Keywords: Adenine nucleotide translocase, ANT, HNE, proton leak, UCP3, uncoupling protein, ANT, adenine nucleotide translocase, BSA, bovine serum albumin, HNE, reactive oxygen species, TPMP, triphenylmethylphosphonium, UCP, uncoupling protein, KO, knockout

Stimulation of mitochondrial proton conductance by hydroxyn (i) Synopsis

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of reactive oxygen species. This proton leak can be induced by lipid peroxidation products of reactive oxygen species, such as 4-hydroxynonenal (HNE). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and the adenine nucleotide translocase, thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We find that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent upon incubation with respiratory substrate. In the presence of HNE, mitochondria energised with succinate become progressively more leaky to protons over time compared to mitochondria in the absence of either HNE or succinate. Energised mitochondria must attain a high membrane potential to allow HNE to activate uncoupling; a drop of 10 -20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), adenine nucleotide translocase (64%) and other pathways (25%). Our findings show that exogenous HNE only activates uncoupling at high membrane potential. They suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.

resolved in [START_REF] Parker | Energisationdependent endogenous activation of proton conductance in skeletal muscle mitochondria[END_REF]). This discrepancy may highlight the condition-dependence of the interaction of HNE with uncoupling pathways and may be caused by differences in energisation regimes between the present work and [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. Such differences may also explain differences in activation of UCP1 by HNE observed in different laboratories [START_REF] Esteves | Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria[END_REF][START_REF] Shabalina | UCP1 and defense against oxidative stress. 4-Hydroxy-2nonenal effects on brown fat mitochondria are uncoupling protein 1-independent[END_REF]. The discrepancy may also reflect possible differences in the requirements for high membrane potential of activation of UCP3 and ANT by HNE -further work is needed to resolve this point. The observation that UCP3 catalyses about 11% of the effect of HNE while ANT catalyses about 64%, even though UCP3 is present at about 4 pmol/mg protein [18], while ANT is present at about 2 nmol/mg protein [19], suggests that the HNE-activated proton conductance of UCP3 is about 100-fold higher than that of ANT, providing a rationale for an important role of UCP3 in regulating proton conductance and ROS production even though ANT is also activatable and present in excess.

The present paper explores the conditions under which HNE stimulates the uncoupling of skeletal muscle mitochondria. Firstly, we find that extent of incubation time during energisation is important. Secondly, we find that a high membrane potential must be achieved during this energisation. Physiologically, uncoupling by HNE is thought to be involved in the negative feedback control of mitochondrial ROS production [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF] which is strongly attenuated if protonmotive force drops [START_REF] Miwa | Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling[END_REF][START_REF] Papa | Reactive oxygen species, mitochondria, apoptosis and aging[END_REF]. We propose that the requirement for a high membrane potential for stimulation of proton conductance by HNE ensures that this feedback uncoupling only occurs at very high membrane potential, and prevents the uncoupling of mitochondria by nonmitochondrial ROS and its peroxidation products produced during high workload when mitochondria are functioning at a lower membrane potential. Energisation-dependent stimulation of proton leak in rat skeletal muscle mitochondria by 50 µM HNE Proton leak kinetics were measured in rat skeletal muscle mitochondria as described in the experimental section. (A) Timings of additions to the incubation chamber. Mitochondria plus inhibitors (M) were added at t = -10 min, 50 µM HNE (where present) was added at t = -9 min, 4 mM succinate (S) was added at the times indicated, then leak kinetics (LK) were measured between t = 0 and t = 3 min by titration of membrane potential and oxygen consumption rate with malonate from 0.14 to 2.

Figure 1

 1 Figure 1Energisation-dependent stimulation of proton leak in rat skeletal muscle mitochondria by 50 µM HNE Proton leak kinetics were measured in rat skeletal muscle mitochondria as described in the experimental section. (A) Timings of additions to the incubation chamber. Mitochondria plus inhibitors (M) were added at t = -10 min, 50 µM HNE (where present) was added at t = -9 min, 4 mM succinate (S) was added at the times indicated, then leak kinetics (LK) were measured between t = 0 and t = 3 min by titration of membrane potential and oxygen consumption rate with malonate from 0.14 to 2.3 mM. Kinetic response of mitochondrial proton leak rate (measured as oxygen consumption rate) to membrane potential measured after energisation with 4 mM succinate for 0.75 (circles), 2.5 (triangles) or 5.0 min (squares) was measured minus (B) or plus (C) 50 µM HNE. (D) Proton leak rate interpolated at 166 mV (solid vertical lines in (B) and (C)); the average highest protonmotive force common to all curves generated in the experiment. Data are means ± S.E.M. of duplicate experiments performed on five separate preparations. One-way ANOVA was performed for (D); p = 0.0001, with Tukey's Multiple Comparison Test used posthoc: *significantly different: p < 0.05; **, p < 0.001.

  Figure 1Energisation-dependent stimulation of proton leak in rat skeletal muscle mitochondria by 50 µM HNE Proton leak kinetics were measured in rat skeletal muscle mitochondria as described in the experimental section. (A) Timings of additions to the incubation chamber. Mitochondria plus inhibitors (M) were added at t = -10 min, 50 µM HNE (where present) was added at t = -9 min, 4 mM succinate (S) was added at the times indicated, then leak kinetics (LK) were measured between t = 0 and t = 3 min by titration of membrane potential and oxygen consumption rate with malonate from 0.14 to 2.3 mM. Kinetic response of mitochondrial proton leak rate (measured as oxygen consumption rate) to membrane potential measured after energisation with 4 mM succinate for 0.75 (circles), 2.5 (triangles) or 5.0 min (squares) was measured minus (B) or plus (C) 50 µM HNE. (D) Proton leak rate interpolated at 166 mV (solid vertical lines in (B) and (C)); the average highest protonmotive force common to all curves generated in the experiment. Data are means ± S.E.M. of duplicate experiments performed on five separate preparations. One-way ANOVA was performed for (D); p = 0.0001, with Tukey's Multiple Comparison Test used posthoc: *significantly different: p < 0.05; **, p < 0.001.

Figure 2

 2 Figure 2Sensitivity of HNE-stimulated proton leak in rat skeletal muscle mitochondria to membrane potential (A-C), effect of malonate. (D-F), effect of KCN. (A,B,D,E), proton leak kinetics. 4 mM succinate was added at t = -1.5 min (see Fig.1A) in the absence (open symbols) or presence (closed symbols) of 50 µM HNE. Proton leak kinetics were measured between t = 0 and t = 3 min after no treatment (A, D) or addition of 430 µM malonate (B) or 43 µM KCN (E) immediately prior to addition of succinate. (C, F), inhibition of HNE-stimulated proton leak by malonate and KCN. The increase in proton conductance at t = 0 caused by addition of 50 µM HNE was interpolated (solid vertical lines, (A,B,D,E)) at the highest common membrane potential, 164 mV in (C) and 169 mV in (F). 'X' indicates the drop in membrane potential measured at t = 0 caused by pre-addition of malonate or KCN. (G), effect of this drop in membrane potential on HNE-stimulated proton conductance. The increases in proton leak rate from (C) and additional experiments (not shown) using 140 µM and 290 µM malonate (circles), and from (F) and additional experiments (not shown) using or 14 µM and 29 µM KCN (triangles) are plotted against the drop ('X') in membrane potential at t = 0 in the absence of HNE. Data are means ± S.E.M. of 5 (A-C) or 6 (D-F) experiments each performed in duplicate. **Significantly different from zero addition by paired Student's t test: p < 0.01; ***, p < 0.001. Lines in (G) are linear regressions.

Figure 3

 3 Figure 3 Contribution of UCP3 to HNE-stimulated proton leak after 2.5 min energisation in mouse skeletal muscle mitochondria (A, B) proton leak kinetics. (A) mitochondria from wild-type mice. (B), mitochondria from Ucp3KO mice. 4 mM succinate was added at t = -2.5 min, minus (open symbols) or plus (closed symbols) 50 µM HNE. (C), proton leak due to UCP3 measured by interpolation at 160 mV (solid vertical lines in (A,B)) as (A) minus (B). Data are means ± S.E.M. of 12 experiments, each performed in duplicate. *Significantly different from control by paired Student's t test: p < 0.05.

Figure 4

 4 Figure 4Contribution of ANT to HNE-stimulated proton leak after 2.5 min energisation in Ucp3KO mouse skeletal muscle mitochondria

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S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

(vi) Acknowledgements

We thank Dr. Mary-Ellen Harper (Ottawa) for Ucp3KO mice and Julie Buckingham and Helen Boysen for technical assistance. This work was supported by the Medical Research Council and the Wellcome Trust (grants 065326/Z/01/Z and 066750/B/01/Z).

(vii) References


Cornelia Kornblum

Gábor Zsurka

Rudolf J Wiesner

Rolf Schröder

Dr Wolfram S Kunz
email: wolfram.kunz@ukb.uni-bonn.de.

Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

Keywords: mitochondrial DNA, chronic progressive external ophthalmoplegia, mitochondrial tRNA, oxidative phosphorylation, mitochondrial disorders, mitochondrial DNA diseases 1

Chronic progressive external ophthalmoplegia (CPEO) 1 is a common mitochondrial disease phenotype in adults which is due to mitochondrial DNA (mtDNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge particularly when several mtDNA sequence variants are present. Here we report on a CPEO patient in whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315, T1658C in tRNA Tyr , tRNA Ile , and tRNA Val genes. In skeletal muscle, tRNA Val and tRNA Ile mutations were homoplasmic, while the tRNA Tyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fiber analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibers, (ii) Northern blot analyses of mitochondrial tRNA Tyr , tRNA Ile , tRNA Val . We demonstrated that both the G5835A tRNA Tyr and del4315A tRNA Ile mutation have serious functional consequences. Single fiber analyses displayed a high threshold of the tRNA Tyr mutation load for biochemical phenotypic expression on the single cell level indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory chain activities, a reduced overall cytochrome c oxidase (COX) staining intensity, and abundant COX-negative fibers. Northern blot analyses showed a dramatic reduction of tRNA Tyr and tRNA Ile levels in muscle with impaired charging of tRNA Ile , whereas tRNA Val levels were only slightly decreased with aminoacylation unaffected. Our findings suggest that the heteroplasmic tRNA Tyr and homoplasmic tRNA Ile mutation act together resulting in a concerted effect on the biochemical and histological phenotype. Thus, homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.

Introduction

Chronic progressive external ophthalmoplegia (CPEO) is one of the most common mitochondrial disease phenotypes in adults. In approximately 50% of CPEO patients, mitochondrial DNA (mtDNA) analysis of skeletal muscle tissue reveals single large-scale mtDNA deletions that usually arise sporadically [START_REF] Biousse | Neuro-ophthalmology of mitochondrial diseases[END_REF]. However, CPEO may as well be due to maternally inherited or sporadic mtDNA point mutations, or to mutations in nuclear genes resulting in autosomal disorders of intergenomic communication [START_REF] Hirano | ANT1, Twinkle, POLG, and TP: new genes open our eyes to ophthalmoplegia[END_REF][START_REF] Mancuso | Mitochondrial DNA-related disorders[END_REF]. The identification of the specific pathogenic disease-defining mtDNA point mutation often remains a challenge, particularly when several mtDNA sequence variants are present. Due to the high variability of the mitochondrial genome some earlier established criteria for pathogenicity of mtDNA mutations, e.g. the absence in a control population, might fail in cases of previously not described "private" mtDNA mutations. Moreover, even heteroplasmy is no longer a clear criterion for pathogenicity since many heteroplasmic polymorphisms have been reported [START_REF] Tully | A sensitive denaturing gradient-gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region[END_REF], and various homoplasmic pathogenic tRNA mtDNA point mutations have been identified to date [START_REF] Mcfarland | Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation[END_REF][START_REF] Mcfarland | Familial myopathy: new insights into the T14709C mitochondrial tRNA mutation[END_REF][START_REF] Taylor | A homoplasmic mitochondrial transfer ribonucleic acid mutation as a cause of maternally inherited hypertrophic cardiomyopathy[END_REF][START_REF] Limongelli | Variable penetrance of a familial progressive necrotising encephalopathy due to a novel tRNA(Ile) homoplasmic mutation in the mitochondrial genome[END_REF][START_REF] Scuderi | Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene[END_REF].

Materials and Methods

Patients

A 22-year-old male patient of Turkish ethnic origin (patient A) presented with a medical history of slowly progressive upper eye lid ptosis first realized at the age of 15 years. Later, he developed incomplete external ophthalmoplegia as well as exercise intolerance. His further past medical history was uneventful. All family members lived in a secluded village in the province of Sakarya (Adapazari), Eastern Turkey, and were reported to be free of neuromuscular symptoms. The patient underwent a neurological investigation, open skeletal muscle biopsy including histochemical, biochemical, and molecular genetic investigations as well as genetic analyses of leukocyte and buccal mucosa DNA. Blood for genetic analysis was obtained from all accessible family members (mother, two sisters, and one brother).

To compare functional features of mtDNA mutations in patient A, we obtained corresponding histological and biochemical data from patient B (female, aged 37 years at time of open skeletal muscle biopsy) presenting with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) due to the tRNA Leu(UUR) A3243G mtDNA point mutation. In patient B, only skeletal muscle tissue was available for analysis. Skeletal muscle biopsies from 43 neurologically unaffected patients (25 male, 18 female, age range 3-77 years) and no histologic evidence for a neuromuscular disorder were used as controls.

All examinations were conducted according to the Declaration of Helsinki (2000) of the World Medical Association and to guidelines of the local ethics committee of the University of Bonn. Informed consent was obtained from all investigated patients.

Skeletal muscle histology

6 m consecutive cryostate sections of open vastus lateralis muscle biopsies were processed for standard histologic and enzyme-histochemical work-up as previously described [START_REF] Dubowitz | Muscle biopsy, a practical approach[END_REF]. Quantitative histochemical single fiber analyses of cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activities were performed using fiber specific grey level determination of 12-bit video images in patient A [START_REF] Varlamov | Metabolic consequences of a novel missense mutation of the mtDNA CO I gene[END_REF][START_REF] Zsurka | Tissue dependent co-segregation of the novel pathogenic G12276A mitochondrial tRNALeu(CUN) mutation with the A185G D-loop polymorphism[END_REF].

Biochemical investigations

Respiratory chain enzyme activities [rotenone sensitive NADH:CoQ1 oxidoreductase (complex I), COX (complex IV)], and citrate synthase (CS) in skeletal muscle homogenate were measured spectrophotometrically as previously described [START_REF] Wiedemann | Evaluation of methods for the determination of mitochondrial respiratory chain enzyme activities in human skeletal muscle samples[END_REF]. Results were given in U/g wet weight and were corrected for potential fiber type variations or adaptive mitochondrial proliferation by normalization of data for the mitochondrial matrix marker enzyme CS.

Molecular genetic analyses

We extracted genomic DNA from about 30 mg skeletal muscle specimens and from buccal mucosa with the QiaAmp DNA Mini Kit (Qiagen, Hilden, Germany). Genomic DNA was isolated from 10 ml aliquots of EDTA anticoagulated blood using a salting out method [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF]. MtDNA was screened for large-scale rearrangements by Southern blotting of PvuII-cleaved total DNA from skeletal muscle using digoxigenin-labeled isolated human mtDNA as full length mtDNA probe (all restriction endonucleases from New England Biolabs, Frankfurt, Germany). The presence of duplications was excluded by digestion with PmeI and BamHI. For direct sequence analysis, 28 overlapping PCR fragments were amplified from the whole mitochondrial genome of skeletal muscle DNA and sequenced using a commercial sequencing service (MWG Biotech, Ebersberg, Germany).

To quantitate A3243G and G5835A mutation loads, we used different polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays. For quantification of G5835A, we used a forward primer 5'-GCACCCTAATCAACTGGCTTC-3', and a reverse mismatch primer (mismatch underlined) 5'-CTAAAGACAGGGGTTAGGGCT-3' to introduce a novel BanII restriction site in the wild-type allele. To introduce a novel BceAI restriction site in the mutant allele, we designed a forward primer 5'-AGCTAAGCACCCTAATCAACTG-3', and a mismatch reverse primer with a T7-extension 5'-TAATACGACTCACTATAGGGAAATCTAAAGACAGGGGTTAGGCCG-3' (italic, T7extension). For quantification of A3243G, a PCR product was amplified using a forward primer 5'-GAAAGGACAAGAGAAATAAGGCCT-3', and a reverse primer, 5'-GGAATTGAACCTCTGACTGTAAAG-3'. PCR products were digested with HaeIII.

Amplification conditions were as follows: 95°C for 10 min; 33 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 40 s; and finally 72°C for 7 min. We separated restriction fragments on 10% polyacrylamide gels and visualized them by SYBR Green I staining (Sigma-Aldrich, Steinheim, Germany). The proportions of mutant and wild-type mtDNA were calculated from band intensities with local area background subtraction (Image J analysis software, rsb.info.nih.gov/ij).

Single fiber PCR analysis

We used 10 m sections of unstained frozen skeletal muscle tissue of patient A located inbetween the muscle sections stained for COX and SDH activity. Fiber types of single fibers were distinguished based on myosin ATPase stainings at pH 4.6 and 9.4. For single fiber PCR analysis, we randomly selected 20 type I fibers with comparable SDH activity quantified by fiber specific grey level determination to examine the dependency of the cellular biochemical function on the mutation load. Laser microdissection of muscle fibers was performed as previously described [START_REF] Zsurka | Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy[END_REF]. To determine the G5835A mutation load in single fibers, we used a nested PCR technique. First, we amplified a PCR product spanning the mutation site using the forward primer 5'-CCTTACCACGCTACTCCTACCTATCTCC-3' (nucleotides 5462-5489), and the reverse primer 5'-CTCCAGCTCATGCGCCGAATAATAG-3' (nucleotides 5985-5961). In the second round of nested PCR, we subjected PCR products of the first round to mismatch PCR-RFLP with digestion of the wild-type allele as described above.

