Ecto-ATPase Activity of α-Sarcoglycan (Adhalin)*

α-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of α-sarcoglycan. α-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3′-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin·dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that α-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of α-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.

Dystrophin is a large cytoskeletal protein associated with a complex of integral and peripheral membrane proteins collectively termed DAPs. 1 Dystrophin is a long filamentous protein comprising four distinct structural domains: the amino-terminal domain, which binds F-actin, the rod-like central domain; the cysteine-rich domain, which binds the cytoplasmic portion of ␤-dystroglycan and syntrophins; and the carboxyl-terminal domain (1). The DAPs complex is composed of three subcomplexes: syntrophins, dystroglycans, and sarcoglycans (2,3). Syntrophins are peripheral membrane proteins of unknown function that bind the carboxyl terminus of dystrophin (4,5). Dystroglycans consist of two proteins derived from a common precursor protein: ␣-dystroglycan, a peripheral glycoprotein that binds extracellular matrix proteins like laminin-2 (merosin) and, in the neuromuscular junction, laminin-4 (agrin); and ␤-dystroglycan, an intrinsic membrane protein that binds dystrophin at its cytoplasmic tail and ␣-dystroglycan at the opposite end (6,7). Therefore, the dystroglycans represent the link between the subsarcolemmal actin cytoskeleton and the extracellular matrix through dystrophin. Five sarcoglycans have been described: ␣-sarcoglycan (adhalin, 50 kDa), ␤-sarcoglycan (43 kDa), ␥and ␦-sarcoglycans (35 kDa) (8 -10), and ⑀-sarcoglycan (11,12). The function of the sarcoglycans remains unknown.
Dystrophin is defective in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. In patients with DMD and in the mdx mouse, an animal model for DMD, all of the components of the DAPs are severely reduced at the sarcolemma (13,14), even though they are almost normal at the neuromuscular junction (15).
Mutations in the ␣-sarcoglycan gene, which is located on chromosome 17q21 (10), were demonstrated in limb girdle muscular dystrophy-2D (LGMD-2D), an autosomal recessive muscular dystrophy that affects both females and males (16,17). In LGMD-2D, ␤and ␥-sarcoglycan were also absent or greatly reduced, whereas dystrophin and the dystroglycan complex were preserved (18). Similar modifications were also found in the skeletal muscle of the cardiomyopatic hamster, an animal model of this disease (19). Recently, mutations in the genes that encode for ␤-, ␥-, and ␦-sarcoglycan, located on chromosomes 4q12, 13q12, and 5q33-34, were discovered in LGMD-2E, -2C, and -2F, respectively (9,20,21). These mutations caused the absence not only of the respective protein product but also of the other three components of the sarcoglycan complex. Thus, mutations causing loss of one component result in the disruption of the whole sarcoglycan complex, although dystrophin and the dystroglycan complex are preserved. These findings suggest that the subcomplexes of the dystrophin-DAPs have distinct physiological roles. Dystrophin and the dystroglycan complex, by linking the actin membrane cytoskeleton to the extracellular matrix, organize the membrane cytoskeleton and protect the sarcolemma from mechanical stress during muscle contraction; syntrophins and the sarcoglycan complex, apart from a suggested stabilizing effect on the dystrophin-glycoprotein complex, have a function that is as yet unknown.
Here we show that ␣-sarcoglycan is a sarcolemma ecto-ATPase and that sarcolemma expresses a P2X-type purinergic receptor, a nonspecific cationic channel. We speculate that ␣-sarcoglycan, by controlling the extracellular concentration of ATP, may modulate the activity of these receptors providing an attractive pathogenetic mechanism for cell death in sarcoglycanopathies.
