Effect of Hailey-Hailey Disease Mutations on the Function of a New Variant of Human Secretory Pathway Ca2+/Mn2+-ATPase (hSPCA1)*

Rebecca J. Fairclough {ddagger} §, Leonard Dode {ddagger}, Jo Vanoevelen ¶, Jens Peter Andersen ||, Ludwig Missiaen ¶, Luc Raeymaekers ¶, Frank Wuytack ¶ and Alain Hovnanian {ddagger} **

From the {ddagger}Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Dr., Oxford OX3 7BN, United Kingdom, the Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium, and the ||Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark

Received for publication, January 16, 2003 , and in revised form, April 9, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ATP2C1, encoding the human secretory pathway Ca2+/Mn2+ ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey Disease (HHD), an autosomal dominant skin disorder characterized by persistent blisters and erosions. To investigate the underlying cause of HHD, we have analyzed the changes in expression level and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into hSPCA1d, a novel splice variant expressed in keratinocytes, described here for the first time. Encoded by the full-length of optional exons 27 and 28, hSPCA1d was longer than previously identified splice variants. The protein competitively transported Ca2+ and Mn2+ with equally high affinity into the Golgi of COS-1 cells. Ca2+- and Mn2+-dependent phosphoenzyme intermediate formation in forward (ATP-fuelled) and reverse (Pi-fuelled) directions was also demonstrated. HHD mutant proteins L341P, C344Y, C411R, T570I, and G789R showed low levels of expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. P201L had little effect on the enzymatic cycle, whereas I580V caused a block in the E1~P -> E2-P conformational transition. D742Y and G309C were devoid of Ca2+- and Mn2+-dependent phosphoenzyme formation from ATP. The capacity to phosphorylate from Pi was retained in these mutants but with a loss of sensitivity to both Ca2+ and Mn2+ in D742Y and a preferential loss of sensitivity to Mn2+ in G309C. These results highlight the crucial role played by Asp-742 in the architecture of the hSPCA1 ion-binding site and reveal a role for Gly-309 in Mn2+ transport selectivity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
At present, three distinct classes of phosphorylation-type Ca2+ transport ATPases have been identified in mammalian cells: plasma membrane (PMCA),1 sarco(endo)plasmic reticulum (SERCA), and Golgi-associated secretory pathway (SPCA) Ca2+ transport ATPases. These proteins serve to actively pump Ca2+ out of the cytoplasm, thus contributing to the maintenance of a low cytosolic Ca2+ concentration in resting conditions (reviewed in Ref. 1). Relatively little is known about the SPCA class. ATP2C1, the gene encoding the human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1), was recently identified as the defective gene in Hailey-Hailey disease (HHD, OMIM 16960), an autosomal-dominant skin disorder characterized by abnormal keratinocyte adhesion in the suprabasal layer of the epidermis (2, 3). Sequence analysis suggests that the protein retains the remarkable conservation in structure and function that exists across the Ca2+-ATPase class and that, like PMCA, the SPCA class possesses only one of the two high affinity Ca2+-binding sites present in SERCA. Most of our knowledge of the SPCA class comes from studies of the yeast Saccharomyces cerevisiae homologue, named somewhat confusingly PMR1 for plasma membrane ATPase-related, which shares 49% amino acid identity with hSPCA1. Following the initial identification of PMR1 as a probable Ca2+-ATPase (4), the protein was localized to the Golgi or one of its subcompartments (5). Studies of null strains defective in PMR1 illustrated a pleiotropic effect on Golgi function, including impaired proteolytic processing, incomplete glycosylation, and defective pre-, post-, and intra-Golgi translocation of secreted proteins (5). These defects resulted in defective cell wall morphogenesis, which is interestingly reminiscent of abnormal keratinocyte adhesion in HHD. Moreover, in yeast these phenotypes could be reversed by addition of Ca2+ (10 µM) to the medium, implicating a direct role for Ca2+ in Golgi function (5). Evidence suggests that PMR1 can also transport Mn2+ (6). Although being an essential cofactor for a wide range of enzymes (79), high concentrations of Mn2+ are toxic, interfering with Mg2+-binding sites on proteins and compromising the fidelity of DNA polymerases (10). PMR1-type pumps appear to be the principal route for Mn2+ detoxification, via the secretory pathway, and are important in maintaining both cytosolic and luminal Mn2+ homeostasis.

The first functional study on SPCA in animals was conducted on Caenorhabditis elegans. C. elegans SPCA was shown to transport Ca2+ and Mn2+ with high affinity into the Golgi following heterologous expression in COS-1 cells (11). Overexpression of human SPCA1 in yeast and Chinese hamster ovary cells was restricted to the Golgi compartment and, moreover, was able to complement the PMR1 null mutation, as demonstrated by the ability to transport Ca2+ and Mn2+ in yeast (12).

Key insights into structure/function relationships have been provided by functional analysis of over 250 point mutations in SERCA1a. This has led to identification of specific domains important in Ca2+ and ATP binding and phosphorylation, together with residues important in the conformational changes associated with the various stages of the catalytic cycle (13, 14) (Scheme 1). In contrast, mutagenic studies of the SPCA class have been limited and have only involved the S. cerevisiae protein. Mutations in the active phosphorylation site, Asp-372, had the expected effect of abolishing Ca2+ transport activity (15). Residues in the putative N-terminal EF hand-like motif were reported to have a modulatory effect on ion transport, through their importance in ion binding and participation in long range interactions involved in ATP binding and phosphorylation (16). Other reports have targeted the oxygen-containing side chains of the predicted transmembrane domains M4–M8, likely to be involved in the coordination of Ca2+ and Mn2+ ions (17), and have defined Gln-783 as crucial in Mn2+ selectivity of PMR1 (18).



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SCHEME 1.
The SERCA minimal reaction cycle modified from de Meis and Vianna (47). In the reversible enzymatic cycle, the binding of Ca2+ from the cytosol to high affinity binding sites located in the transmembrane domains is closely associated with the binding of ATP to the cytosolic domain and the subsequent formation of a phosphoenzyme intermediate. In this high energy E1~P state, the enzyme goes through an extensive series of conformational changes to move the ion(s) through the transmembrane pore. The release of Ca2+ to the other side of the membrane is closely associated with transfer of the phosphoenzyme to the lower energy state, E2-P, and the subsequent dephosphorylation of the enzyme. Note that, although SERCA transports two Ca2+ ions during one cycle, hSPCA1 is postulated to transport only one Ca2+ or Mn2+ ion per cycle.

 

We have previously described a spectrum of mutations scattered throughout the ATP2C1 gene in HHD patients (2, 19). Although nonsense, frameshift, and splice-site mutations are predicted to cause nonsense-mediated mRNA decay (20) or aberrant splicing, the underlying cause of disease in patients carrying missense mutations cannot be predicted a priori. In this study we investigated the molecular and physiological basis of HHD in patients carrying these mutations, providing new insights into SPCA structure/function relationships. Site-directed mutagenesis was used to introduce these disease-causing point mutations into the cDNA sequence of a novel splice variant of hSPCA1, which was identified and functionally characterized in this study. More than 50% of the mutants studied here showed low levels of protein expression, despite normal levels of mRNA and correct localization to the Golgi compartment. Other mutants are characterized by lack of ion transport, caused by specific alterations to the partial reactions of the catalytic cycle, such as defects in Ca2+ and Mn2+ binding and inability of the phosphoenzyme intermediate to undergo the energy-transducing E1~P -> E2-P conformational transition.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Radiochemicals and chemicals were obtained from Amersham Biosciences (UK), PerkinElmer Life Sciences (Boston, MA), and Sigma (Dorset, UK). DNA sequencing was performed using the ABI Prism 377 sequencer and the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE-Applied Biosystems, Cheshire, UK). Unless otherwise stated, PCR was carried out using the ExpandTM Long Template PCR system (Roche Applied Science, E. Sussex, UK). Anti-mouse/rabbit alkaline phosphate-conjugated secondary antibodies were from Amersham Biosciences. Sheep anti-TGN46 was purchased from Serotec (Oxford, UK); fluorescein isothiocyanate-conjugated secondary antibodies (donkey anti-sheep, goat anti-rabbit) were from Molecular Probes (Leiden, The Netherlands). Free ion concentrations were calculated using the CaBuf program (available at ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) based on the stability constants for oxalate, EGTA, and ATP, as described before (11).