Northern Blot

Total RNA was isolated from a small piece of skeletal muscle tissue of patient A, 30-50 mg pieces of muscle tissue of four healthy control subjects, and cultured human osteosarcoma cells (143B cell line). To maintain charging of the tRNAs with amino acid and separate tRNA from aminoacylated tRNA, RNA was isolated and electrophoresed under acidic conditions on long gels as previously described in detail [START_REF] Maniura-Weber | Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNA Leu(UUR) ) gene[END_REF]. Regions of mtDNA encompassing mitochondrial tRNA genes were amplified from 143B cell DNA by PCR and used as probes. For each tRNA, two different probes were used, either specifically designed for wild-type or mutant tRNA. Primer sequences used for the design of tRNA Leu(UUR) , tRNA Val , tRNA Tyr , and tRNA Ile probes can be obtained from the corresponding author by request. For the normalisation of tRNAs to a non-mitochondrial small RNA species, blots were hybridised to a 5S rRNA probe (5'-GGGTGGTATGGCCGTAGAC-3') endlabeled using (γ-32 P) ATP (5000 Ci/mmol, Hartmann Analytic, Braunschweig, Germany) and T4-PNK [START_REF] Yasukawa | A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile)[END_REF]. Purified PCR products (QIAquick PCR purification columns, Qiagen, Hilden, Germany) were radiolabeled with (α-32 P)-dCTP (5000 Ci/mmol; Hartmann Analytic, Braunschweig, Germany) by the random-primer method, and unincorporated nucleotides were removed from labeling reactions by gel filtration through ChromaSpin columns (Clontech, Heidelberg, Germany). Prehybridisation, hybridisation and analysis of the data by phosphoimaging was carried out as described [START_REF] Maniura-Weber | Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNA Leu(UUR) ) gene[END_REF]. The two bands corresponding to aminoacylated and non-acylated tRNA were identified by comparison of native to alkali-treated, deacylated RNA samples from 143B cells and quantitated individually in order to estimate the degree of loading with amino acid. Finally, the sum of both values was divided by the signal for 5S rRNA to obtain tissue levels of tRNAs.

Results

Histochemical and biochemical evaluation of skeletal muscle shows an exceptionally severe mitochondrial pathology

As shown in Fig. 1, back-to-back stained muscle biopsy sections from patient A revealed a weaker overall COX staining, abundant SDH-positive COX-negative fibers (indicated by circles, "mosaic" COX staining pattern), and ragged red fibers (stars) in comparison to patient B with a similar mutation load of the heteroplasmic A3243G mtDNA point mutation (Fig. 1b,e). The low overall COX staining of muscle fibers from patient A is not a result of a lowered mitochondrial content as evidenced from the SDH staining intensities in Figs. 1c andf. Mitochondrial respiratory chain enzyme activities in skeletal muscle homogenate of patient A were compared to controls and to patient B (Table 1). In line with the histological stainings the data showed a dramatic decline of absolute activities of complex I and complex IV in the biopsy of patient A, but also when normalized for CS activity.

MtDNA sequence analysis reveals three novel and potentially pathogenic tRNA mtDNA point mutations

MtDNA large-scale rearrangements were ruled out. Direct sequence analysis of the mitochondrial genome isolated from skeletal muscle tissue of patient A revealed three novel and putative pathogenic tRNA mtDNA point mutations among a multitude of mutations when compared to the reference sequence [START_REF] Andrews | Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA[END_REF]. For mutation details, please refer to Fig. 2. a) We identified a novel G to A exchange at position 5835 of mtDNA which was found to be heteroplasmic in skeletal muscle. The G5835A mutation is located in the mitochondrial tRNA Tyr gene and causes a C→U transition at an evolutionary highly conserved position in the T-stem of the tRNA Tyr (Fig. 2a). The mutation has not been previously described in human mtDNA databases and is not present in more than 2500 controls (cf. MITOMAP, www.mitomap.org; Human Mitochondrial Genome Database, www.genpat.uu.se/mtDB).

b)

We also detected a previously not described homoplasmic mutation, del4315A, in the mitochondrial tRNA Ile gene affecting the tRNA TC loop (Fig. 2b). c) Finally, a not yet reported homoplasmic U→C transition was detected at position 1658 in the mitochondrial tRNA Val gene affecting the tRNA T-stem at an evolutionary not well conserved position (Fig. 2c). Further sequencing revealed two novel homoplasmic mutations in protein-coding genes: a C11972T transition in the ND4 gene, and a A14539G transition in the ND6 gene (cf. MITOMAP; Human Mitochondrial Genome Database), both genes encoding subunits of complex I. With high probability C11972T and A14539G are without functional consequences since they do not result in amino acid changes. A known heteroplasmic D-loop polymorphism was detected at position 72 resulting in a T→C transition in skeletal muscle mtDNA. Sequencing revealed the following homoplasmic polymorphisms being in line with the mitochondrial haplogroup "H": 73A, 2706A, 7028C. More than 400 of the publicly available complete mitochondrial coding region sequences belong to haplogroup "H". None of the five novel mutations mentioned above has been reported in these phylogenetically related individuals, or in more than 2100 other, not related sequences. The further identified polymorphisms C64T, A263G, ins310C, A750G, A1438G, A4769G, A8860G, A15326G, A16269G, and T16519C did not allow a more precise classification of the sample to the subgroups of the "H" haplogroup so far described [START_REF] Achilli | The molecular dissection of mtDNA haplogroup H confirms that the Franco-Cantabrian glacial refuge was a major source for the European gene pool[END_REF].

Mismatch PCR-RFLP analysis demonstrated in accordance with the sequence analysis (Fig. 3a) the presence of G5835A in 69  3% of skeletal muscle bulk mtDNA in patient A (Figs. 3b,c, lane 2). This mutation was restricted to muscle and not present in buccal mucosa and blood (Figs. 3b,c, lanes 3-4). Analysis of blood samples of two sisters, one brother, and the patient's mother did not reveal detectable amounts of the heteroplasmic mutation (Figs. 3b,c, lanes 5-8). These findings suggest a mutational event after germ-layer differentiation, however a mutation arisen early in embryogenesis or in the maternal germ line cannot be excluded. All other mutations were found to be homoplasmic in the blood of the patient, one sister, and their mother (other relatives were not analysed for these mutations).

Skeletal muscle single fiber PCR analysis in patient A showed that all SDH-positive COXnegative fibers as evidenced by comparison of COX-and SDH-activity (Fig. 1b,c) harboured at least 98% of the G5835A mutation, which demonstrates a high threshold for phenotypical expression on the single cell level (Fig. 4).

The levels of the mitochondrial tRNAs for tyrosine and isoleucine are severely reduced

In comparison to the nuclear encoded 5S rRNA, which served as a loading control, we observed a 95% reduction of the tRNA Tyr amount in total skeletal muscle RNA of patient A (Fig. 5a,b). tRNA Ile levels were found to be decreased by about 85%. In contrast, the tRNA Val amount was reduced by only about 60%. The levels of tRNA Leu(UUR) were even slightly increased. The same results were independently obtained for each tRNA with two different probes designed for wild-type (Fig. 5a) or mutant tRNA (data not shown). This approach rules out the possibility that lower tRNA signals in the patient`s muscle might be caused by poorer hybridization of wild-type probes to mutant tRNAs. Evaluation of the ratio of aminoacylated vs. non-acylated tRNA showed that tRNA Tyr and tRNA Val were predominantly loaded with amino acid in control muscles, while tRNA Ile and tRNA Leu(UUR) were equally present in acylated and deacylated forms. As shown in Fig. 5a, charging of tRNA Ile with amino acids was clearly impaired in the patient`s muscle, with less than 15% compared to app. 40% in controls.

Discussion

The diagnosis of mitochondrial disorders often requires a complex approach including clinical evaluation, skeletal muscle histology, biochemical and finally molecular genetic analyses [START_REF] Siciliano | Functional diagnostics in mitochondrial diseases[END_REF]. However, assigning pathogenicity to mtDNA point mutations is often a challenge [START_REF] Dimauro | Mitochondrial DNA mutations in human disease[END_REF][START_REF] Mcfarland | Assigning pathogenicity to mitochondrial tRNA mutations: when "definitely maybe" is not good enough[END_REF], especially when novel mutations are present in individuals with an ethnical background that has not been extensively studied with respect to haplogroup defining mutations. Moreover, since various homoplasmic tRNA mtDNA point mutations have been proved to be pathogenic [START_REF] Mcfarland | Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation[END_REF][START_REF] Mcfarland | Familial myopathy: new insights into the T14709C mitochondrial tRNA mutation[END_REF][START_REF] Taylor | A homoplasmic mitochondrial transfer ribonucleic acid mutation as a cause of maternally inherited hypertrophic cardiomyopathy[END_REF][START_REF] Limongelli | Variable penetrance of a familial progressive necrotising encephalopathy due to a novel tRNA(Ile) homoplasmic mutation in the mitochondrial genome[END_REF][START_REF] Scuderi | Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene[END_REF] and many apparently neutral polymorphisms are heteroplasmic [START_REF] Tully | A sensitive denaturing gradient-gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region[END_REF], the heteroplasmic state of a mutation is no longer a major criterion for pathogenicity. Equally problematic is the criterion of degree of conservation among species, since e.g. the A3243G tRNA Leu(UUR) mtDNA mutation with proven pathogenicity in humans is the normal genotype in Canis familiaris. This diagnostic challenge is amply illustrated in the genetic analysis of our CPEO patient in whom we detected five novel mtDNA mutations. Two mutations were located in proteincoding genes and were silent mutations, not changing amino acids. However, all further three novel tRNA mtDNA point mutations are potentially pathogenic. To address the pathogenic relevance of each of these mutations, we performed (i) single skeletal muscle fiber analysis, and (ii) Northern blot analyses as functional tests.