isolated as described previously (22). The purification of the dystrophin-DAPs complex was performed according to the digitonin, 0.5 M NaCl, wheat germ agglutinin protocol of Ervasti et al. (23), as described previously (24), with the only difference being that the dystrophin-DAPs purification was terminated after the DEAE-cellulose column chromatography. The 175 mM NaCl and the first 500 mM NaCl eluates were collected, concentrated by filtration using an Amicon system (model 75 PSI), and stored at Ϫ80°C until used. The obtained dystrophin-DAPs preparation displays a protein pattern very similar to that published elsewhere ( Fig. 1; see Refs. 4,7,and 23). To verify the composition and the level of purification of the dystrophin-DAPs preparation, commercial antibodies against dystrophin, ␤-dystroglycan, and ␣and ␥-sarcoglycans were used (not shown).
[␣-32 P]ATP Photoaffinity Labeling-Photoaffinity labeling was carried out by the UV irradiation method previously described (25). Dystrophin-DAPs preparations (14 -21 g of protein) or sarcolemma membranes (28 g of protein) were equilibrated with 0.4 M ATP (containing 2 and 4 Ci of [␣-32 P]ATP, respectively) in 100 l of the following different assay environments: 1) Ca-Mg buffer: 25 mM Tris, pH 7.4, 2.5 mM MgCl 2 , 10 M CaCl 2 , and 1 mM dithiothreitol; 2) magnesium buffer: 25 mM Tris, pH 7.4, 2.5 mM MgCl 2 , 2.5 mM EGTA, and 1 mM dithiothreitol; 3) Ca/Mg-free buffer: 25 mM Tris, pH 7.4, 2.5 mM EGTA, 2.5 mM EDTA, and 1 mM dithiothreitol. Competitive binding assays were carried out on the dystrophin-DAPs preparation (20 g of protein) equilibrated with 0.4 M [␣-32 P]ATP either in Ca-Mg buffer in the presence of one of the following nucleotides, each at 50 M: 3Ј-O-(4-benzoyl)benzoyl ATP (BzATP), adenosine 5Ј-triphosphate-2Ј,3Ј-dialdehyde, ADP, UTP, and GTP; or in calcium-free buffer with 50 M BzATP. The inhibition of ATP binding by the monoclonal anti-adhalin antibody was measured by incubating 37.5 l of the antibody with 10 g of protein from a dystrophin-DAPs preparation in 100 l of Ca-Mg buffer without DTT. After incubation at room temperature for 2 h with constant stirring, 0.4 M [␣-32 P]ATP was added. UV irradiation was carried out by direct exposure to UV light (254 nm) for 20 min in a flat dish refrigerated on ice. The UV lamp (8 watts) was kept at a distance of about 5 cm from the dish surface. The reaction was stopped by the addition of 30 l of SDS-PAGE sample buffer.
Immunological Methods-A polyclonal antiserum against the Ser-Ala-Gln-Val-Pro-Leu-Ile-Leu-Asp-Gln carboxyl-terminal peptide of adhalin (Chiron Mimotopes, Clayton, Australia) was raised in New Zealand White rabbits by subcutaneous injections. For the first injection, 500 g of peptide in PBS mixed 1:1 (v/v) with Freund's complete adjuvant was used. After 2 weeks, rabbits were boosted four times at 1-week intervals. Specificity of the polyclonal antibody was checked onto the dystrophin-DAPs preparation ( Fig. 1) by immunoblotting as described below.
One hundred g of protein from a dystrophin-DAPs preparation were incubated with 0.4 M [␣-32 P]ATP (20 Ci/ml) in 500 l of Ca-Mg buffer without DTT and photoactivated as described above. After photoactivation, 500 l of polyclonal anti-adhalin antibody cross-linked to immobilized protein A resin were added and incubated at 0°C for 2 h. The mixture was then centrifuged to sediment the protein bound to the resin. The pellet was washed exhaustively with Ca-Mg buffer without DTT and finally solubilized in the SDS-PAGE sample buffer.