Reverse Transcriptase-PCR Analyses—Following extraction of total RNA from human keratinocytes with TRIzol (Invitrogen), both reverse transcription of ATP2C1 and 3' rapid amplification of cDNA ends (RACE)-PCR, were performed using the MarathonTM cDNA amplification kit (Clontech, Palo Alto, CA). The gene-specific primer, ATP2C13'GSP (5'-CCAAGTCTGTGTTTGAGATTGGACTCTGC-3'), corresponded to nucleotides (nt) 2618–2646 in the ATP2C1d sequence. Products were subcloned into the PCR-cloning vector pGEM-T (Promega, Madison, WI) and sequenced in both directions. Splice variants identified by sequencing were subsequently amplified by PCR using ATP2C13'GSP and antisense primers Ex27R (5'-TTGCCCTTCTAAATGATCCTC-3') or Ex28R (5'-GGAAGAGCTGCAGGAAGATG-3') in standard reaction conditions. Ex27R and Ex28R, respectively, represent the inverse complement of nt 3039–3059 and 3048–3067 in the ATP2C1a and ATP2C1d sequences. PCR conditions were as follows: 4 min of denaturation at 95 °C and 30 cycles of 30 s at 95 °C,30sat58 °C, and 30 s at 72 °C.

First strand cDNA was prepared for LightCyclerTM-based real-time PCR analysis (Roche Applied Science) following total RNA extraction from transfected COS-1 cells using the Absolutely RNATM RT-PCR Miniprep kit (Stratagene). A 276-bp PCR product was generated using ATP2C1 primers C7F (5'-TTGGTTGGCTGGTTACTGGG-3') and C7R (5'-AGCATGCAGACCATCTGAAGT-3'), corresponding to nt 973–992 and the inverse complement of nt 1228–1248, respectively, in the ATP2C1d sequence. GAPDH amplification was performed, to control for equal RNA loading between samples, using primers GAPDHF (5'-ATCATCCCTGCCTCTACTGG-3') and GAPDHR (5'-TGCTGTAGCCAAATTCGTTG-3') to generate a 349-bp product. Sequences correspond to nt 120–139 and the inverse complement of nt 449–468 in the GAPDH sequence. Real-time PCR amplification was performed with 1 µl of cDNA product in a 20-µl reaction containing 0.5 µM of each primer and QuantiTectTM SYBR® Green PCR Master Mix (Qiagen, W. Sussex, UK). The PCR cycle consisted of 15 min of denaturation at 95 °C, followed by 45 cycles of 15 s at 95 °C, 20 s at 60 °C, and 20 s at 72 °C. Fluorescence data was acquired at the end of each extension cycle. To confirm amplification specificity, PCR products were subjected to a melting curve analysis, as described previously (21). Analysis of real-time PCR data was performed using the LightCyclerTM software (Roche Applied Science). Crossing points, defined as the cycle number at which all samples are exponentially amplifying, were recalculated into relative log concentrations of RNA according to PCR efficiency calculated from standard curves generated using serially diluted cDNA for GAPDH and ATP2C1 primer sets. ATP2C1 amplification was then corrected for equal GAPDH expression between samples. PCR products were isolated from the LightCyclerTM capillaries after cycling and visualized following electrophoresis on 1.5% agarose gels.

cDNA Construction, Site-specific Mutagenesis, COS-1 Cell Culture, and Transfection—A full-length cDNA clone encoding ATP2C1d was constructed using cDNA fragments amplified by 5' and 3' RACE-PCR and was subcloned into vector pSPORT1 (Invitrogen). The full-length ATP2C1d cDNA was then transferred into the mammalian expression vector pMT2, with the insert corresponding to the entire coding sequence (nt 125-end) of the deposited ATP2C1d nucleotide sequence. Missense mutations originally reported in HHD patients by Sudbrak et al. (2) and Dobson-Stone et al. (19) were introduced into the wild type ATP2C1 expression clone using the QuikChangeTM XL site-directed mutagenesis kit (Stratagene). The full-length cDNA clones were sequenced in both directions to ensure no additional sequence changes had been introduced during mutagenesis. Details of mutations introduced into the hSPCA1 protein sequence are given in Table I. COS-1 cells for immunocytochemistry, microsome preparation, and isotope flux were cultured and transiently transfected with FuGENETM 6 transfection reagent (Roche Applied Science) as previously described (11). For total RNA or protein extraction 1 x 106 COS-1 cells were seeded in 60-mm culture dishes 24 h before transfection.


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TABLE I
Summary of mutant proteins studied

 

Preparation of Antiserum to hSPCA1 Protein—Antibodies were raised essentially as described before (11). ATP2C1 cDNA was amplified using primers ATP2C1CYTF (5'-GCATGCTGTGAAAAAGCTGCCTATTG-3'; ATP2C1d nt 1122–1141) and ATP2C1CYTR (5'-GTCGACGCAACCTTTGGTACTATTTG-3'; ATP2C1d nt 1954–1973) containing, respectively, SphI and SalI sites at their 5'-end. PCR cycling conditions were as follows: 94 °C for 2 min, followed by 30 cycles of 10 s at 94 °C, 30 s at 57 °C, and 60 s at 68 °C. After 10 cycles the extension time was increased to 90 s. The PCR amplification product encoded a fragment of hSPCA1 corresponding to the large cytoplasmic loop between transmembrane segments 4 and 5. The SphI/SalI-cut PCR fragment was ligated into the corresponding sites in the pQE-31 bacterial expression vector (Qiagen). Following expression in Escherichia coli using the QIAexpress Type IV System (Qiagen), the recombinant protein migrated at the expected size of 33 kDa on a 12% SDS-polyacrylamide gel.

Protein Preparation and Immunostaining Analyses—For total protein extractions COS-1 cells were washed twice in phosphate-buffered saline before lysis in freshly prepared buffer (150 mM NaCl, 50 mM Tris HCl (pH 8), 5 mM EDTA, 2% SDS, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of leupeptin, pepstatin, chymostatin, and antipain). After 30-min incubation on ice, insoluble proteins were pelleted at 6000 x g. Soluble protein was retained in the supernatant. Microsomes were isolated from COS-1 cells as described by Verboomen et al. (22). Protein concentrations were determined by using the bicinchoninic acid method (Pierce, Rockford, IL). For Western blotting, proteins were separated and transferred onto Immobilon-P membranes (Millipore, Edinburgh, UK) using the NuPAGE® system with 4–12% Bis-Tris pre-cast gels (Invitrogen). For immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. Cells and membranes were quenched with 3% bovine serum albumin and 1% goat serum, followed by incubation with primary and secondary antiserum as described in the figure legends. Membranes were then incubated with ECF substrate (Amersham Biosciences) and fluorescent imaging was performed using a Storm 840 PhosphorImager in combination with ImageQuaNTTM software (Amersham Biosciences).

45Ca2+/54Mn2+ Fluxes—These radioactive isotope fluxes were essentially performed as described earlier (11). Cells were loaded with 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM ATP, 0.44 mM potassium EGTA (pH 6.8), 10 mM NaN3, and 2 µM thapsigargin to inhibit SERCA activity. Unless otherwise stated, cells were loaded for 90 min and 5 mM potassium oxalate was included in the loading medium. MgCl2, CaCl2, and MnCl2 were added to obtain a free Mg2+ concentration of 0.5 mM (unless otherwise stated) and the indicated concentrations of free Ca2+ and Mn2+. Calcium ionophore A23187 [GenBank] (10 µM) was included in the efflux medium as indicated.