Demonstrating a correlation between biochemical phenotype and mutation loads of heteroplasmic mtDNA point mutations in individual cells has been long used as a strong evidence for pathogenicity [START_REF] Mcfarland | Assigning pathogenicity to mitochondrial tRNA mutations: when "definitely maybe" is not good enough[END_REF]. In our patient, we found that those muscle fibers that displayed a failure of oxidative phosphorylation (as indicated by negative COX staining in SDH-positive fibers) harboured very high levels of the heteroplasmic G5835A tRNA Tyr mutation. Thus, the heteroplasmic tRNA Tyr mutation is proven to be functionally relevant and plays a primary role in creating the mosaic pattern in our histochemical COX muscle stainings. Its 98% mutation load threshold for cellular biochemical impairment is however higher than described for the A3243G tRNA Leu(UUR) mutation [START_REF] Petruzzella | Extremely high levels of mutant mtDNAs co-localize with cytochrome c oxidasenegative ragged-red fibers in patients harboring a point mutation at nt 3243[END_REF] and the G12276A tRNA Leu(CUN) mtDNA mutation [START_REF] Zsurka | Tissue dependent co-segregation of the novel pathogenic G12276A mitochondrial tRNALeu(CUN) mutation with the A185G D-loop polymorphism[END_REF][START_REF] Cardaioli | A novel heteroplasmic tRNA(Leu(CUN)) mtDNA point mutation associated with chronic progressive external ophthalmoplegia[END_REF], which suggests a mild functional effect of the mutation. This contrasts to the severe biochemical alterations present in the patient's skeletal muscle homogenate (Table 1) and to the histological findings with reduced overall COX staining intensity, a mosaic pattern of COX activity, and abundant ragged red fibers (Fig. 1). The biochemical phenotype and general decrease in COX activity (including fibers with low G5835A mutation loads) apart from the mosaic pattern cannot be explained by the presence of the heteroplasmic G5835A tRNA Tyr mtDNA mutation alone. Functional analyses of both further tRNA mtDNA mutations demonstrated that the tRNA Ile mutation plays an addition role for pathogenicity. Since this mutation is homoplasmic, it affects all cells, which readily explains the overall decrease of the COX staining in skeletal muscle biopsy. In this respect, Northern blot analyses revealed that apart from tRNA Tyr , also tRNA Val and tRNA Ile showed reduced steady state levels, leaving a remaining 40% and 15% of the respective tRNAs as compared to controls. The latter value is similar to residual tRNA amounts reported in previous studies on homoplasmic pathogenic tRNA mtDNA mutations (9-15% and 5-10%, respectively [START_REF] Mcfarland | Familial myopathy: new insights into the T14709C mitochondrial tRNA mutation[END_REF][START_REF] Taylor | A homoplasmic mitochondrial transfer ribonucleic acid mutation as a cause of maternally inherited hypertrophic cardiomyopathy[END_REF]). Impaired aminoacylation of tRNA Ile was also observed further stressing the pathogenic relevance. However, the presence of the homoplasmic tRNA Ile mutation alone appears not to cause serious clinical symptoms. The patient's maternal relatives carried all homoplasmic mutations present in the patient, but no family member showed apparent signs of a neurological disorder. We suggest that the G5835A tRNA Tyr and del4315A tRNA Ile mutation act in concert and perform a "double trouble" of translation of mitochondrially encoded proteins. Since Tyr and Ile codons are present at high frequencies in most protein coding mitochondrial genes, low levels of these tRNAs in combination with a defect in charging of tRNA Ile with amino acids readily explain the severe biochemical and histological phenotype. In contrast, the 60% reduction of tRNA Val is probably not sufficient to contribute to an additional impairment of translation. This interpretation is in line with the conservation of sites among speciesthe tRNA Tyr mutation is located at a strictly conserved site, the tRNA Ile mutation is located at a site which is conserved among primates, while the tRNA Val mutation occurred at a non-conserved site. Not surprising, Northern blot analysis confirmed the considerable pathogenic role of the heteroplasmic G5835A tRNA Tyr mutation since tRNA Tyr steady state levels were found to be decreased by 95%. This is higher than the skeletal muscle bulk mtDNA mutation load which might indicate an effect of mutant tRNAs on the stability of wild-type tRNAs. Interaction between tRNA molecules can be a possible mechanism for this effect and has been previously described for dimerized A3243G tRNA Leu(UUR) [START_REF] Wittenhagen | Dimerization of a pathogenic human mitochondrial tRNA[END_REF].

In conclusion, we demonstrated that two of five not previously described mtDNA mutations, the G5835A tRNA Tyr and del4315A tRNA Ile mutation, are functionally relevant. For both mutations, this was proven by Northern blot analyses, for the heteroplasmic mutation by single fiber analysis as well. Single fiber data suggested a rather mild effect of the G5835A tRNA Tyr mutation, which was in contrast to severe histological and biochemical skeletal muscle findings. Concerted pathogenic effects of both mutations on the skeletal muscle phenotype can readily explain these findings. Our study underlines the importance of assessment of both (i) the complete sequence information, and (ii) the biochemical effects of each candidate pathogenic mutation in this process. The interaction of tRNA mtDNA point mutations illustrated here provides a basis for understanding how homoplasmic mutations, specific for a family or even an entire haplogroup, may influence the functional effects of pathogenic heteroplasmic mtDNA point mutations. b) del4315A in the mitochondrial tRNA Ile gene affects the tRNA TC loop. The nucleotide composition of the tRNA Ile TC loop is highly variable among species, whereas its size is moderately conserved at least among mammals. In the majority of mammals including primates, the number of nucleotides in the tRNA Ile TC loop is fixed to 7 [21, www.ncbi.nlm.nih.gov/genomes/ORGANELLES/mitoabout.html, mitochondrial genomes of 143 mammals], however its size may vary from 4 to 8 nucleotides. A 6-nucleotide tRNA Ile TC loop similar to our patient is present in some mammalian species belonging to various orders like Cetacea, Insectivora, or Artiodactyla.

c) The mitochondrial T1658C tRNA Val gene mutation results in a U→C transition at nucleotide 61 in the T-stem of the mitochondrial tRNA Val at an evolutionary not well conserved position. The mutation can be found in various mammalian species also comprising primates, e.g. Pongo pygmaeus (orangutan). Molecular weight marker (lane 1), patient's skeletal muscle (lane 2), patient's buccal mucosa (lane 3), patient's blood (lane 4), sister A's blood (lane 5), sister B's blood (lane 6), brother's blood (lane 7), mother's blood (lane 8), control skeletal muscle (lane 9).

Fig. 4

Correlation of COX activities with G5835A tRNA Tyr mtDNA mutation loads in single skeletal muscle fibers of patient A (each data point represents a single type I skeletal muscle fiber). Note the high threshold for phenotypic expression of this mutation.

Bioscience

  Reports Immediate Publication. Published on 03 Apr 2008 as manuscript BSR20080004 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080004 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 03 Apr 2008 as manuscript BSR20080004 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080004 S t a g e 2 ( a ) P O S T -P R I N T

Fig. 1

 1 Fig. 1

Fig. 2 Bioscience

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 5 Bioscience

 5 Fig. 5

Table 1 Mitochondrial respiratory chain enzyme activities in skeletal muscle homogenate.

 1

	Bioscience Reports Immediate Publication. Published on 03 Apr 2008 as manuscript BSR20080004
	Respiratory chain enzyme activity in U/g wwt total mutation load in skeletal muscle bulk mtDNA citrate synthase (CS) NADH:CoQ1 reductase (complex I) complex I / CS cytochrome c oxidase (complex IV) complex IV / CS Patient A with CPEO harbouring the novel G5835A tRNA Tyr , del4315A tRNA Ile , and T1658C controls (N = 43: 25 m, 18 f, 3-77 years) G5835A patient A A3243G patient B 0% 69  3% 75  4% 13.9  4.5 6.7 10.0 1.42  0.82 0.09 0.68 0.11  0.045 0.01 0.068 9.41  2.87 1.34 4.31 0.68  0.15 0.2 0.43 tRNA Val mtDNA mutations. Patient B with MELAS harbouring the A3243G tRNA Leu(UUR) mtDNA mutation. CS, citrate synthase; m, male; f, female; wwt, wet weight; U, Units; g, gramme S t a g e 2 ( a ) P O S T -P R I N T	THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080004

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080004 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft (KU-911/15-1, SCHR-562/4-3) and the Center for Molecular Medicine Cologne (CMMC, C9). The skilful technical assistance of Maria Bust (Cologne), Ulrike Strube and Karin Kappes-Horn (Bonn) is gratefully acknowledged. We wish to thank the patient and family members in Sakarya (Adapazari), Eastern Turkey, for their cooperation.