SDS-PAGE was carried out according to Laemmli (26) using 5-15% polyacrylamide linear gels. The gels were either stained with Coomassie Brilliant Blue or dried. For Western blotting, the proteins were transferred overnight to nitrocellulose sheets at 300 mA in 25 mM Tris, 192 mM glycine, 0.03% SDS, and 10% methanol. The nitrocellulose was stained with Ponceau Red (0.2%, w/v) in 3% (v/v) trichloroacetic acid, photographed, and destained in distilled H 2 O. For staining with the monoclonal antibody against adhalin, nitrocellulose was first saturated for 1 h in 50 mM Tris, pH 8.0, 85 mM NaCl, and 2% bovine serum albumin. The saturating solution was discarded, and the nitrocellulose was incubated with a 1:300 dilution of the anti-adhalin antibody in the same buffer. For staining with monoclonal anti-nNOS antibody, nitrocellulose membranes were saturated for 1 h in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% low fat milk and incubated for 2 h with monoclonal anti-nNOS antibody diluted 1:500 in the same buffer. In both cases, after three washes with 50 mM Tris, pH 8.0, 85 mM NaCl, 0.1% bovine serum albumin, and 0.2% Tween 20, the nitrocellulose was incubated for 1 h with anti-mouse antibodies conjugated with peroxidase diluted 1:2000 in saturation buffer. After three washes, the reaction was developed with the Luminol-based Boehringer BM chemiluminescence kit. Autoradiography was carried out at Ϫ80°C by exposing dried gels or nitrocellulose sheets to Kodak XAR-5 films.
ATPase Activity-ATPase activity was measured spectrophotometrically at 37°C with an enzyme-coupled ADP release assay (27) by measuring the oxidation of NADH at 340 nm (28). The assay solution contained in 1 ml: 20 mM histidine, pH 7.2, 0.1 M KCl, 5 mM MgCl 2 , 2 mM ATP, 0.15 mM NADH, 0.5 mM phospho(enol)pyruvate, 5 units of pyruvate kinase/L-lactate dehydrogenase, and 10 g of dystrophin-DAPs. For Ca 2ϩ -ATPase activity measurements, basal ATPase activity was checked first in the presence of 1 mM EGTA. Then, 1 mM CaCl 2 (to obtain a final free Ca 2ϩ concentration of 10 M) was added. The inhibition of ATPase activity by BzATP was performed by UV irradiation of 10 g protein from a dystrophin-DAPs preparation in the Ca-Mg buffer (see above) without DTT for 5 min, in the presence of 100 M BzATP. In control experiments, the dystrophin-DAPs preparation was irradiated in the absence of [␣-32 P]ATP. ATPase activity was measured as above. Inhibition of ATPase activity by antibodies was performed by preincubating the dystrophin-DAPs preparation with either monoclonal or polyclonal anti-adhalin antibody in the Ca-Mg buffer for 2 h at room temperature. 2 mM ATP was then added, and the incubation was continued for a further 10 min. P i release was measured according to Lanzetta et al. (29).

RESULTS AND DISCUSSION
The physiological role of ␣-sarcoglycan (adhalin), the protein of the sarcoglycan complex that is missing in LGMD-2D, is unknown. In the present study, we demonstrate that ␣-sarcoglycan 1) binds ATP, 2) has an ATPase activity not inhibitable by known inhibitors of endo-ATPases, and 3) is not a purinergic receptor.
