May 19, 1993 - and in revised form, August 6, 1993). Triad vesicles from frog skeletal muscle exhibited calcium-induced calcium release highly sensitive to ex-.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268, No. 34, Isme of December 5, pp. 25432-25438 1993 Printed in d.S.A.
pH-sensitive Calcium Release in Triads from Frog Skeletal Muscle RAPID FILTRATIONSTUDIES* (Received for publication, May 19,
Triad vesicles from frog skeletal muscle exhibited calcium-induced calcium release highly sensitive to extravesicular pH; calcium induced release at pH > 7.4 butnotat pH 6.8. In contrast, triads isolated from rabbit skeletal muscle exhibited significant calciuminduced calcium release at pH 6.8. At pH 7.4, there was no stimulation of calcium release in triads from frog at pCa 7, maximum stimulation at pCa 5, and complete inhibition atpCa 3. Addition of ATP at pCa 6,pH 6.8, induced calciumrelease with the same high rate constantsin bothpreparations. In triadsfrom frog, ATP-induced calcium release at pCa 5 had the same kinetics at pH 6.8 and 7.4, whereas ATP-induced calcium releaseat pCa > 8, pH 6.8, was partial, with a decrease in the amounts released but not inrate constants. In contrast, triads from rabbit displayed the opposite behavior, with a decrease in rate constants but not in the amounts of calcium released at pCa > 8, pH 6.8. In triads from frog ATP-induced calcium release decreased at &a < 4, to reach total inhibitionat pCa 2; addition of magnesium (free > 0.5 mM) completely abolished ATP-induced calcium release at pH 6.8, pCa 7 or 5 . The potential physiological relevance of these resultsis discussed.
Calcium release from sarcoplasmic reticulum (SR)’ is akey process in excitation-contraction (E-C) coupling in skeletal muscle (Endo, 1977; Martonosi, 1984). Physiological release is mediated by calcium channels localized in the SR terminal cisternae regions (Fleischer and Inui, 1989). It is generally accepted that thechannel activity resides in a high molecular weight protein associated in tetramers that form part of the feet structures that span thetriadic gap between the SR and the transverse tubules (Kawamoto et al., 1986; Lai et al., 1988; Smith et al., 1988).The channel protein acts as high a affinity receptor for ryanodine (Laiand Meissner, 1989), aplant alkaloid known to interfere with muscle contraction. While only one ryanodine receptor protein is present in SR from rabbit fast skeletal muscle (Lai et al., 1988), two ryanodine
* This study was supported by FONDECYT Grants 1108/91 and 193/1053 and by a grant from COPEC to the Centro de Estudios Cientificos de Santiago. 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. To whom correspondence should be addressed Dept. de Fisiologia y Biofisica, Facultad de Medicina, Universidad de Chile, Casilla 70005,Santiago 7,Chile. Fax: 56-2-777-6916. $ Recipient of a John S. Guggenheim Fellowship. The abbreviations used are: SR, sarcoplasmic reticulum; AMPexcitation-conPCP, ~,y-methyleneadenosine-5‘-trisphosphate;E-C, traction; HEDTA, N-hydroxyethylethylenediaminetriaceticacid; MOPS, 3-(N-morpholino)propanesulfonicacid.