Note

Nucleotide sequence data reported are available in the DDBJ/ EMBL/GenBank databases under the accession number DQ473537

Steady state tRNA levels in the patient's skeletal muscle. a) Northern blot. RNA from 143B osteosarcoma cells and from muscle biopsies of patient A and controls was isolated and run under acidic denaturing conditions, blotted and probed for tRNA Tyr , tRNA Ile , tRNA Val , tRNA Leu(UUR) , and for 5S rRNA. A sample of deacylated RNA from 143B cells obtained by alkaline treatment was also loaded for comparison. b) tRNA / 5S rRNA ratios in the skeletal muscle of the patient (white bars) and of four control skeletal muscle biopsies (grey bars). Values for tRNAs are the sum of signals for aminoacylated and deacylated form. The control data (grey bars) are expressed as mean  SD of four biopsies; all data were analysed using a phosphoimager.


Adama Kamagate

Abdullah Sener

Philippe Courtois

Willy J Malaisse
email: malaisse@ulb.ac.be

André Herchuelz

Prof W J Malaisse

Effects of plasma membrane Ca 2+ -ATPase overexpression upon D-glucose metabolism in insulin-producing BRIN-BD11 cells

Keywords: Ca 2+ -ATPase, BRIN-BD11 cells, D-glucose metabolism, cell transfection, insulin release, ATP cell content Bioscience

In order to investigate the possible link between plasma membrane Ca 2+ -ATPase (PMCA) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with plasma membrane Ca 2+ -ATPase (PMCA2yb and PMCA2wb) was documented by RT-PCR, Western blot analysis, indirect immunofluorescence microscopy and 45 Ca 2+ uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-3 H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-14 C]oxidation and D-[5-3 H]glucose utilization prevailing at high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of 14 C-labelled acidic metabolites and amino acids and that of 3 HOH from D-[5-3 H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (as linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentration. The net uptake of 45 Ca 2+ by intact cells was also decreased in the transfected cells, but to a greater extent than apparently attributable to the change in ATP generation rate. These findings document the relevance of plasma membrane Ca 2+ -ATPase activity to both Dglucose metabolism and Ca 2+ handling in insulin-producing cells, with emphasis on the key role for both cytosolic and mitochondrial Ca 2+ concentration in the regulation of D-glucose catabolism. They also reveal that over expression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.

INTRODUCTION

The plasma membrane Ca 2+ -ATPase (PMCA) is responsible in part for the ejection of Ca 2+ from all eukaryotic cells [START_REF] Carafoli | The Ca 2+ pump of the plasma membrane[END_REF]. Glucose, the major physiological stimulus for insulin release, initiates the voltage-dependent entry of Ca 2+ into pancreatic islet β-cells [START_REF] Hellman | Cytoplasmic Ca 2+ and insulin secretion[END_REF]. This regulatory effect of glucose on insulin secretion involves intracellular metabolism of the sugar by oxidative glycolysis and an increase in the ATP/ADP ratio [START_REF] Henquin | Triggering and amplifying pathways of regulation of insulin secretion by glucose[END_REF][START_REF] Wollheim | Regulation of insulin release by calcium[END_REF][START_REF] Herchuelz | Calcium and insulin release. ln The Role of Calcium in Biological Systems[END_REF]. A rise in intracellular Ca 2+ is the key trigger of insulin secretion [START_REF] Wollheim | Regulation of insulin release by calcium[END_REF][START_REF] Herchuelz | Calcium and insulin release. ln The Role of Calcium in Biological Systems[END_REF][START_REF] Prentki | Ca 2+ , cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion[END_REF][START_REF] Hellman | Glucoseinduced modifications of the calcium movements regulating insulin and glucagon release[END_REF]. The maintenance of normal Ca 2+ homeostasis during sustained cellular activity involves inter alia plasma membrane Ca 2+ -ATPase and Na/Ca exchanger [START_REF] Rorsman | Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells[END_REF][START_REF] Nadal | Metabolic regulation of intracellular calcium concentration in mouse pancreatic islets of Langerhans[END_REF].

One approach to study the physiological function of plasma membrane Ca 2+ -ATPase isoforms is to develop systems which would allow modulation of their expression. Successful overexpression of different isoforms has been achieved in different systems [START_REF] Liu | Consequences of functional expression of the plasma membrane Ca 2+ pump isoform 1a[END_REF][START_REF] Hilfiker | Cloning and expression of isoform 2 of the human plasma membrane Ca 2+ ATPase. Functional properties of the enzyme and its splicing products[END_REF][START_REF] Guerini | Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane Ca(2+)-ATPase[END_REF]. Overexpression of PMCA2wb was recently achieved in BRIN-BD11 cells; an insulinsecreting cell line established by electrofusion of RINm5F cells with NEDH rat pancreatic islets cells [START_REF] Kamagate | Plasma membrane Ca 2+ -A TPase overexpression reduces Ca 2+ oscillations and increases insulin release induced by glucose in insulin secreting BRIN-BDll cells[END_REF][START_REF] Mcclenaghan | Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BDll, produced by electrofusion[END_REF]. BRIN-BD11 cells were selected for such a purpose because they display improved metabolic and secretory response to D-glucose, when considering the difference otherwise found between normal and tumoral islet cells [START_REF] Mcclenaghan | Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BDll, produced by electrofusion[END_REF][START_REF] Rasschaert | D-glucose metabolism in BRlN-BDll islet cells[END_REF]. In response to D-glucose, overexpressing cells showed markedly reduced rise in [Ca 2+ ] i and suppression of [Ca 2+ ] i oscillations, despite increased D-glucose metabolism and insulin release [START_REF] Kamagate | Plasma membrane Ca 2+ -A TPase overexpression reduces Ca 2+ oscillations and increases insulin release induced by glucose in insulin secreting BRIN-BDll cells[END_REF].

The present study extends such investigations to BRIN-BD11 cells transfected with either PMCA2wb or PMCA2yb. It aims mainly at exploring the feedback control of Dglucose catabolism resulting from changes in Ca 2+ handling, with emphasis on the measurement of ATP cell content, rate of ATP generation attributable to the catabolism of Dglucose, and relationship between the latter rate and the net uptake of 45 Ca in control and transfected cells. The results reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and utilization.

MATERIALS AND METHODS Materials

RPMI, L-glutamine, penicillin, streptomycin, G418, foetal calf serum, Ca 2+ and Mg 2+ -free HBSS, cell dissociation buffer, phosphate buffered saline, NotL AlwNI were from Gibco BRL (Merelbeke, Belgium); SgrAI KpnI were from Boehringer Mannheim (Brussels, Belgium); oligomycin, thapsigargin and calmodulin were from Calbiochem (Bierges, Belgium); and ouabain from ICN Biomedicals Inc. (Aurora, Ohio).

Construction of the expression vectors

Pancreatic islet isolation, total RNA preparation, reverse transcription, polymerase chain reaction, cloning and sequencing of PCR products have been described elsewhere [START_REF] Kamagate | Expression of multiple plasma membrane Ca 2+ -ATPases in rat pancreatic islet cells[END_REF]. In order to construct full-length PMCA2wb and PMCA2yb cDNA, three different fragments were amplified by polymerase chain reaction, based on rat PMCA2 cDNA sequences [START_REF] Shull | Molecular cloning of two isoforms of the plasma membrane Ca 2+ -transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na + , K + -and other cation transport ATPases[END_REF]. The fragment (I) contains the ATG starting docon and a 93 bp exon (for PMCA2yb) or 135 bp exon (for PMCA2wb) at site A (Figure 1). The fragments (II) and (III) are the same for PMCA2yb and PMCA2wb. The fragment (III) contains TAG stop codon. The fragments I-III were subcloned in the vector pCR®-Blunt (Invitrogen, Leek, The Netherlands), sequenced and cut with NotI-AlwNL AlwNI-SgrAI and SgrAI-KpnI respectively. The complete fulllengthy clone corresponding to the alternatively spliced PMCA isoforms 2yb or 2wb were obtained as follows. The 5'-end 1087 bp NotI-AlwNl fragment containing the alternatively spliced region site A, the 1107 bp AlwNI-SgrAI central portion fragment and the 1542 bp SgrAI-KpnI 3'-end fragment were ligated to each other. The final full-length PMCA2wb was subcloned into the multicloning site of the pcDNA3.1 (-) mammalian expression vector (Invitrogen) digested by NotI and KpnI. The positive clones were verified by restriction enzyme mapping and sequencing.

Culturing of BRIN-BD11 cells and stable transfection

Cells were grown at 37°C in RPMI-1640 medium supplemented with 10 % foetal calf serum (vol/vol), 2 mM L-glutamine, 100 lU/ml penicillin, 100 µg/ml streptomycin and equilibrated against 5 % CO 2 and 95 % air. The D-glucose concentration of the culture medium was 11.1 mM unless otherwise stated. Cells were gently washed with Ca 2+ and Mg 2+ -free HBSS (5 ml per T75 flask) for 30 s at room temperature before detachment from culture flask by incubation with cell dissociation buffer (5 ml per T75 flask) for 1-2 min. They were then immediately used for other application. To establish cell lines that stably express rat PMCA2yb or PMCA2wb, 5 µg/ml PMCA construct was transfected into BRIN-BD11 by Lipofectamin method (Gibco BRL). The neomycin-resistant colonies were selected with 250 µg/ml G418 in RPMI medium containing 10 % foetal calf serum. Colonies were picked and grown as individual cell lines in the presence of 250 µg/ml G418. Each cell line was examined to confirm PMCA expression.