Photoaffinity labeling experiments using [␣-32 P]ATP demonstrate that one protein in a purified dystrophin-DAPs preparation bound ATP. As shown in Fig. 1, the incubation of a dystrophin-DAPs fraction with 0.4 M Mg-[␣-32 P]ATP followed by To confirm the identity of the 50-kDa ATP-labeled protein as ␣-sarcoglycan, we have used different immunological approaches with monoclonal and polyclonal anti-␣-sarcoglycan antibodies ( Fig. 2A, lanes c and d, respectively). Among the dystrophin-DAPs, the same protein band that bound [␣-32 P]ATP (Fig. 2B, lane f) was also selectively stained by the monoclonal anti-␣-sarcoglycan antibody (Fig. 2B, lane g). Furthermore, the binding of [␣-32 P]ATP to the 50-kDa protein was inhibited by incubation of the dystrophin-DAPs preparation with the monoclonal anti-␣-sarcoglycan antibody prior to photoactivation (Fig. 2C, lane i). Finally, polyclonal anti-␣-sarcoglycan antibodies immunoprecipitated the 50-kDa [␣-32 P]ATPlabeled protein (Fig. 2C, lane j).
Analysis of the deduced amino acid sequence of rabbit ␣-sarcoglycan (19) for ATP binding consensus sequences revealed the presence of two sequences at amino acids 163-171 (Gly-(Leu-Trp-Glu-Pro)-Gly-Glu-Leu-Lys) and amino acids 221-234 (Arg-(Cys-Ala-Arg)-Gly-(Gln-Pro-Pro)-Leu-(Leu-Ser-Cys-Tyr)-Asp) that are similar to the consensus sequences Gly-(X) 4 -Gly-Lys-(Thr) and Arg/Lys-(X) 3 -Gly-(X) 3 -Leu-(hydrophobic) 4 -Asp present in several ATPases (30). Both sequences are located in the extracellular domain of the protein. Interestingly, the peptide used to produce the monoclonal antibody is a fusion protein encompassing amino acids 217-289 of the rabbit ␣-sarcoglycan sequence. It should be noted that this sequence is conserved between rabbit, mouse, hamster, and human ␣-sarcoglycans and that it contains residues 221-234 of the putative ATPbinding domain, a finding that explains the ability of the monoclonal antibody to prevent the binding of ATP to ␣-sarcoglycan.
Several extracellular proteins are known to bind ATP.
Among these are the ecto-ATPases, the protein kinases, and the purinergic receptors. Ecto-ATPases are transmembrane enzymes that catalyze the hydrolysis of extracellular ATP. They have been identified at the surface of numerous cell types in many different species (31)(32)(33). When purified, these ectoenzymes generally show an activity that is dependent on Mg 2ϩ or Ca 2ϩ , although it is insensitive to specific inhibitors of endo-ATPases (33). ATP binding to ␣-sarcoglycan was Mg 2ϩ -dependent and Ca 2ϩ -independent (Fig. 3), because after incubation of the dystrophin-DAPs preparation in the absence of Mg 2ϩ (Fig.  3A, lane e) ␣-sarcoglycan was not labeled. It can be noted that, in the presence of Mg 2ϩ (2.5 mM), a protein of about 150 kDa was also labeled by [␣-32 P]ATP (Fig. 3A, lanes c and d). This protein was tentatively identified as neuronal-type nitric oxide synthase (nNOS) by immunoblot using monoclonal anti-nNOS antibody (data not shown). In fact, this enzyme is known to be a nonstructural component of the dystrophin complex, so that in some preparations it may copurify with dystrophin because of a direct interaction with ␣1-syntrophin (34) (compare, for example, Fig. 2, lane h with lane f). It has been demonstrated that P2-type purinergic receptors in several cell systems bind ATP 4Ϫ (35,36). To determine whether the 50-kDa protein is a muscle isoform of the P2-type purinergic receptor, we have incubated a dystrophin-DAPs preparation with 0.4 M [␣-32 P]ATP 4Ϫ in the absence of both Ca 2ϩ and Mg 2ϩ (calculated according to Fabiato (37)). Fig. 3A, lane e, shows that, under these conditions, two proteins of about 260 and 130 kDa, but not the 50-kDa species, were intensely labeled. Boiling the [␣-32 P]ATP-labeled samples in SDS sample buffer before electrophoresis resulted in a great reduction of the intensity of the 260-kDa protein band, suggesting that the 260-kDa protein is probably a dimer of the 130-kDa species (data not shown). Interestingly, no protein corresponding to this 130-kDa band was visible in the Coomassie Bluestained gel (Fig. 3), suggesting that a minor protein component with very high affinity for [␣-32 P]ATP has been labeled.   -e and h-j). Lanes a and f, molecular mass standards. Lane k, immunoblot staining with the monoclonal anti-␣sarcoglycan antibody of a sarcolemma membrane preparation.