Analysis of Phospho-intermediates—Phosphorylation from [{gamma}-32P]ATP, processing of the acid-precipitated microsomal proteins, and acidic SDS-PAGE electrophoresis were essentially performed as described before (11). The rate of dephosphorylation of the phosphoenzyme intermediate formed from ATP was analyzed after treatment with EGTA and ADP for serial time intervals prior to acid quenching (23, 24). Phosphorylation was performed in 100 µl of a solution containing 160 mM KCl, 17 mM potassium Hepes (pH 7), 5 mM NaN3, 1 mM dithiothreitol, and 2 µM thapsigargin. Phosphorylation from 0.5 mM 32Pi was performed at 25 °C as before (23). Quantification of the separated phosphoenzyme band was performed by imaging using the Packard CycloneTM storage phosphor system (Packard Bioscience, Berkshire, UK). Appropriate background phosphorylation levels were subtracted before data analysis as described (24).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alternative Splicing of ATP2C1 Primary Transcript—The primary aim of this study was to explore the stability and functional properties of a selected panel of mutated hSPCA1 proteins (see Table I), with each mutant incorporating one of the documented ATP2C1 missense mutations previously identified in HHD patients in our laboratory (2, 19). We have chosen to assess the effect of these disease mutations when introduced into a new splice variant, ATP2C1d, described here for the first time. This variant was identified following the rapid amplification of cDNA ends (RACE)-PCR analysis, performed on cDNA reverse-transcribed from isolated human keratinocyte total RNA. As shown in Fig. 1A, 3' RACE-PCR produced three strong bands of 450, 420, and 336 bp. Subcloning and subsequent sequencing confirmed that the 336- and 420-bp fragments, respectively, corresponded to the previously described splice variants ATP2C1a (2, 3) and ATP2C1b (2), whereas the 450-bp band corresponded to a novel splice variant, designated ATP2C1d. A weaker 325-bp band, corresponding to a variant previously identified by Hu et al. (3), was also amplified by PCR and was designated ATP2C1c in this study. However, the corresponding protein, hSPCA1c, is unlikely to play an important functional role in keratinocytes (see "Discussion"). The scheme in Fig. 1B depicts the four modes of alternative splicing, and Fig. 1C illustrates the structure of the corresponding mature mRNAs. ATP2C1d (hSPCA1d), identified in this study, results from activation of a novel internal 5' donor splice site (designated D2) in exon 27 within codon Val-919 (nt 2756 relative to the ATG start codon). Val-919 is found immediately upstream of the exon 27 TGA translation stop codon, meaning that exon 27 reaches a maximum size of 128 bp. ATP2C1d mRNA is expressed to a high level in keratinocytes and is encoded by the full-length of optional exons 27 and 28, making ATP2C1d the longest alternatively spliced variant. In view of this, all our further experiments were performed using this variant, either in its wild type form or following the introduction of nine missense mutations identified in HHD patients (Table I).



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FIG. 1.
Gene structure of ATP2C1 and alternative transcript processing. A, shows the results of PCR amplification from human keratinocyte double-stranded cDNA using a gene-specific primer ATP2C13'GSP located in exon 26, and primers specific for the 3'-untranslated regions of exon 28 (Ex28R; lane 1), and exon 27 (Ex27R; lane 2), following size fractionation by 2.5% agarose gel electrophoresis. The position and size (bp) of DNA markers (lane M) is given to the left of the image. The splice variant represented by each band is given in brackets next to the fragment size. Panel B shows the 3' organization of the ATP2C1 gene and provides an overview of alternative splicing events. Exons are represented by boxes, with wide boxes depicting the open reading frame. The thin horizontal line represents the position of introns. Sa, b, c, and d represent the position of the translation stop codon for each splice variant. The internal 5' donor splice sites, D1 (nt 2726; Val-909) and D2 (nt 2756; codon Val-919), are also represented. Diagonal lines illustrate the splicing patterns generating splice variants ATP2C1a–d. Panel C describes the four ATP2C1 splice variants found in keratinocytes. The perforated line with breaks represents exon 2 to exon 25. ATG denotes the translation start codon. TGA or TAA are the stop translation codons, depending on the pattern of splicing. ATP2C1a results from splicing of exon 26 to exon 27, and its translation stop codon is located in exon 27; ATP2C1b arises from splicing of exons 27 to 28 following activation of internal 5' splice donor site D1; splicing of exons 26 to 28 gives rise to ATP2C1c; activation of splicing at internal site D2 in exon 27 to exon 28 gives rise to ATP2C1d. The relative sizes of each splice variant are as given in the figure.

 

Characterization of the Transport Capacity for Ca2+ and Mn2+ of Novel Splice Variant hSPCA1d—For functional characterization, hSPCA1d was transiently expressed in COS-1 cells. First, its ability to transport Ca2+ and Mn2+ was investigated. Because the fraction of Golgi-derived membranes accounts for only a small proportion of total ER in microsomal fractions, conventional techniques used to measure SERCA-mediated Ca2+ transport activity into isolated microsomes (22) could not be employed here. Alternatively, all Ca2+ uptake measurements were performed using detergent-permeabilized whole cells (25). COS-1 cells transfected with empty vector (control) or wild type hSPCA1d cDNA were first permeabilized with saponin in a medium mimicking cytosolic composition and then loaded with 45Ca2+ in the presence of sodium azide (NaN3), to inhibit mitochondrial pump activity, and in the presence of thapsigargin, to inhibit SERCA pump activity. Efflux of the Ca2+ taken up by active pumps was then followed for 16 min in Ca2+-free medium, with the ionophore A23187 [GenBank] being added after 8 min. Fig. 2A shows that hSPCA1d-transfected cells exhibit a significantly higher thapsigargin-insensitive Ca2+-uptake compared with control cells. The addition of A23187 [GenBank] enhanced the release of stored Ca2+, suggesting that the Ca2+ had been transported into a membrane-delineated compartment. These results support the theory that hSPCA1d represents a thapsigargin-insensitive Golgi pump. Furthermore, the addition of 5 mM potassium oxalate to the loading medium stimulated an increase in the Ca2+-uptake capacity of hSPCA1 by more than 50%, when compared with uptake in the absence of oxalate. Oxalate acts to increase the ability of hSPCA1d to transport Ca2+ into the Golgi by precipitating Ca2+ and thus reducing the free Ca2+ concentration within the Golgi lumen. Fig. 2B compares the time-dependent loading of 45Ca2+ in hSPCA1d-expressing COS-1 cells versus control cells. A higher Ca2+ content was observed in transfected cells, compared with control cells, with increasing loading time. However, because more cells are prone to detach from the culture plate with prolonged loading times, subsequent experiments were all maintained at 90-min loading. Fig. 2C shows that half-maximal activation of hSPCA1d occurred at 0.20 µM free Ca2+, which is equivalent to that of 0.25 µM Ca2+, previously reported for the orthologue from C. elegans (11) and 0.26 µM reported for hSPCA1a, following heterologous expression in yeast (12).



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FIG. 2.
Ca2+ transport activity of hSPCA1d. A, effect of oxalate (5 mM) on 45Ca2+ loading of COS-1 cells transfected with hSPCA1 and empty vector. Cells were loaded at 0.2 µM free Ca2+ for 90 min in the presence of 2 µM thapsigargin. Following two washes with efflux medium, the passive efflux of Ca2+ was measured for 16 min. Ionophore A23187 [GenBank] was administered after 8 min of efflux. B, time-dependent 45Ca2+ loading of COS-1 cells transfected with hSPCA1 and non-transfected control cells. Cells were loaded for 2, 10, 20, 30, 60, or 90 min at 0.24 µM free Ca2+ in the presence of 2 µM thapsigargin and 5 mM potassium oxalate. Results in A and B represent the mean ± S.E. of three experiments. C, Ca2+ uptake (90 min) into COS-1 cells transfected with hSPCA1 or empty vector was measured at different free Ca2+ concentrations. Results show the Ca2+ content of the stores, plotted as a function of the Ca2+ concentration in the loading medium. Data points correspond to the difference between cells transfected with hSPCA1 and empty vector control cells. They represent a mean ± S.E. of six experiments.