Indirect immunofluorescence microscopy

The cell were plated on coverslips and analysed by indirect immunofluorescence microscopy 48-60 h after plating. The cells were washed with TBS (Tris-buffered saline, 20 mM Tris, 137 mM NaCl, pH 7.2), fixed for 20 min in 4 % formol at pH 7.4 (4°C) and washed with TBS. The cells were permeabilized in a solution containing 0.01 % Triton X-100, 197 µM MgCl 2 , 19.5 µM DTT and 10 % glycerol (pH 7.4, 4°C), washed twice with TBS and incubated in blocking buffer containing 1 % horse serum (Vector Laboratories Inc, Burlingame, CA) in TBS for 20 min. The coverslips were overlaid with the primary antibody (SWant, Rabbit anti-Ca 2+ -ATPase isoform 2, Bellinzona, Switzerland) diluted 1/1000 in TBS-1 % BSA buffer for 1 h. The control cells were incubated in TBS-1 % BSA buffer without antibody and washed three times with TBS. The cells were treated with secondary antibody (Alexa Fluor TM 594 goat anti-rabbit IgG (H + L) conjugate, Molecular Probes, Eugene, OR) diluted 1/400 in TBS-1 % BSA for 45 min and washed four times with TBS. The cells were incubated with 300 nM DAPI solution (DAPI: 4', 6-diamino-2-phenylindole, dihydrochloride; Molecular Probes, Eugene, OR) and washed twice with TBS. The coverslips were mounted with Vectashield® (Vector Laboratories Inc, Burlingame, CA) and the cells were observed with Axioplan microscope (Zeiss, Germany) equipped with microscope illuminator with HBO 100 W or XBO 100 W and a x 40 objective, and photographed with Dual-mode cooled CCD Camera C4880 (Hamamatsu, Japan).

Isolation of microsomes from cultured BRIN-BD11 cells

Crude microsomal membranes were prepared by a modified version of a procedure previously described [START_REF] Varadi | Characterisation of endoplasmic reticulum and plasma membrane Ca 2+ -ATPases in pancreatic beta-cells and in islets of Langerhans[END_REF]. Cells (5-10.10 6 cells per ml) were suspended in a hypotonic solution consisting of a 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM MgCl 2 , 0.1 mM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 mM dithiothreitol. The cells were swollen for 15 min on ice and then homogenized with 40 strokes in Dounce homogeniser. The homogenate was diluted with an equal volume of 0.5 M sucrose, 0.3 M KCl, 2 mM dithiothreitol, 10 mM Tris-HCl, pH 7.5, homogenized again with 20 strokes and centrifuged at 5,000 g for 15 min at 4°C. The supernatant was made 0.6 M in KCI and, in order to remove calmodulin, an excess of EDTA (1.5 mM) was also added. The suspension was centrifuged at 110,000 g for 1 h at 4°C to sediment the microsomal fraction. The final pellet was resuspended in a solution containing 0.25 M sucrose, 0.15 M KCI, 10 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol and 20 µM CaCl 2 at a protein concentration of 1-3 mg/ml, aliquoted, frozen immediately in liquid nitrogen and stored at -70°C. Protein concentration was determined using Bio Rad Dc Protein Assay (Bio-Rad, Nazareth, Belgium) with BSA as standard.

Ca 2+ uptake in microsomes

Calcium influx into microsomal vesicles was measured at 37°C by rapid filtration through filters as previously described with some modification [START_REF] Enyedi | A highly active 120-kDa truncated mutant of the plasma membrane Ca 2+ pump[END_REF]. The filters were presoaked in 150 mM KCI, 1 mM CaCl 2 to reduce the background. Twenty µg of microsomal proteins were resuspended in the uptake buffer containing 100 mM KCI, 25 mM TES-triethanolamine (pH 7.2), 5 mM NaN 3 , 4 µg/ml oligomycin, 0.5 mM ouabain, 7 mM MgCl 2 , 40 mM KH 2 P0 4 , 1 mM EGTA and sufficient CaCl2 (labelled with 45Ca2+ to obtain the desired free Ca2+ using the Max Chelator program (Stanford University). Thapsigargin (200 nM) was also included to inhibit the activity of the SERCA pumps. Immediately after the addition of 45 Ca 2+ , reaction was started by addition of 6 mM ATP. The incubation time was 10 min, and the transport activity was determined in the absence or presence of 240 nM calmodulin. At given times, 100 µl of the mixture were rapidly filtered through 0.45 µM nitrocellulose filters (Millipore, Brussels, Belgium), which were washed three time with cold 150 mM KCI and 1 mM CaCl 2 . The filters were then counted in Beckman scintillation counter.

Measurement of glucose metabolism

The methods used to measure D-[5-3 H]glucose metabolism, D-[U-14 C]glucose oxidation, and the generation of acidic metabolites (e.g. lactic acid) and amino acids from D-[U-14 C]glucose have been previously described [START_REF] Malaisse | Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events[END_REF][START_REF] Hutton | The metabolism of 4-methyl-2oxopentanoate in rat pancreatic islets[END_REF][START_REF] Malaisse | 3-O-methyl-D-glucose transport in tumoral insulin-producing cells[END_REF].

A TP measurement BRIN-BD11 cells (4.10 6 cells/ml) were incubated at 37°C for 90 min in bicarbonate-buffered medium [START_REF] Malaisse-Lagae | Insulin release by pancreatic islets. ln Methods in Diabetes Research[END_REF] containing bovine serum albumin (5 mg/ml) and 1.1 or 16.7 mM D-glucose.

The incubation was halted by addition of PCA (perchloric acid) to a final concentration of 3 %. The tubes were then mixed by sonication, 3 x l0 s, on ice, and centrifuged for 10 min at 15,000 g and 4°C. An aliquot of the supernatant was mixed with an equal volume of 0.5 M KOH-0.2 M imidazole solution and left at -70°C for at least 1 h. Thereafter, the extracts were thawed on ice and centrifuged for 10 min at 15,000 g and 4°C. The supernatants were transferred to new tubes and an aliquot of each tube was diluted 10 times with a buffer containing 5 mM MgCl 2 , 50 mM KCl, and 0.1 M imidazole (pH 7.75). ATP content was determined by luciferin-luciferase method using ATP Bioluminescence assay adapted from [START_REF] Gallez | Salivary biomasse assessed by bioluminescence ATP assay related to (bacterial and somatic) cell counts[END_REF]. Briefly, 0.5 ml of the diluted extract was mixed with 0.5 ml luciferin-luciferase reagent from the ATP Bioluminescence Assay Kit CLSII (Roche Diagnostics GmbH, Mannheim, Germany). Bioluminescence was measured on an LKB Wallace 250 luminometer (LKB-Pharmacia, Uppsala, Sweden). The main characteristics of the method are the following: linearity of ATP concentration between 1 and 9 nM; within-day precision for repeated assay (n = 6) yielding a coefficient of variation ≤ 5.7 %. The recovery of a standard after the whole extraction procedure was 62.9 ± 1.1 % (n = 8).

DNA measurement

DNA content was measured by fluorometric assay according to Labarca & Paigen [START_REF] Labarca | A simple, rapid and sensitive DNA assay procedure[END_REF]. Washed cells were ultrasonicated in 950 µl of 50 mM pH phosphate buffer (7.40) containing 2 M NaCl. After addition of 50 µl of the fluorochrome reagent bisbenzimidazole (20 µg/ml in phosphate saline) (Hoechst 33258, Sigma, St Louis, MO), relative fluorescence intensity was measured using a spectrophotofluorometer at excitation and emission wavelengths of 356 and 448 nm respectively. Standard curves were prepared from calf thymus DNA type 1 (Sigma); standard concentration was exactly determined by measurement of the absorbance at 260 nm. Samples and standards were assayed in duplicate.

45

Ca uptake by intact cells

The media used to incubate the BRIN-BD11 cells consisted in a Krebs-Ringer bicarbonate buffered solution (pH 7.4, 37°C) with the following composition (in mM) : NaCl 115, CaCl 2 1, KCI 5, MgCl 2 l, HEPES/NaOH 10. The media were gassed with ambient air. The different media contained 1.1 or 11.1 mM D-glucose. The method used for the measurement of 45 Ca 2+ uptake was performed as described previously [START_REF] Van Eylen | Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells[END_REF]. In brief, BRIN-BD11 cells were preincubated in 1 ml of a non-radiaoctive solution during 30 min and then incubated for 90 min in 1 ml of the same medium containing in addition 45 Ca 2+ (10 µCi/ml). At the end of the incubation, the cells were separated from the incubation medium using a combined lanthanum and oil technique [START_REF] Plasman | Characterisation of the process of sodium-calcium exchange in pancreatic islet cells[END_REF].

Presentation of results

All results, including those already mentioned are presented as mean values (± SEM) together with either the number of individual determinations (n) or degree of freedom (d.f.). The statistical significance of differences between mean values was assed by analysis of variance followed by Tuckey's post test and/or by Student's t-test.