To verify whether native ␣-sarcoglycan is also able to bind ATP, we performed photoaffinity labeling experiments using a sarcolemma membrane preparation. In agreement with the results obtained by using the dystrophin-DAPs preparation, photoaffinity labeling of sarcolemmal vesicles with [␣-32 P]ATP caused labeling of the ␣-sarcoglycan protein band. Again, the labeling was Mg 2ϩ -dependent and Ca 2ϩ -independent (Fig. 3B,  lanes h and i, respectively). On the other hand, when the sarcolemma vesicles were incubated with [␣-32 P]ATP 4-, ␣-sarcoglycan was not labeled (Fig. 3B, lane j). It should be noted that under the latter condition, in addition to the high molecular mass proteins identified in the dystrophin-DAPs preparations (i.e. the 260-and 130-kDa proteins in Fig. 3A, lane e), an additional protein of about 100 kDa was also labeled.
Extracellular ATP can be either the agonist of purinergic P2 receptors or the substrate of ecto-ATPases. To elucidate the protein family to which ␣-sarcoglycan belongs, we have analyzed the effects of a number of agonists and antagonists of P2-type receptors, and also of nucleotides that are substrates of ecto-nucleotidases, on the binding of [␣-32 P]ATP to ␣-sarcoglycan. As indicated by the results shown in Fig. 3, because ␣-sarcoglycan is not labeled by ATP 4Ϫ , it is not a P2-type purinergic receptor. This fact is further demonstrated by the inability of adenosine 5Ј-triphosphate-2Ј,3Ј-dialdehyde, a P2X 7 -type receptor antagonist (38), to affect the binding of [␣-32 P]ATP to ␣-sarcoglycan (Fig. 4, lane e). BzATP is an agonist of P2X 7 purinergic receptors (35,36,39), but it is also a photoaffinity probe that binds covalently to the nucleotide sites of ATPases (40,41). As shown in Fig. 4, lane d, preincubation of the dystrophin-DAPs preparation with BzATP completely inhibited the binding of [␣-32 P]ATP to ␣-sarcoglycan, further suggesting that ␣-sarcoglycan is an ATPase. On the other hand, BzATP also inhibited the binding of [␣-32 P]ATP 4Ϫ to the 130-kDa protein (Fig. 4, lane  j). This result suggests that the 130-kDa protein is a P2X 7 purinergic receptor. It is well known that GTP, UTP, and ADP may be substrates of ecto-nucleotidases (33,42). Fig. 4, lane f, shows that ADP completely prevented the binding of [␣-32 P]ATP to ␣-sarcoglycan, whereas GTP had a very little effect (Fig. 4, lane i). The inability of UTP to influence the labeling of [␣-32 P]ATP to ␣-sarcoglycan suggests it has a different specificity to nucleotides relative to other ecto-ATPases (31,33). On the other hand, the inability of UTP, a P2Y receptors agonist (43), to affect the binding of [␣-32 P]ATP to ␣-sarcoglycan (Fig. 4, lane h) indicates that this protein is not a P2Y receptor.
Plesner et al. (44) have demonstrated that increasing concentrations of monovalent cations up to 20 mM caused the increase of the ecto-ATPase activity of an enzyme isolated from mesenteric arteries. Further increase of monovalent cation salts decreased this activity. We have therefore tested the effects of varying concentrations of NaCl on the photoaffinity labeling of ␣-sarcoglycan by [␣-32 P]ATP. Fig. 5 shows that ATP labeling of ␣-sarcoglycan was increased by NaCl up to 20 mM, whereas higher concentrations were inhibitory. These data indicate that ␣-sarcoglycan share the same sensitivity to monovalent cations as known ecto-ATPases.