 

In the next series of experiments, the ability of hSPCA1d to transport Mn2+ was assessed. First, the ability of Mn2+ to inhibit 45Ca2+ uptake was evaluated, as an indirect indicator of Mn2+-uptake capacity. Fig. 3A clearly shows that, at higher Ca2+ concentrations, more Mn2+ was needed to inhibit Ca2+ uptake. Half-maximal inhibition was observed at 0.86 and 0.36 µM Mn2+ when loaded with 1 or 0.1 µM Ca2+, respectively. The respective values reported for C. elegans SPCA were 1 and 0.2 µM Mn2+. Fig. 3 (B and C) provide direct evidence that hSPCA1d can transport Mn2+. COS-1 cells transfected with hSPCA1d showed an increased level of 54Mn2+ uptake relative to control cells (Fig. 3B). Following the addition of ionophore A23187 [GenBank] after 4 min of efflux, the rate of Mn2+ release was increased. Fig. 3C shows efflux curves from cells loaded with 1 µM Mn2+ at various free Ca2+ concentrations in the loading medium. The Mn2+ uptake became increasingly inhibited by the increased Ca2+ concentration, thus demonstrating the competition between Ca2+ and Mn2+.



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FIG. 3.
Mn2+ transport activity of hSPCA1d and competition between Mn2+ and Ca2+ for uptake. A, inhibition of hSPCA1 45Ca2+ uptake by Mn2+. Cells were loaded at 1 µM or 0.1 µM free Ca2+ in the presence of a range of free Mn2+ concentrations. Results show the Ca2+ content of the stores plotted as a function of the free Mn2+ concentration and represent the mean ± S.E. of four independent experiments. B, 54Mn2+ loading of COS-1 cells transfected with hSPCA1 or empty vector control. Cells were loaded for 90 min at 1 µM free Mn2+. Following three washes with efflux medium, passive efflux of Mn2+ was measured for 12 min. Ionophore A23187 [GenBank] was added after 4 min. Results represent the mean ± S.E. of six experiments. C, inhibition of hSPCA1 54Mn2+ uptake by Ca2+. Cells were loaded at 1 µM free Mn2+ and nominal, 1 µM, or 10 µM free Ca2+.Mn2+ efflux was followed for 10 min. Values represent the difference between hSPCA1 and empty vector-transfected COS-1 cells. Points represent the average of two experiments.

 

Expression and Cellular Localization of Wild Type and HHD Mutant hSPCA1d—As a first step in the analysis of the HHD mutants, expression and cellular localization were compared with that of the wild type, because even minor mutation-induced structural abnormalities could, in principle, lead to protein degradation and/or cellular mistargeting.

The expression of wild type hSPCA1d in COS-1 cells transfected with the corresponding cDNA was clearly demonstrated by both immunocytochemistry and Western blot analysis (Fig. 4, A and B) using a newly generated rabbit polyclonal antibody raised against the large cytoplasmic loop of the hSPCA1 protein. The low level of hSPCA1 reactivity seen in control COS-1 cells indicated that our antiserum was able to detect the low level of endogenous COS-1 SPCA1 homologue. Immunocytochemical analysis of wild type hSPCA1-expressing COS-1 cells illustrates that the protein was correctly targeted to the Golgi. This result was further confirmed by co-localization of hSPCA1d with the key Golgi markers TGN46 (Fig. 4A) and mannosidase II (data not shown). Additionally, all nine HHD mutants studied were also correctly targeted to the Golgi, as exemplified by the cellular localization of D742Y. Wild type and mutant protein expression levels were compared by Western blot analysis of total protein extracted from transfected COS-1 cells (Fig. 4B, top row). The hSPCA1-specific immunoreactive bands migrated slightly below the predicted molecular mass of 104 kDa. P201L, G309C, I580V, and D742Y expression was comparable to wild type, whereas L341P, C344Y, C411R, T570I, and G789R were only marginally expressed above background. Equal protein loading was confirmed by re-staining of the stripped membrane with an antibody against {alpha}-tubulin (Fig. 4B, bottom row). The low levels of hSPCA1d protein observed for L341P, C344Y, C411R, T570I, and G789R were not accompanied by correspondingly low mRNA levels, as documented by real-time PCR, performed in the presence of SYBR Green 1. ATP2C1 mRNA copy numbers were calculated to be 250-fold higher in COS-1 cells transfected with the wild type cDNA, compared with endogenous levels. The mRNA levels for all nine HHD mutants did not differ significantly from the wild type ATP2C1 mRNA level (data not shown). PCR products isolated from the LightCyclerTM capillaries after cycling are shown in Fig. 4C.



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FIG. 4.
Wild type and HHD mutant hSPCA1 protein and mRNA expression in COS-1 cells. A, immunocytological staining of COS-1 cells (x100 magnification) transiently transfected with empty vector (control), D742Y, and wild type hSPCA1. Primary incubation was with anti-hSPCA1 antiserum (1:1500) and an antibody against the resident Golgi marker TGN46 (1:300). Secondary antibodies were fluorescein isothiocyanate-conjugated donkey anti-sheep and goat anti-rabbit (1:1000). The merged image illustrates the superimposable localization of hSPCA1 and TGN46 in a juxtanuclear concentration characteristic of Golgi. D742Y distribution is representative of that found for all nine HHD mutant proteins. B, Western blot of COS-1 protein (25 µg) isolated from cells transiently transfected with wild type or HHD mutant hSPCA1 and empty vector (control). Primary incubation was with anti-hSPCA1 (1:500) antibody. Secondary antibody was alkaline phosphatase-conjugated goat anti-rabbit (1:8000). The blot was then stripped and immunostained with mouse anti-{alpha}-tubulin (1:2000) followed by anti-mouse secondary antibody as above. Blots were analyzed using a PhosphorImager model Storm 840 (Amersham Biosciences). C, products generated with ATP2C1 primers C7F and C7R were isolated following LightCyclerTM real-time PCR cycling and visualized after electrophoresis on 1.5% agarose gels. Control samples, where no reverse transcriptase was added (–RT enz), were included in all experiments to show that all products were RNA-derived and not the result of genomic contamination. Also represented are negative controls included during reverse transcription (RT-ve) and PCR (PCR-ve). Results represent those obtained from three closely similar independent experiments.

 