RESULTS

Generation and selection of BRIN-BD11 stable cell line expression the PMCA pump

The full-length clone for PMCA2 pump isoform 2yb and 2wb contains the 93 or 135 bp exon at site A, respectively, without a 227 bp exon at site C. These two isoforms were constructed from polymerase chain reaction amplified cDNA. Stably transfected BRIN-BD11 cell colonies were picked and grown as individual cell lines in the presence of G418. Quantitative RT-PCR and Western blot analysis confirmed overexpression of PCMA2wb [START_REF] Kamagate | Plasma membrane Ca 2+ -A TPase overexpression reduces Ca 2+ oscillations and increases insulin release induced by glucose in insulin secreting BRIN-BDll cells[END_REF] and PCMA2yb (Figure 2).

The overexpression of PMCA2yb and PMCA2wb protein at the level of the plasma membrane of transfected cells was also visualized by immunofluorescence using rabbit anti-Ca 2+ -ATPase isoform 2 antibody, PMCA2 and nuclei being stained in red and blue, respectively. Results showed that higher levels of Ca 2+ pump were expressed in the PMCA2yb-and PMCA2wb-transfected cells; as compared with control BRIN-BD11 cells (Figure 3). A pattern of peripheral distribution of stained material was quite obvious in cells overexpressing PMCA2yb and PMCA2wb, suggesting that a major portion of the overexpressed protein was associated with the plasma membrane. In control cells, minimal staining of the endogenous proteins was observed. These experiments support the view that the largest fraction of the overexpressed PMCA2 pump was correctly targeted to the plasma membrane.

Ca 2+ uptake by microsomes

Additional support for correct targeting of PMCA2 came from the assay of PMCA activity in isolated microsomes, as judged from ATP-dependent Ca 2+ uptake. In these experiments, an excess concentration of thapsigargin was used to ensure complete inhibition of SERCA pump activity, without affecting the calmodulin-dependent Ca 2+ uptake mediated by the PMCA. As shown in Table 1, the PMCA activity was higher (P < 0.05 or less) in transfected cells than in the control BRIN-BD11 cells, whether in the absence or presence of calmodulin. The enzymic activity in the PMCA2wb-transfected cells exceeded (P < 0.01 or less) that in the PMCA2yb-transfected cells both in the absence or presence of calmodulin. Calmodulin increased (P < 0.05 or less; paired comparison) the mean value for 45 Ca uptake by the microsomes. The relative extent of such an increase was not significantly different in control BRIN-BD11 and transfected cells, with an overall mean value of 84.1 ± 7.4 % (n = 3; P < 0.01). The expressed isoforms PMCA2yb and PMCA2wb thus displayed characteristic dependence on calmodulin for activation.

D-glucose metabolism

In both the control and transfected BRIN-BD11 cells, the rise in D-glucose concentration augmented significantly (p < 0.01 or less) the generation of 3 H 2 O from D-[5-3 H]glucose and that of 14 C-amino acids and 14 C-acidic metabolites (e.g. lactic acid) from D-[U-14 C]glucose, whilst failing to affect significantly the production of 14 CO 2 from D-[U- 14 C]glucose (Table 2). Hence, in these tumoral cells, the 14 CO 2 / 3 H 2 O ratio was always lower (p < 0.001) at 16.7 mM that at 1.1 mM D-glucose.

The utilization of D-[5-3 H]glucose and oxidation of D-[U- 14 C]glucose were, on occasion, significantly lower, in transfected cells than in control BRIN-BD11 cells. However, the most substantial difference between control and transfected cells consisted in the higher generation of 14 C-acidic metabolites in the latter cells than in the former ones. Whether at 1.1 or 16.7 mM D-glucose, the net production of 14 C-acidic metabolites was indeed significantly higher (p < 0.02 or less) in BRIN-BD11 cells transfected with PMCA2yb than in control cells. At both 1.1 and 16.7 mM D-glucose, it was further increased (p < 0.001) in the PMCA2wbtransfected cells. In this respect, there was thus a close parallelism between the generation of 14 C-acidic metabolites (Table 2) and Ca 2+ -dependent ATPase activity (Table 1).

Three further differences between control and transfected BRIN-BD11 cells merit to be mentioned. First, the rise in D-glucose concentration augmented to a greater relative extent (p < 0.02 or less) D-[5-3 H]glucose utilization in PMCA2yb-transfected cells (multiplication factor : 2.664 ± 0.168; d.f. = 79) and PMCA2wb-transfected cells (multiplication factor : 2.600 ± 0.205; d.f. = 80) than in control BRIN-BD11 cells (multiplication factor : 1.998 ± 0.064; d.f. = 54). Second, whether at 1.1 or 16.7 mM Dglucose, the mean generation of 14 C-labelled acidic metabolites, expressed relative to D-[5-3 H]glucose utilization, was always higher in the transfected cells than in the control cells, averaging in the former cells 198.3 ± 10.8 % (p < 0.005) of the mean corresponding value found at the same D-glucose concentration in the control BRIN-BD11 cells. When the net generation of both 14 C-labelled acidic metabolites and amino acids was taken into account, the latter percentage averaged 159.7 ± 7.9 % and, as such, remained significantly higher than unity (p < 0.005). Last, the rise in glucose concentration decreased to a greater absolute or relative extent the ratio between D-[U- 14 C]glucose oxidation and D-[5-3 H]glucose utilization in PMCA2-transfected cells than in control BRIN-BD11 cells. Such a difference achieved statistical significance (p < 0.05) in the PMCA2wb-transfected cells, but not so in PMCA2ybtransfected cells, once again paralleling the changes in Ca 2+ -dependent ATPase activity.

Cell ATP content

Table 3 provides the absolute values for the ATP content of control BRIN-BD11 cells and PMCA2yb-or PMCA2wb-transfected cells as measured after 90 min incubation in the presence of either 1.1 or 16.7 mM D-glucose. In the three cell types, the ATP content was lower (P < 0.02 or less) at 16.7 mM D-glucose than at 1.1 mM D-glucose. Thus, at 16.7 mM D-glucose, the values averaged, in control BRIN-BD11 cells and PMCA2yb-or PMCA2wbtransfected cells, respectively 87.6 ± 2.7 % n = 9), 90.2 ± 2.3 % (n = 27) and 87.8 ± 1.8 % (n = 27) of the corresponding mean values found within the same experiment(s) and same cell type at 1.1 mM D-glucose (i.e. 100.0 ± 1.2 %, n= 8; 100.0 ± 2.9 %, n = 27; and 100.0 ± 2.3 %, n = 27). Whether at 1.1 or 16.7 mM D-glucose, the ATP content of the PMCA2yb and PMCA2wb-transfected cells was significantly lower (P < 0.005 or less) than that recorded at the same hexose concentration and within the same experiment(s) in control BRIN-BDl1 cells. Relative to the latter control values, the readings made in the transfected cells were not significantly different at 1.1 or 16.7 mM D-glucose and in PMCA2yb-or PMCA2wbtransfected cells, with an overall mean value of 81.3 ± 1.5 % (n = 108).

45

Ca uptake by intact cells

The measurements of 45 Ca net uptake made at low and high D-glucose concentration are summarized in Table 4. In all cell types, the net uptake of 45 Ca was significantly higher (P < 0.005 or 1ess) at high, rather than low, hexose concentration. At the two concentrations of Dglucose, the net uptake of 45 Ca was lower (P < 0.001) in transfected cells than in the control BRIN-BDII cells.

Figure 4 illustrates the relationship between 45 Ca net uptake and the estimated rate of ATP generation. The latter rate was calculated from the data listed in Table 2 as follows. The ATP generation rate was taken as the sum of (i) the rate of D-[U- 14 C]glucose oxidation (multiplied by 36) and (ii) the differences between the rate of D-[5-3 H]glucose utilization and that of D-[U- 14 C]glucose oxidation (multiplied by 2). Figure 4 documents that, in all three cell types, a given increase in ATP generation (as resulting from a rise in D-glucose concentration) coincided with a comparable increase in 45 Ca net uptake. However, for any given ATP generation rate, the net uptake of 45 Ca was lower in transfected cells than in the control BRIN-BDl1 cells. Such a difference appeared most pronounced in the PMCA2wbtransfected cells, in which the activity of PMCA2 was also the highest (see Table 1). The present data document that, in BRIN-BD11 cells transfected with either PMCA2yb or PMCA2wb, the estimated ATP yield resulting from the catabolism of D-glucose is decreased, this coinciding with a lower ATP content and lower 45 Ca net uptake in intact transfected cells. The latter cationic anomaly is likely to be directly caused by overexpression of PMCA2 in the transfected cells. It coincides with a lesser increase in cytosolic Ca 2+ concentration in these cells, when exposed to insulin secretagogues [START_REF] Kamagate | Plasma membrane Ca 2+ -A TPase overexpression reduces Ca 2+ oscillations and increases insulin release induced by glucose in insulin secreting BRIN-BDll cells[END_REF].

DISCUSSION

The alteration of the ATP content in the transfected cells is likely to be attributable mainly to the increased consumption of ATP caused by enhanced activity of PMCA2, rather than to any major change in D-glucose catabolism, at least in the PMCA 2wb-trasfected cells.

The metabolism of D-glucose in both the control and transfected BRIN-BD11 cells displayed the typical features of a Crabtree effect, namely inhibition of cellular respiration at high concentration of D-glucose, as previously documented in tumoral insulin-producing cells of the RINm5F line [START_REF] Sener | Crabtree effect in tumoral pancreatic islet cells[END_REF].