These photoaffinity labeling experiments suggested that ␣-sarcoglycan might be an ecto-ATPase. To prove this point conclusively, we tested the ATPase activity of our dystrophin-DAPs preparation. We found that the dystrophin-DAPs preparations had an ATPase activity (0.39 Ϯ 0.01 mol/min/mg protein, n ϭ 6) that was Mg 2ϩ -dependent and Ca 2ϩ -independent and that was highly reduced upon covalent binding with BzATP after UV irradiation (Table I). Furthermore, like other ecto-ATPases (33,45,46), the ATPase activity of dystrophin-DAPs preparations was not inhibited by inhibitors of ion-translocating ATPases such as thapsigargin, cyclopiazonic acid, and vanadate (data not shown). It appears likely that the relatively low specific activity is because of the presence of digitonin, used to purified the DAP complex (22,23). Indeed, it has been demonstrated that detergents inhibit the activity of other ecto-ATPases (31,33). To ascertain whether the ATPase activity could be attributed to ␣-sarcoglycan only, we incubated the dystrophin-DAPs preparation with the monoclonal anti-␣-sarcoglycan antibody (that is that raised against the putative ATP binding site of the protein) before measuring the ATPase activity. Under these conditions this antibody was able to reduce the ATPase activity (Fig. 6). The inhibitory action of the monoclonal antibody was not the result of nonspecific effects, because the polyclonal antibody raised against the last 10 amino acids of the C terminus of ␣-sarcoglycan (a portion of the protein without critical sites for the ATPase activity) had no effect. At variance from the almost complete inhibition of the binding of [␣-32 P]ATP to ␣-sarcoglycan, inhibition of ATPase activity by the monoclonal anti-␣-sarcoglycan antibody was only partial. One possible explanation is that our preparation was contaminated by trace amounts of T-tubule ecto-ATPase, a 56-kDa protein which is characterized by a high specific activity (6.6 mmol/min/mg protein, Ref. 32). Although labeling of a 56-kDa protein was not detected, the rabbit T-tubule ecto-ATPase has a relatively low affinity for ATP (the apparent K m at 25°C for Mg-ATP is 170 M (31)). Therefore, amounts of the contaminant below the threshold of detection in [␣-32 P]ATP binding could still be responsible for the fraction of ATPase activity that is not inhibited by the anti-␣-sarcoglycan antibody. Consistent with this explanation, polyclonal antibodies directed against the T-tubule Mg 2ϩ -ATPase (a generous gift of T. Kirley, University of Cincinnati) have revealed the presence of a 56-kDa protein in our preparations (data not shown).
Extracellular ATP is an important neurotransmitter in a wide variety of tissues. Its action is regulated by enzymatic degradation by several ecto-ATPases (33,36,42,47). Thus, ecto-ATPases appear to have an important role in modulating purinergic neurotransmission. In skeletal muscle a functional role of extracellular ATP in modulating the opening time of acetylcholine receptors at the end plate region has been de-scribed (48). Our results indicate that a P2X-type purinergic receptor is expressed in skeletal muscle, and this is likely to represent the hypothetical receptor for ATP suggested by Lu and Smith (48). Furthermore, our results indicate that ␣-sarcoglycan is an ecto-ATPase. Thus, it appears possible that ␣-sarcoglycan modulates the activity of P2X-type purinergic receptors.