1993, and in revised form, August 6, 1993)
receptor isoforms are present in SR isolated from avian and frog skeletal muscle (Airey et al., 1990; Olivares et al., 1991; Lai et al., 1992; Murayama and Ogawa, 1992). The physiological mechanism(s) whereby these channels proteins open in uiuo in response to transverse tubule depolarization remain unknown (Rios et al., 1992; Dulhunty, 1992). Calcium release in vitro has been investigated mostly in heavy SR vesicles or in triadpreparations isolated from mammalian muscle; it presents similar characteristics when studied at the single channel level in planar lipid bilayers (Smith et al., 1985, 1986), or when measuring calcium release fluxes from calcium-loaded vesicles (Meissner, 1984; Meissner et al., 1986; Sumbilla and Inesi, 1987; Moutin and Dupont, 1988, Ikemoto et al., 1989). Both approaches have shown that the channel is activated by micromolar calcium concentrations andmM [ATP], and inhibitedby mM [ M e ] and micromolar ruthenium red (Smith et al., 1985,1986;Meissner, 1984; Meissner et al., 1986; Sumbilla and Inesi, 1987; Moutin and Dupont, 1988; Ikemoto et al., 1989). All these compounds exert their effect from the cytoplasmic side of the channel; scant information is available regarding channel regulation from the luminal side. Most studies of the physiology of E-C coupling and the regulation of calcium release in whole or skinned cells have been done in frog skeletal muscle. Yet limited information exists regarding calcium release in vesicles isolated from frog muscle (Ogawa, 1970; Ogawa and Ebashi, 1976; Volpe et al., 1988; Dettbarn and Palade, 1991), and there are no reports concerning fast release kinetics. For this reason, in this work we studied the effects of calcium, pH, ATP, and magnesium on the fast kinetics of calcium release in triads isolated from frog skeletal muscle. In contrast toprevious reports concerning release from SR vesicles isolated from mammalian muscle (Kim et al., 1983; Meissner, 1984; Moutin and Dupont, 1988) and to our present results with triads from rabbit, we found calcium-induced calcium release in triads from frog only at pH values > 7.0 but not at pH 6.8. However, in both triad preparations 2 mM ATP activated calcium release at pH 6.8 to the same extent as at pH 7.4. In triads from frog, ATPinduced release was most prominent in the pCa range 7-4 and was inhibited by magnesium at the concentrations normally present in resting muscle. ATP-induced release rates were fast enough to be compatible with the in uiuo rates of calcium release. A preliminary account of some of these findings has been presented elsewhere (Donoso et al., 1992). EXPERIMENTAL PROCEDURES
Preparation of Triads-Triads were isolated from frog and rabbit skeletal muscle as described elsewhere (Hidalgo et al., 1993).Briefly, finely minced muscles from the legs of the frog Cadiverbera caudiverbera, or from the back muscles of white albino rabbits, were homogenized in 0.15 M KCI, 5 mM MgSO,, 20 mM MOPS-Tris, pH 6.8, and the following protease inhibitors: leupeptin (1 rg/ml), Pep-
25432
pH-sensitive Calcium Release in Isolated Triads statin (1 pg/ml), benzamidine (0.4 mM), and phenylmethylsulfonyl fluoride (1mM). Vesicles sedimenting between 1,500-17,000 X g were collected by differential centrifugation and were resuspended in the same buffer used for homogenization. After discarding contaminating contractile proteins from this suspension by sedimentation at 1,500 X g, triads were collected by sedimentation at 17,000 X g, and were resuspended in a small volume of 0.3 M sucrose, 20 mM MOPS-Tris, pH 6.8, with the same protease inhibitors as above. Small aliquots of the triad preparations were quickly frozen in liquid NO and were stored at -80 “C for up to 1 month. Fast Filtration Studies-Vesicles were passively loaded with calcium by incubation in 3 mM “CaCI2 (4 mCi/mmol), 0.1 M KC], 40 mM MOPS-Tris, pH 6.8, at 0.6 mg of protein/ml. After incubation for 3 h at 18-20 “C, 50 pl of vesicles were diluted in 1 ml of the same loading solution but containing only non-radioactive calcium. Vesicles were immediately filtered through Millipore filters (AA, 0.8 pm) and were washed with 5 ml of non-radioactive loading solution. Release was induced in a fast filtration system (Biologic) essentially as described by Moutin and Dupont (1988). The time elapsed between dilution of the loaded vesicles, washing, and initiation of release was 30 s and was kept constant throughout the experiments. The composition of the releasing solutions are described in the legend for the figures. Releasing times between 20 ms to 1 s were routinely used. Other Procedures-Nitrendipine and ouabain binding were measured as previously described (Jaimovich et al., 1986). Ryanodine binding was measured as described in Bull et al. (1989). Protein was determined as described by Hartree (1972) using bovine serum albumin as standard. Free calcium and magnesium concentrations were calculated with a computer program (Goldstein, 1979) using the binding constants for EGTA, HEDTA, and ATP from Martell and Smith (1974). The denomination pCa > 8 describes solutions containing 10 mM EGTA and no added calcium. Free calcium concentrations were always checked with a calcium electrode. Materials-The radioactive compounds 46CaC12,[3H]ouabain, and [‘Hlnitrendipine (5-methyL3H), were obtained from DuPont-New England Nuclear Corp.; [3H]ryanodine and ryanodine were a kind gift from Dr. John Sutko. Protease inhibitors were obtained from Sigma. All other reagents used were of analytical grade. RESULTS
Calcium-induced Calcium Release: Effect of p H
25433
.-C
0
0.2 0.4 0.6 OB 1.0 Tim,..