Formation of hSPCA1 Phosphoenzyme Intermediate from ATP and PiA characteristic of the reaction mechanism of phosphorylation-type ion transport ATPases, cycling under normal (forward) conditions, is the transient transfer of the {gamma}-phosphate of ATP to the protein. This phosphorylation reaction requires that one or more of the transported ions are bound to the enzyme. Conversely, in the SERCA enzymes, phosphorylation from inorganic phosphate in the reverse reaction is inhibited by the presence of Ca2+ (24, 26), presumably because Ca2+ binding transforms the Pi-reactive E2 conformation into the E1 conformation (see Scheme 1). It has not previously been determined whether the hSPCA1 enzyme responds in a similar way and whether Mn2+ exerts similar effects as Ca2+ on this enzyme. An interesting difference between the SERCA and SPCA is that, although the transport stoichiometry in the former is two per cycle, the latter is likely to bind and transport only one Ca2+ or Mn2+ per cycle, because only the residues binding one of the two Ca2+ ions in SERCA (corresponding to the so-called "site II") are conserved in SPCA. In the next series of experiments, we investigated the ability of the overexpressed wild type and HHD mutant hSPCA1d to phosphorylate from [{gamma}-32P]ATP or 32Pi in a Ca2+- and Mn2+-dependent way. Phosphorylation from ATP was performed using conditions known to favor accumulation of the E1~P form of SERCA in the presence of Ca2+ (0 °C, neutral pH, presence of alkaline metal ions) (23). Fig. 5B clearly shows that as little as 5 µM Ca2+ or Mn2+ is required to activate the phosphoryl transfer from ATP in wild type hSPCA1d. Phosphorylation from Pi was performed in conditions known to favor SERCA E2-P accumulation in the absence of Ca2+ (acidic pH, absence of alkali metal ions, and presence of the organic solvent dimethyl sulfoxide (26)). Fig. 5C shows that these conditions also allow formation of phosphoenzyme from Pi in the SPCA class. Furthermore, 2.5 mM Ca2+ or Mn2+ completely prevented phosphorylation from Pi in wild type hSPCA1d, indicating that Ca2+ and Mn2+ have a similar effect on the E1-E2 conformational equilibrium of the SPCA class to that shown for Ca2+ with SERCA. These phosphorylation assays furthermore permitted assessment of the intactness of the ion binding sites in the SPCA mutants, following the same principles as those previously applied to SERCA1a mutants (13, 14). Phosphorylation reactions were performed with equal amounts of hSPCA1 protein, taking into account any small differences in the level of hSPCA1 expression found among the different microsome preparations by Western blot analysis (Fig. 5A). This enabled the direct comparison between wild type and mutant phosphoenzyme levels. Our results indicate that Ca2+ or Mn2+ bind with normal affinity to P201L and I580V, causing activation of phosphorylation from ATP (Fig. 5B) and inhibition of phosphorylation from Pi (Fig. 5C). As seen in Fig. 5B, P201L phosphorylation from ATP was stimulated to a similar level as the wild type by 5 µM Ca2+ and Mn2+, whereas a higher level of phosphorylation was observed for I580V. The latter increase can be explained by the reduced rate of E1~P -> E2-P conversion observed for this mutant, as described below, because the amount of phosphoenzyme accumulated at a given Ca2+ concentration depends on the rate of phosphoenzyme turnover as well as the Ca2+-dependent rate of phosphoenzyme formation.



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FIG. 5.
Wild type and HHD-mutated hSPCA1 phosphorylation from ATP or Pi. A, shows an immunoblot of the microsomal fraction (10 µg) prepared from COS-1 cells transfected with WT or HHD mutant hSPCA1. Primary incubation was with polyclonal anti-hSPCA1 antibody (dilution 1:500); secondary antibody was alkaline phosphatase-conjugated goat anti-rabbit (1:8000). B, autoradiograph showing the phosphoenzyme intermediate formed from 5 µl of 0.5 µCi/µl[{gamma}-32P]ATP using 1 µg of microsomal protein in the presence of 5 µM Ca2+, 5 µM Mn2+, or 2 mM EGTA (as a measure of background phosphorylation), and 160 mM KCl, 17 mM potassium Hepes (pH 7), 0.5 mM MgCl2, 5 mM NaN3, 1 mM dithiothreitol, and 2 µM thapsigargin. Free Ca2+ and Mn2+ concentrations were buffered with 0.5 mM potassium EGTA (pH 7). The radioactively labeled phosphoprotein was preserved by maintaining acidic conditions during SDS electrophoresis. C, phosphorylation from 32Pi (0.5 mM) was performed using 7.5 µg of microsomal protein in the presence of 100 mM MES/Tris (pH 6), 10 mM MgCl2, 2 mM EGTA, and 30% (v/v) Me2SO at 25 °C for 10 min prior to acid quenching. To demonstrate the binding of Ca2+ or Mn2+ (resulting in background phosphorylation levels in wild type) 2.5 mM of these ions was included in the phosphorylation medium. Results are shown as an autoradiogram of dried SDS-polyacrylamide gels.

 

In contrast, G309C and D742Y were unable to form significant amounts of phosphoenzyme from ATP in the presence of 5 µM Ca2+ or Mn2+ (Fig. 5B), although a high level of phosphoenzyme was produced from Pi (Fig. 5C). Because these mutations involve residues within the transmembrane sector, located far from the nucleotide binding and phosphorylation domains, the lack of phosphoenzyme formation from ATP is not likely to be caused by a direct effect on nucleotide binding or catalysis but is, rather, due to defective ion binding and/or signal transduction from the ion binding site to the phosphorylation domain. The inability of D742Y to bind either of the transported ions is further verified by the observation that phosphorylation of D742Y from Pi remained high in the presence of 2.5 mM Ca2+ or Mn2+; a concentration that completely prevented phosphorylation from Pi in the wild type (Fig. 5C). Interestingly, phosphorylation of G309C from Pi remained high in the presence of 2.5 mM Mn2+, but was inhibited by 2.5 mM Ca2+ (Fig. 5C). This shows that, although this mutation entirely abolishes the ability to bind Mn2+, the enzyme can still bind Ca2+, albeit with a reduced affinity, because the phosphorylation from ATP was not activated by 5 µM Ca2+.

Dephosphorylation from E1~P—The decay of the E1~P phosphoenzyme intermediate formed from ATP can occur either in the forward direction, via the conformational change to E2-P and subsequent E2-P hydrolysis, or in the reverse direction, through transfer of the phosphoryl group to ADP, yielding ATP (see Scheme 1). The rate of phosphoenzyme decay in the forward direction can be monitored by determination of residual phosphoenzyme levels upon treatment with EGTA (1 mM, to remove Ca2+ and, thereby, prevent further phosphorylation) for serial time intervals, prior to acid quenching. This assay can therefore be used to identify mutants in which the high energy, phosphorylated E1~P conformation can only move forward slowly, if at all, into the E2-P conformation. By addition of ADP (1 mM) to dephosphorylate E1~P in the reverse direction, the amount of residual phosphoenzyme in the E2-P (ADP-insensitive) form can also be measured. Therefore, mutants defective in either the E1~P -> E2-P conversion or E2-P hydrolysis can be identified. The slow decay of I580V phosphoenzyme relative to wild type shown in Fig. 6 demonstrates the limited ability of this mutant to go through the E1~P -> E2-P conformational change. Approximately 80% of I580V phosphoenzyme remained after 120 s treatment with EGTA, compared with only 10% of the wild type. The amount of phosphoenzyme in the ADP-insensitive (E2-P) conformation was similar to wild type levels, demonstrating that this mutant was not defective in E2-P hydrolysis. Conversely, the rate of dephosphorylation and the ratio of E1~P to E2-P for P201L were similar to wild type levels.



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FIG. 6.
Autoradiographs showing dephosphorylation from El~P in wild type and HHD mutant hSPCA1. Phosphorylation from ATP was performed as described in the legend to Fig. 5B using 15–25 µg of microsomal protein, 5 µl of 0.5 µCi/µl [{gamma}-32P]ATP, and 0.1 mM Ca2+ or 2 mM EGTA. The presence of alkali metal ions in the reaction medium was maintained to promote accumulation of E1~P. Ionophore A23187 [GenBank] (2 µM) was included in the reaction medium to prevent inhibition of dephosphorylation arising from Ca2+ accumulation in microsomes. Following a 20-s phosphorylation, dephosphorylation was monitored by addition of 1 mM EGTA to terminate phosphorylation by chelation of Ca2+. Acid quenching was performed at the times indicated, after addition of EGTA, for determination of residual phosphoenzyme. ADP (1 mM) was added with EGTA, where indicated, 5 s before acid quenching, to demonstrate the ADP sensitivity of the phosphoenzyme. The phosphoenzyme remaining after a 5-s reaction with ADP represents E2-P, whereas that observed in the absence of ADP represents the sum of E1~P and E2-P. Numbers given underneath each autoradiograph represent the amount of phosphoenzyme remaining as a percentage of that formed in the absence of EGTA treatment (i.e. 0-s EGTA) in each individual experiment, following quantification of radioactive bands as described under "Experimental Procedures."