Mutations in the ␣-sarcoglycan gene have been demonstrated in LGMD-2D (10), a group of diseases that shares some features of DMD. Although in both cases the disease is characterized by muscle fiber necrosis, LGMD and DMD are caused by mutations of different genes leading to different alterations in the two subcomplexes of the dystrophin-associated proteins: the sarcoglycans and the dystroglycans (49,50). In LGMD, only the sarcoglycan complex is lacking (18). In DMD, dystrophin and the dystroglycan complex are missing, and the sarcoglycans are greatly reduced in amount (14,15). Therefore, it is possible that the primary molecular mechanisms involved in the degeneration and necrosis of the muscle fibers are different in the two diseases.
Today, the more widely accepted theory on the role of dystrophin in skeletal muscle fibers is the mechanical theory. Dystrophin, by acting as a link between the actin membrane cytoskeleton and the extracellular matrix via ␣and ␤-dystroglycan, could transmit the local stresses generated during contraction across the sarcolemma to the extracellular matrix. The absence of dystrophin, by weakening the mechanical resistance of the membrane, could therefore predispose to physical disruption of the sarcolemma during muscle activity (2,51,52) allowing the entry of Ca 2ϩ . The elevated intracellular free Ca 2ϩ level could then activate intracellular degradation processes (53,54).
In LGMD-2D, -2C, -2E, and -2F, on the other hand, mutations cause the absence of the sarcoglycans, whereas dystrophin and dystroglycans are preserved at the sarcolemma (3,49,50). Thus, the mechanical resistance of the membrane should not be affected, suggesting that cell necrosis in these diseases has a different origin. One physiological role of ecto-ATPases is  (41,42), on the basal ATPase activity (see "Experimental Procedures") of the dystrophin-DAPs preparation (10 g) was determined. Preincubation of the preparation with 100 M BzATP (ϩBzATP) before measuring ATPase activity was without effect, whereas irradiation for 5 min at 254 nm (ϩUV) partially reduced the activity. On the contrary, irradiation in the presence of 100 M BzATP (ϩBzATP/UV) almost abolished the ATPase activity. Data are from two different dystrophin-DAPs preparations. The Mg 2ϩ dependence and Ca 2ϩ independence of the ATPase activity of ␣-sarcoglycan was determined in five different dystrophin-DAPs preparations (mean Ϯ S.E.) incubated under basal conditions followed either by the stepwise addition of 5, 10, and 20 mM EDTA or by the removal of Ca 2ϩ by the addition of 1 mM EGTA (*, p Ͻ 0.001; **, p Ͻ 0.002).  6. Inhibition of the ATPase activity of dystrophin-DAPs preparation by monoclonal anti-␣-sarcoglycan antibody. Inhibition of ATPase activity was performed by preincubating the dystrophin-DAPs preparation for 2 h at room temperature with either the monoclonal (q) or the polyclonal (E) anti-␣-sarcoglycan antibodies (Ab) at the indicated ratio in 0.025 M Tris, pH 7.4, 2.5 mM MgCl 2 , and 10 M CaCl 2 . The reaction was started by the addition of 2 mM ATP. After 10 min the P i released was measured by the method of Lanzetta et al. (29). DGC, dystrophin-glycoprotein complex. the control of ATP concentration at the surface of cells that express ATP receptors, thereby attenuating the magnitude and/or the duration of ATP-induced signals (41,46). We have demonstrated that skeletal muscle fibers express P2X 7 -type receptors that are activated by extracellular ATP in the micromolar range, forming nonselective pores that mediate a generalized bidirectional increase in plasma membrane permeability to molecules up to 900 Da (35,36). It appears that the absence of ␣-sarcoglycan, and therefore the absence of ecto-ATPase activity of the sarcolemma, could cause a persistent increase of extracellular ATP concentration. The consequent prolonged stimulation by ATP of P2X 7 receptors may in turn lead to intracellular Ca 2ϩ overload and cell death, as has been demonstrated for most mammalian cells that express P2X 7 /P2Xsubtype receptors (55,56). In DMD the low amount of the sarcoglycan complex may represent an additional molecular mechanism that contributes to muscle fiber necrosis.