiated myoblasts express only the mRNAs of the1b and 4b isoforms, whereas in differentiated cells, splic-ing variants 1c, 1d and 4a, having spliced-in exonsequences in their calmodulin-binding region, are alsoexpressed [40]. The PMCA expression pattern here foundin the β-cell, is in agreement with these observations.Thus, the β-cell appears to express two housekeepingenzyme variants (PMCA1b and PMCA4b) and two tissuetype-specific variants (PMCA3a and PMCA3c). Indeed, thepancreatic β-cell is electrically excitable and displays, inresponse to glucose, a characteristic pattern of electricalactivity consisting in slow oscillations of membrane poten-tial onto which spikes are superimposed. These bursts ofaction potentials are attended by slow [Ca2+]i oscillations,and have been postulated to represent long-lasting actionpotentials with properties reminiscent of the cardiacaction potential [41]. Here, it is interesting to notice thatthe sole tissue type-specific or ‘differentiated’ variant (‘a’ and ‘c’, [42]) were of the ‘3’ type, namely splice variantsof the isoform displaying the most restricted distribu-tion [42–44], and hence presumably the most specialized



function. In addition, the PMCA3 gene displays, in its 5’untranslated region, extensive trinucleotides repeatsequences [45]. Such sequences have been implicated inseveral disease states including Fragile X Syndrome,Myotonic Dystrophy and Huntington’s Disease [45].However, there appears to be no link between any pancre-atic β-cell disease state (e.g. diabetes) and the X-chromo-some to which PMCA3 has been mapped [46], except theexceedingly rare congenital absence of the β-cell or islet,which in some cases is an X-linked trait, resulting in severeinsulin-dependent diabetes mellitus in the neonate [47]. Inaddition, the potential implication of PMCA3 in inheritedneuromuscular disorders [45] is of particular interest inview of the similar ontogeny of neurons and β-cells, whichshare many of the enzyme systems, neurotransmitters andelectrophysiological characteristics usually consideredspecific to neural cells [47–48]. The β-cell also appears toexpress two additional isoforms of the 2b type (with differ-ence at site A): PMCA2yb, PMCA2wb, found in specializedand restricted tissues like kidney, liver, heart, brain andlung [13,26].

Interestingly, the splice variants found in the β-cellwere spliced in (or spliced out) at one site only at a time(A or C), a situation not found in brain where splicing inand out occur simultaneously at both sites [42]. Thispoints to the presence of a larger number of specializedsplice variants in brain.

The splicing option of the housekeeping enzymePMCA1 at site A is of the ‘x’ type, because PMCA1 isnever spliced out at site A [18,29,30]. However, the splic-ing option of the housekeeping enzyme PMCA4 at thelatter site has not yet been defined. From the presentstudy, it can be deduced to be, at least, of the spliced-intype, giving PMCA4xb. This is in agreement with previ-ous observations in human brain, where the 4b variantsfound in 14 different brain regions were all of the ‘x’ type[42]. The tissue type-specific splice variants found in theβ-cell were contrary to the spliced-out type (PMCA3zaand PMCA3zc), a picture not identical to that found inbrain (3za but not 3zc), whilst the PMCA2 variants wereof the spliced-in type (PMCA2wb and PMCA2yb), a pic-ture showing less complexity than in brain [42]. This,indeed, points to a very specialized function of the splicevariants of PMCA2 and three isoforms.

At the protein level, the most striking observation wasthat islet cells expressed substantial amounts of onePMCA2 and two PMCA3 splice variants. The presence ofthese ‘specialized’ isoforms in islet cells again points tospecial demands on the regulation of Ca2+ homoeostasisin these cells. Our data also provide the first evidence ofthe presence of PMCA2 and PMCA3 isoforms at the pro-tein level in non-neuronal tissue.

In conclusion, the β-cell expresses to a significantextent a variety of PMCAs, some that would have normal


autoinhibitory and regulatory behaviour, some thatcould display higher basal activity and/or come in to playat high cytosolic-free Ca2+ concentration, some havinghousekeeping activity, some other having more special-ized function. This variety would help the β-cell to main-tain appropriate Ca2+ homoeostasis as required foroptimal cellular activity and insulin secretion.

ACKNOWLEDGEMENTS

The authors thank M. Heinderyckx for initiation to DNAsequencing. This work was supported by the BelgianFund for Scientific Research (FRSM Nr 3.4545.96, LN9.4514.93 and LN 9.4510.95) of which FVE is SeniorResearch Assistant.

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expression of multiple plasma membrane ca2+-atpases in rat pancreatic islet cells

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