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have investigated the underlying cause of HHD by analyzing the changes in expression and function of hSPCA1 caused by mutations found in HHD patients. Mutations were introduced into ATP2C1d, a novel splice variant expressed in keratinocytes, described for the first time in this study. Encoded by the full-length of optional exons 27 and 28, ATP2C1d is longer than previously identified splice variants. We have provided evidence for the existence of up to four distinct ATP2C1 splice variants in human keratinocytes, generated by alternative processing at the 3'-end of the ATP2C1 pre-mRNA. Similar to ATP2C1, internal 5' donor splice sites are involved in generating five alternatively spliced human ATP2A3 (SERCA3) transcripts (27, 28) and four alternatively processed ATP2B1 (PMCA1) transcripts (2931) due to the alternative exclusion, inclusion, or partial inclusion of exons at the 3'-end of the gene. So far, no evidence has been provided for the existence of alternatively spliced transcripts in the S. cerevisiae or C. elegans SPCA orthologues. Interestingly, the cryptic 5' donor splice site D2, identified here, is also conserved in rat (32).

The ubiquitous expression of ATP2C1 mRNA has previously been demonstrated in several tissues (3). We have further studied the mRNA tissue distribution pattern in 76 fetal and adult human tissues by dot blot hybridization analysis and confirmed that hSPCA1 is ubiquitously expressed.2 However, the physiological significance of the presence of four alternatively spliced transcripts in keratinocytes remains to be investigated. Studies of SERCA and PMCA have revealed that divergence in the extreme C-terminal can be responsible for some functional differences between the splice variants (22, 3336). Unfortunately, due to the high degree of sequence overlap at both the DNA and protein levels, it is impossible to generate hSPCA1 variant-specific DNA probes or antibodies that would be required to study any subtle differences in function or expression resulting from divergence in C-terminal sequence. Interestingly, some indications that functional differences do exist between the splice variants can be drawn from a previous study, where we identified a splice acceptor mutation in intron 26 of an HHD patient (19). With the likely effect of impaired splicing to exon 27, it can be inferred that hSPCA1a, hSPCA1b, and hSPCA1d cannot be produced from the mutant allele, because these splice variants contain amino acid stretches encoded by different parts of exon 27 (see Fig. 1). Although the reported mutation should not affect the formation of the ATP2C1c splice variant, the manifestation of classic HHD symptoms in the patient carrying this mutation does not suggest any functional compensation brought about by hSPCA1c. This lack of compensation is most likely due to the disruption of transmembrane segment 10, resulting from the exclusion of exon 27, and suggests that hSPCA1c has very limited functional capability, if any. For this reason, together with the low level of ATP2C1c mRNA expressed in keratinocytes, we have not attempted further characterization of this variant. Instead we have focused on the longest variant expressed in keratinocytes, ATP2C1d (hSPCA1d), which encodes optional 3' exons 27 and 28 in their entirety and, therefore, was thought most appropriate for the consecutive study of disease-causing point mutations.

Recently, the first functional evidence that hSPCA1 is a bona fide member of the SPCA class was reported following heterologous expression of hSPCA1a in yeast (12). Using an isotope flux-based procedure, we show here that hSPCA1d can transport both Ca2+ and Mn2+ into the Golgi apparatus of COS-1 cells with high affinity and in a thapsigargin-insensitive manner. A novelty of the experimental system used here relative to that previously applied in studies of C. elegans SPCA1 (11), is the addition of 5 mM potassium oxalate to the loading medium. We have shown that the addition of oxalate greatly increases the uptake capacity of the hSPCA1-containing store. The ability of oxalate to penetrate the Golgi membrane has been a point of some debate; however, Ca2+ oxalate precipitations have previously been reported in the Golgi of smooth muscle (37). The Ca2+ buffering capacity of oxalate enabled the loading time during isotope flux to be extended to 90 min without saturation of internal stores. This increased the observed uptake of hSPCA1d relative to background levels, thus strengthening the credibility of the data.

By overexpression of hSPCA1d in COS-1 cells, we have demonstrated that the enzyme has a high affinity for Ca2+ equal to that previously demonstrated for hSPCA1a (12) and C. elegans SPCA1 (11). By comparison, S. cerevisiae PMR1 was shown to have an even higher affinity for Ca2+ (K0.5 = 70 nM) (15). We have shown for the first time the direct transport of Mn2+ by hSPCA1 and provide evidence for the competitive uptake of Ca2+ and Mn2+, through inhibition of Ca2+ uptake by increasing concentrations of Mn2+ and vice versa. Half-maximal inhibition of Ca2+ uptake occurred at a Mn2+ concentration approximately equivalent to the Ca2+ concentration required for half-maximal stimulation of the uptake, indicating that the affinity of the hSPCA1d transport ATPase for Ca2+ and Mn2+ is approximately the same. In contrast, similar studies performed on S. cerevisiae PMR1 showed this latter protein to have a somewhat lower affinity for Ca2+ than Mn2+ (17). Conclusions drawn from the isotope fluxes were further verified by the results of phosphoenzyme studies, which indicated that maximal phosphoprotein formation from ATP was achieved at micromolar concentrations of Ca2+ and Mn2+, demonstrating the high affinity nature of the binding sites. Furthermore, phosphorylation was activated to an equal extent by Ca2+ and Mn2+, supporting the notion that these ions are bound with equal affinities.

The large number of nonsense, frameshift, and splice site mutations identified in the ATP2C1 gene of HHD patients support the theory that haploinsufficiency of hSPCA1, via nonsense-mediated mRNA decay of the mutant allele, is a prevalent mechanism for the dominant inheritance of HHD. However, the precise functional consequence of the missense mutations found in patient ATP2C1 cannot be predicted a priori. All nine of the HHD missense mutations studied here occur at residues that are invariant across a range of hSPCA1 orthologues. This high degree of conservation suggests that the residues involved are essential for correct protein function. In this study we have provided evidence that these missense mutations are responsible for low expression levels, or defects in Ca2+ and Mn2+ binding, or the energy-transducing E1~P -> E2-P conformational change. By looking at the precise effect of the mutation on protein function, these results outline the underlying cause of HHD in these patients. Furthermore, this work provides an insight into structure/function relationships in the SPCA class.

We have shown that the low expression level of mutant proteins L341P, C344Y, C411R, T570I, and G789R is not the result of impaired mRNA levels. Instead, we suggest that these non-conservative mutations introduce structural perturbations into the hSPCA1 protein, resulting in either abnormal protein folding or destabilization of the correctly folded protein, thus making it sensitive to endoplasmic reticulum-mediated quality control. Indeed, such an effect was previously observed when deletions or specific substitutions of a few residues were made in the N-terminal region of SERCA1a (38). In this study of SERCA1a, the mutations were shown to have no effect on transcription, translation, or integration of the protein into the membrane but, rather, induced protein degradation at a rate substantially faster than the wild type. The detection of some mutant L341P, C344Y, C411R, T570I, and G789R proteins, which had correctly localized to the Golgi in COS-1 cells, supports the hypothesis that these amino acid substitutions might induce an abnormally rapid rate of degradation of the mutant proteins, because it indicates that at least some of the mutant protein does escape endoplasmic reticulum-mediated quality control. Moreover, these results further support the theory of haploinsufficiency as a mechanism for the dominant inheritance of HHD, by suggesting that epidermal cells are sensitive to levels of hSPCA1.

Only one of the hSPCA1 mutations (T570I) resulting in a low level of protein expression occurred at an amino acid residue that has been studied at its conserved site in SERCA1a (Thr-625). It has been proposed that Thr-625 in SERCA1a, which is an integral part of the highly conserved TGD motif, might participate in the formation of the nucleotide-binding site (39). Conservative mutations in Thr-625 were shown to result in a Ca2+ transport deficiency and impaired abilities to form a phosphorylated intermediate with ATP or Pi (40). Interestingly, as shown here for T570I, the change T625V in SERCA1a also resulted in a marked decrease in expression compared with the level observed for the wild type. Analogous to conclusions drawn from this study, the authors suggested that such a large change in amino acid polarity might have led to a structural alteration.