In the BRIN-BD11 cells overexpressing PMCA2 activity, changes in D-glucose metabolism, when present, are probably attributable, in part at least, to the modulation by mitochondrial Ca 2+ of the activity of FAD-linked glycerophosphate dehydrogenase [START_REF] Rasschaert | Hexose metabolism in pancreatic islets. Glucose-induced and Ca 2+ -dependent activation of FAD-glycerophosphate dehydrogenase[END_REF][START_REF] Sener | Hexose metabolism in pancreatic islets. Ca 2+dependent activation of the glycerol phosphate shuttle by nutrient secretagogues[END_REF]. Such a proposal is supported by three sets of findings. First, the rise in glucose concentration indeed decreased to a greater absolute or relative extent the ratio between D-[U-14 C]glucose oxidation and D-[5-3 H]glucose utilization in PMCA2-transfected cells than in control BRIN-BD11 cells. Second, the anaerobic modality of glycolysis as judged by the generation of 14 Cacidic metabolites from D-[U-14 C]glucose, was indeed higher in PMCA2-transfected cells than in control BRIN-BD11 cells. In these two cases, the hierarchy in mean values paralleled that of microsomal Ca 2+ -dependent ATPase activity. Last, the generation of 14 C-acidic metabolites, considered alone or together with that of 14 C-amino acids, represented a greater fraction of total glycolytic flux, as judged from the production of 3 H 2 O from D-[5-3 H]glucose, in the PMCA2-transfected cells than in the control BRIN-BD11 cells.

In conclusion, the present work extends to changes in PMCA activity the concept of a feedback control by cationic variables of D-glucose catabolism in insulin-producing cells [START_REF] Malaisse | Hexose metabolism in pancreatic islets. Feedback control of D-glucose oxidation by functional events[END_REF]. It indeed documents alterations of both glycolysis and mitochondrial oxidative events in cells overexpressing PMCA, such alterations impeding the rate of ATP generation attributable to the catabolism of the hexose to match the accelerated consumption of ATP caused by increased activity of this Ca 2+ pump.. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. MW : 1 kb DNA ladder molecular weight marker (sizes in bases). (B) Membrane proteins (100 µg) were separated by SDS-PAGE on 7.5 % gels. The gel was electroblotted onto nitrocellulose membrane which was then cut and incubated with primary antibodies (Swant, rabbit anti-Ca 2+ -ATPase isoform 2 raised against PMCA2, diluted 1:1000). The bound antibody was detected with a POD-labelled secondary antibody as described by the manufacturer. Lines 1-4 : control BRIN-BD11 cells, the stable transfected cells clones 1, 2 and 5, respectrively. MW : Kaleidoscope Prestained Standard (Bio-Rad), molecular mass.

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

BioscienceFigure 1 .

 1 Figure 1. Scheme summarizing the splice variants of the plasma membrane Ca 2+ -ATPases isoform 2 found in insulin-producing cells. cDNA organization of PMCAs is shown on top. Putative transmembrane segments 1-10 are indicated by black bars. Exons involved in the alternative splicing at sites A, B and C are represented by grey boxes. Flanking sequences that are conserved are represented by white boxes.

Figure 2 .

 2 Figure 2. RT-PCR amplification and Western blot analysis of PMCA2yb in control and PMCA2yb transfected BRIN-BD11 cells. (A) RT-PCR amplification of PMCA2yb at cycles 32 yielded a bands of 300 and 589 for PMCA2 and ß-actin, respectively. Lines 1-4 : control RT-PCR (BRIN-BD11 non transfected), BRIN transfected with PMCA2yb clone 1, clone 2 and clone 5, respectively.The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. MW : 1 kb DNA ladder molecular weight marker (sizes in bases). (B) Membrane proteins (100 µg) were separated by SDS-PAGE on 7.5 % gels. The gel was electroblotted onto nitrocellulose membrane which was then cut and incubated with primary antibodies (Swant, rabbit anti-Ca 2+ -ATPase isoform 2 raised against PMCA2, diluted 1:1000). The bound antibody was detected with a POD-labelled secondary antibody as described by the manufacturer. Lines 1-4 : control BRIN-BD11 cells, the stable transfected cells clones 1, 2 and 5, respectrively. MW : Kaleidoscope Prestained Standard (Bio-Rad), molecular mass.

Figure 3 .

 3 Figure 3. Immunological localization of the overexpressed pump protein.Immunofluorescence was performed as outlined in Materials and Methods using rabbit anti-Ca 2+ -ATPase isoform 2 raised against PMCA2. Ctrl BRIN refers to experiments without the first antibody on BRIN-BD11 cells. NT BRIN, PMCA2yb and PMCA2wb refer to experiments on BRIN-BD11 cells, BRIN-BD11 transfected with PMCA2yb and BRIN-BD11 transfected with PMCA2wb, respectively, using the first antibody. PMCA2 protein and nuclei were staining by immunofluorescence in red and blue, respectively.

Figure 4 .Figure 1 BioscienceFigure 2 BioscienceFigure 3 Bioscience

 4123 Figure 4. Mean values for 45 Ca net uptake and the estimated ATP generation rate in control BRIN-BD11 cells and in either PMCA2yb-or PMCA2wb-transfected cells incubated in the presence of 1.1 mM D-glucose (open circles) or 16.7 mM D-glucose (closed circles).

Figure 4 Bioscience

 4 Figure 4

Table 1 .

 1 Ca 2+ -dependent ATPase activity in microsomes from control and PMCA2 -

	Bioscience Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042
	transfected cells
		__________________________________
	Calmodulin	Nil	240 nM
	BRIN-BD 11	0.513 ± 0.143 (4)	0.885 ± 0.130 (4)
	BRIN-BD 1l transfected with PMCA2yb	1.537 ± 0.166 (4)	3.041 ± 0.276 (4)
	BRIN-BD11 transfected with PMCA2wb	2.585 ± 0.213 (8)	4.705 ± 0.147 (4)

Table 2 .

 2 D-[U-14 C]glucose and D-[5-3 H]glucose metabolism in control and PMCA2 gene transfected BRIN-BDII cells

	Cells	BRIN-BD11		BRIN-BD11 transfected with PMCA2yb	BRIN-BD11 transfected with PMCA2wb
				_________________________________	_________________________________
	D-glucose	1.1 mM	16.7 mM	1.1 mM	16.7 mM	1.1 mM	16.7 mM
	D-[U-14 C]glucose oxydation 1.54 ± 0.07 (27) a 1.82 ± 0.12 (28)	1.11 ± 0.08 mM (41) 1.35 ± 0.10 (39)	1.37 ± 0.09 (37)	1.46 ± 0.10 (38)
	D-[5-3 H]glucose utilization 9.66 ± 0.11 (28) 19.30 ± 0.58 (28à 6.23 ± 0.27 (42)	16.47 ± 0.77 (39) 8.74 ± 0.51 (41)	22.72 ± 1.21 (41)
	14 CO 2 / 3 H 2 O ratio (%)	15.92 ± 0.73 (27) 9.48 ± 0.60 (28)	17.43 ± 0.91 (41)	8.89 ± 0.66 (34)	15.13 ± 0.43 (36)	6.47 ± 0.23 (39)
	14 C-amino acids	0.95 ± 0.04 (28) 1.33 ± 0.03 (21)	0.81 ± 0.02 (41)	1.33 ± 0.04 (21)	1.08 ± 0.02 (42)	1.44 ± 0.03 (40)
	14 C-acidic metabolites	1.04 ± 0.08 (27) 1.51 ± 0.14 (28)	1.25 ± 0.04 (41)	2.44 ± 0.09 (42)	2.17 ± 0.03 (41)	3.32 ± 0.08 (39)

a All results are expressed as pmol of D-glucose equivalent per 90 mg and ng DNA Bioscience Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 3

 3

	.	ATP	content	in	BRIN-BDII	non-transfected	or	transfected	cells
						ATP content
					1.1 mM D-Glucose	16.7 mM D-Glucose
	BRIN-BD11				606 ± 67 (8) a		541 ± 49 (9)
	BRIN-BD11 transfected with PMCA2yb	489 ± 27 (27)	432 ± 13 (27)
	BRIN-BD11 transfected with PMCA2wb	500 ± 29 (27)	438 ± 25 (27)
	a All results are expressed as pmol of ATP per µg DNA

Bioscience Reports Immediate Publication. Published on 21 Jul 2008 as manuscript BSR20080042 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T

Table 4 .

 4 Effect of D-glucose on45 Ca net uptake by BRIN-BD11 non-transfected or transfected cells All results are expressed as fmol per 10 3 cells at the 90th min of incubation

	D-glucose	1.1 mM	16.7 mM
	BRIN-BD11	696 ± 23 (12) a	814 ± 24 (12)
	BRIN-BD11 transfected with PMCA2yb	495 ± 19 (30)	591 ± 24 (30)
	BRIN-BD11 transfected with PMCA2wb	487 ± 11 (30)	563 ± 11 (30)

*

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BSR20080042 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Ca 2+ uptake (nmol/mg protein per 10 min)

ACKNOWLEDGEMENTS

This study was supported by the Belgian Foundation for Scientific Medical Research (grants 3.4562.00 and 3.4520.07).