The evidence presented in this study suggests that Asp-742 is equally important for the binding of Ca2+ and Mn2+, whereas Gly-309 seems to influence Mn2+ binding more profoundly than Ca2+ binding. These conclusions are based on the observations that D742Y and G309C phosphorylation from ATP was not activated by 5 µM Ca2+ or Mn2; ion concentrations known to almost saturate the wild type enzyme, as judged by the results of our isotope fluxes. Phosphorylation of D742Y from Pi was not inhibited at 2.5 mM of either ion, whereas phosphorylation of G309C from Pi was inhibited at 2.5 mM Ca2+, but not at 2.5 mM Mn2+. The latter finding shows that G309C is unable to bind Mn2+ even at 2.5 mM, whereas Ca2+ is bound with a moderately reduced affinity, intermediate between 5 µM and 2.5 mM. An equivalent assay was originally used to demonstrate the inability of SERCA1a mutants with respect to Ca2+ binding (41, 42). The importance of the hSPCA1 residue Asp-742 in ion binding is not surprising, in view of the results obtained in intensive mutagenic and structural studies of Asp-800, the equivalent amino acid in SERCA1a. All evidence points toward this residue forming an integral part of the Ca2+-binding site, by acting as a Ca2+-binding ligand through the donation of side-chain oxygen atoms (4145). More recently, Ca2+ and Mn2+ uptake has been shown to be inhibited by mutations at the equivalent residue, Asp-778, in S. cerevisiae PMR1 (17), supporting our evidence that ion binding is conserved at this site within the SPCA class. It is interesting to note that, although Asp-800 donates a side-chain oxygen atom to each of the two Ca2+-binding sites in SERCA1a (41, 45), this residue still plays a crucial role in SPCA ion binding, where only one ion-binding site, corresponding to site II in SERCA1a (45), appears to be conserved (based on comparison with the SERCA1a sequence). Furthermore, our studies show that, in the wild type hSPCA1, Ca2+ or Mn2+ binding inhibits phosphorylation from Pi, similar to the effect of Ca2+ binding in SERCA1a (41, 42), despite the fact that in SERCA1a this effect is caused by Ca2+ binding at the site (site I) that apparently does not exist in SPCA. Hence, it can be concluded that the inhibition of Pi phosphorylation occurs irrespective of which Ca2+ site is being occupied, supporting the notion that inhibition is caused by displacement of the E1-E2 conformational equilibrium away from the Pi-reactive E2 form (see Scheme 1).

The difference between the effects of HHD mutant G309C on Ca2+ and Mn2+ binding observed here suggest that Gly-309 is important for Mn2+ transport selectivity. Gly-309 is located immediately adjacent to the putative ion-binding residue, Glu-308, in transmembrane segment M4. Studies of the equivalent residue, Gly-310, in SERCA1a infer that mutations could sterically hinder ion binding, with the Ca2+ affinity of the enzyme decreasing as the size of the substituted side chain in this position was increased (41). Recently, a mutation in the putative transmembrane segment M4 Ca2+-binding site of S. cerevisiae was shown to have a more deleterious effect on the transportation of Mn2+ in comparison to Ca2+ (17). This observation supports our conclusion that this region of the protein plays an important role in hSPCA1 Mn2+ transport selectivity. Furthermore, structural information has highlighted the close interaction between transmembrane segments M4 and M6 in SERCA1a (45). With the recent identification of Gln-783 in transmembrane segment M6 as an important residue in Mn2+ selectivity of S. cerevisiae PMR1 (18), it seems conceivable that specific residues in M4 and M6 could interact to control uptake of this ion in the SPCA class.

The relatively conservative mutation, I580V, has been identified as a conformational change mutant in this study. No information regarding this or adjacent residues in the phosphorylation domain has been reported for the equivalent site in SERCA1a. However, it now seems that the I580V mutation disrupts the crucial conformational change associated with the transformation of the enzyme from the "high energy" state (E1~P), to the "low energy" state (E2-P). Interestingly, in the crystal structure of SERCA1a (45) the equivalent residue Ile-635 has been shown to be located close to the hinge between the phosphorylation and nucleotide-binding domains, which could explain the involvement in the conformational change if a hinge-bending movement occurs in relation to the E1~P -> E2-P transition.

The fact that the P201L mutation was not detected in 100 control chromosomes suggests that it does not represent a benign polymorphism. However, we found little evidence that the hSPCA1 enzymatic cycle was directly affected in this mutant protein, which correctly localized to the Golgi and was expressed to a similar level as the wild type. These results are concurrent with previous observations made following investigation of the equivalent mutation, P195L, in SERCA1a (46). Interestingly, a 15% reduction in the Vmax for Ca2+ transport was observed for P201L in isotope fluxes, whereas the apparent affinity for Ca2+ was close to wild type levels (data not shown). However, the validity of comparing the ion transport capacity of different proteins by isotope flux was questioned in view of the differential levels of toxicity exerted on cells by the transfection of individual constructs. The potentially variable loss of cells from culture plates transfected with different constructs would have a direct effect on the ion uptake figures obtained during each experiment. Nevertheless, in view of this result, it is tempting to speculate that such a reduction could be the cause of the HHD phenotype in patients carrying this mutation. Alternatively, the reduction of the Vmax for Ca2+ transport could be explained by the existence of slower steps in the catalytic cycle compared with the wild type, such as those associated with the ion-binding transition (E2 -> E1(Ca2+/Mn2+)). However, it is also possible that the mutation might affect an important interaction with a regulatory protein present in keratinocytes.


    FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) ATP2C1a (hSPCA1a), AF181120 [GenBank] ; ATP2C1b (hSPCA1b), AY268374 [GenBank] ; ATP2C1c (hSPCA1c), AF181121 [GenBank] ; ATP2C1d (hSPCA1d), AY268375 [GenBank] ; GAPDH, CA942187 [GenBank] .

* This work was supported in part by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Wellcome Trust Prize Studentship. Back

** Holder of a Wellcome Trust Senior Clinical Fellowship. To whom correspondence should be addressed: INSERM U563, Pavillon Lefebvre, Purpan Hospital, Place du Dr Baylac, 31059 Toulouse cedex 03, France. Tel.: 33-561-158-432; Fax: 33-561-499-036; E-mail: alain.hovnanian{at}toulouse.inserm.fr.

1 The abbreviations used are: PMCA, plasma membrane calcium adenosine triphosphatase; (h)SPCA1, (human) secretory pathway Ca2+/Mn2+-adenosine triphosphatase; HHD, Hailey-Hailey disease; SERCA, sarco(endo)plasmic reticulum calcium adenosine triphosphatase; PMR, plasma membrane ATPase-related; cDNA, complementary DNA; RACE, rapid amplification of cDNA ends; GSP, gene-specific primers; nt, nucleotide(s); D, donor; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid. Back

2 R. J. Fairclough, L. Dode, J. Vanoevelen, J. P. Andersen, L. Missiaen, L. Raeymaekers, F. Wuytack, and A. Hovnanian, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Jerry Renders for initial help with the isotope flux, Yves Parijs, Sylvie De Swaef, Marleen Schuermans, Lea Bauwens, and Anja Floorizone for technical assistance, and Dr. Helen Fletcher and Laura Andrews for providing primers and help with real-time PCR. Further thanks go to Prof. Humbert De Smedt and Dr. Kurt Van Baelen for helpful advice and interesting discussions and to Prof. Anthony P. Monaco for his invaluable role in project supervision.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Møller, J. V., Juul, B., and le Maire, M. (1996) Biochim. Biophys. Acta 1286, 1–51[Medline] [Order article via Infotrieve]
  2. Sudbrak, R., Brown, J., Dobson-Stone, C., Carter, S., Ramser, J., White, J., Healy, E., Dissanayake, M., Larrègue, M., Perrussel, M., Lehrach, H., Munro, C. S., Strachan, T., Burge, S., Hovnanian, A., and Monaco, A. P. (2000) Hum. Mol. Genet. 9, 1131–1140[Abstract/Free Full Text]
  3. Hu, Z., Bonifas, J. M., Beech, J., Bench, G., Shigihara, T., Ogawa, H., Ikeda, S., Mauro, T., and Epstein, E. H., Jr. (2000) Nat. Genet. 24, 61–65[CrossRef][Medline] [Order article via Infotrieve]
  4. Rudolf, H. K., Antebi, A., Fink, G. R., Buckley, C. M., Dorman, T. E., LeVitre, J., Davidow, L. S., Mao, J., and Moir, D. T. (1989) Cell 58, 133–145[Medline] [Order article via Infotrieve]
  5. Antebi, A., and Fink, G. R. (1992) Mol. Biol. Cell 3, 633–654[Abstract]
  6. Lapinskas, P. J., Cunningham, K. W., Lui, X. F., Fink, G. R., and Culotta, V. C. (1995) Mol. Cell. Biol. 15, 1382–1388[Abstract]
  7. Dürr, G., Strayle, J., Plemper, R., Elbs, S., Klee, S. K., Catty, P., Wolf, D. H., and Rudolf, H. K. (1998) Mol. Biol. Cell 9, 1149–1162[Abstract/Free Full Text]
  8. Cottrell, G. S., Hooper, N. M., and Turner, A. J. (2000) Biochemistry (Mosc) 39, 15121–15128[CrossRef][Medline] [Order article via Infotrieve]
  9. Hearn, A. S., Stroupe, M. E., Cabelli, D. E., Lepock, J. R., Trainer, J. A., Nick, H. S., and Silverman, D. N. (2001) Biochemistry (Mosc) 40, 12051–12058[CrossRef][Medline] [Order article via Infotrieve]
  10. Beckman, R. A., Mildvan, A. S., and Loeb, L. A. (1985) Biochemistry (Mosc) 24, 5810–5817[Medline] [Order article via Infotrieve]
  11. Van Baelen, K., Vanoevelen, J., Missiaen, L., Raeymaekers, L., and Wuytack, F. (2001) J. Biol. Chem. 276, 10683–10691[Abstract/Free Full Text]
  12. Ton, V.-K., Mandal, D., Vahadji, C., and Rao, R. (2002) J. Biol. Chem. 277, 6422–6427[Abstract/Free Full Text]
  13. Andersen, J. P., and Vilsen, B. (1995) FEBS Lett. 359, 101–106[CrossRef][Medline] [Order article via Infotrieve]
  14. MacLennan, D. H., Rice, W. J., and Green, N. M. (1997) J. Biol. Chem. 272, 28815–28818[Free Full Text]
  15. Sorin, A., Rosas, G., and Rao, R. (1997) J. Biol. Chem. 272, 9895–9901[Abstract/Free Full Text]
  16. Wei, Y., Marchi, V., Wang, R., and Rao, R. (1999) Biochemistry (Mosc) 38, 14534–14541[CrossRef][Medline] [Order article via Infotrieve]
  17. Wei, Y., Chen, J., Rosas, G., Tompkins, D. A., Holt, P. A., and Rao, R. (2000) J. Biol. Chem. 275, 23927–23932[Abstract/Free Full Text]
  18. Mandal, D., Woolf, T. B., and Rao, R. (2000) J. Biol. Chem. 275, 23933–23938[Abstract/Free Full Text]
  19. Dobson-Stone, C., Fairclough, R., Dunne, E., Brown, J., Dissanayake, M., Munro, C. S., Strachan, T., Burge, S., Sudbrak, R., Monaco, A. P., and Hovnanian, A. (2002) J. Invest. Dermatol. 118, 338–343[Abstract/Free Full Text]
  20. Frischmeyer, P. A., and Dietz, H. C. (1999) Hum. Mol. Genet. 8, 1893–1900[Abstract/Free Full Text]
  21. Gutzmer, R., Mommert, S., Kiehl, P., Wittman, M., Kapp, A., and Werfel, T. (2001) J. Invest. Dermatol. 116, 926–932[Abstract/Free Full Text]
  22. Verboomen, H., Wuytack, F., De Smedt, H., Himpens, B., and Casteels, R. (1992) Biochem. J. 286, 591–596[Medline] [Order article via Infotrieve]
  23. Andersen, J. P., Vilsen, B., Leberer, E., and MacLennan, D. H. (1989) J. Biol. Chem. 264, 21018–21023[Abstract/Free Full Text]
  24. Dode, L., Vilsen, B., Van Baelen, K., Wuytack, F., Clausen, J. D., and Andersen, J. P. (2002) J. Biol. Chem. 277, 45579–45591[Abstract/Free Full Text]
  25. Missiaen, L., De Smedt, H., Parys, J.-B., and Casteels, R. (1994) J. Biol. Chem. 269, 7238–7242[Abstract/Free Full Text]
  26. de Meis, L., Martins, O. B., and Alves, E. W. (1980) Biochemistry (Mosc) 19, 4252–4261[Medline] [Order article via Infotrieve]
  27. Dode, L., De Greef, C., Mountian, I., Attard, M., Town, M. M., Casteels, R., and Wuytack, F. (1998) J. Biol. Chem. 273, 13982–13994[Abstract/Free Full Text]
  28. Martin, V., Bredoux, R., Corvazier, E., van Gorp, R., Kovàcs, T., Gélébart, P., and Enouf, J. (2002) J. Biol. Chem. 277, 24442–24452[Abstract/Free Full Text]
  29. Strehler, E. E., Strehler-Page, M.-A., Vogel, G., and Carafoli, E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6908–6912[Abstract]
  30. Stauffer, T. P., Hilfiker, H., Carafoli, E., and Strehler, E. E. (1993) J. Biol. Chem. 268, 25993–26003[Abstract/Free Full Text]
  31. Keeton, T. P., Burk, S. E., and Shull, G. E. (1993) J. Biol. Chem. 268, 2740–2748[Abstract/Free Full Text]
  32. Gunteski-Hamblin, A.-M., Clarke, D. M., and Shull, G. E. (1992) Biochemistry (Mosc) 31, 7600–7608[Medline] [Order article via Infotrieve]
  33. Verboomen, H., Wuytack, F., Van Den Bosch, L., Mertens, L., and Casteels, R. (1994) Biochem. J. 303, 979–984[Medline] [Order article via Infotrieve]
  34. Lytton, J., Westlin, M., Burk, S. E., Shull, G. E., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 14483–14489[Abstract/Free Full Text]
  35. Elwess, N. L., Filoteo, A. G., Enyedi, A., and Penniston, J. T. (1997) J. Biol. Chem. 272, 17981–17986[Abstract/Free Full Text]
  36. Enyedi, A., Verma, A. K., Heim, R., Adamo, A. G., Filoteo, A. G., and Strehler, E. E. (1994) J. Biol. Chem. 269, 41–43[Abstract/Free Full Text]
  37. Kowarski, D., Shuman, H., Somlyo, A. P., and Somlyo, A. V. (1985) J. Physiol. (Lond) 366, 153–175[Abstract]
  38. Daiho, T., Yamasaki, K., Suzuki, H., Saino, T., and Kanazawa, T. (1999) J. Biol. Chem. 274, 23910–23915[Abstract/Free Full Text]
  39. Taylor, W. R., and Green, N. M. (1989) Eur. J. Biochem. 179, 241–248[Abstract]
  40. Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 22223–22227[Abstract/Free Full Text]
  41. Andersen, J. P., Vilsen, B., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 19383–19387[Abstract/Free Full Text]
  42. Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476–478[CrossRef][Medline] [Order article via Infotrieve]
  43. Rice, W. J., and MacLennan, D. H. (1996) J. Biol. Chem. 271, 31412–31419[Abstract/Free Full Text]
  44. Vilsen, B., and Andersen, J. P. (1992) J. Biol. Chem. 267, 25739–25743[Abstract/Free Full Text]
  45. Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647–655[CrossRef][Medline] [Order article via Infotrieve]
  46. Vilsen, B., Andersen, J. P., Clarke, D. M., and MacLennan, D. H. (1989) J. Biol. Chem. 264, 21024–21030[Abstract/Free Full Text]
  47. de Meis, L., and Vianna, A. L. (1979) Annu. Rev. Biochem. 48, 275–292[CrossRef][Medline] [Order article via Infotrieve]