0
I
0
I
0.8
I
I
I .6
I
I
2.4
I
I
3.2
I
I
4.0
Releasing Time, s FIG. 1. Calcium release at pH 6.8, pCa 5. Triads isolated from frog (0)or rabbit (0)skeletal muscle were equilibrated with 3 mM “CaC12,0.1 M KCl, 40 mM MOPS-Tris, pH 6.8, and release was induced in a fast filtration system as described under “Experimental Procedures.” The releasing solution contained 1.22 mM CaC12,2 mM HEDTA (10PM free calcium), 0.1 M KC], and 40 mM MOPS-Tris, pH 6.8. The experimental points, scaled to 100% at time 0, were adjusted to a double exponential decay plus an offset. The first exponential corresponds tothe ruthenium red-insensitive release component, or basal release, and was adjusted tothethe same equation for triads from frog and rabbit: N ( t ) = 17 exp(-12t), where t is given in s. The second exponential corresponds to specific calcium release through ruthenium red-sensitive channels and had a k value ofCO.01 s-‘ for frog and of 0.23 s-’ for rabbit. The corresponding offsets for the curves shown above were 30% for frog and 25% for rabbit. The offset originated from two sources: from vesicles lacking calcium release channels, which corresponded to 15-30%of the vesicles present in all triad fractions studied, and from the 5-8% of nonspecific bound calcium that was not released byA23187. The effect of 10mM M&12 and 10 p~ ruthenium red added to thereleasing solution is shown in the inset, where the equation used to fit the unscaled experimental points was N ( t ) = 32 exp(-12.9t) + 111.
Most studies of calcium release in vesicles isolated from TABLEI mammalian skeletal muscle have been done at pH 6.8 (Kim Effect of p H and ATP on calcium release et al., 1983; Meissner, 1984; Sumbilla and Inesi, 1987; Moutin Data are given as mean k S.D. In parentheses is the number of and Dupont, 1988). For this reason, the effects of calcium, ATP, and magnesium on calcium release in triads from frog determinations, otherwise the deviations represent the statistical of a single determination as given by the computer fits to the skeletal muscle were initially studied at pH 6.8. Triads from error exponential decay equations. A concentration of 2 mM ATP was used. frog equilibratedwith 3 mM CaC12routinely accumulated 130- Rate constants for ruthenium red-sensitive release are given, calcu180 nmol of calcium/mg of protein, whereastriads from rabbit lated as described in the legend to Fig. 1. accumulated significantlylesscalcium, 70-90 nmol/mgof Rate constant, s-l Releasing solution protein. Addition of ionophore A23187 produced immediate Control ATP release of 93-95% of the calcium associated with the vesicles Triads from rabbit (not shown), indicating its intravesicularlocation. 6.0 +- 1.2 pH 6.8, pCa > 8 Following dilution of triads from frog in a solution at pH pH 6.8, pCa 7 0.12 f 0.01 11.8 +- 1.8 6.8 and pCa 5.0, we observed fast release of a small fraction pH 6.8, pCa 5 0.23 f 0.01 (2) 11.6 f 2.4 (17%) of the vesicular calcium, with a time constant i n the range of 12-18 s-*; negligible calcium release was observed Triads from frog 8 thereafter even when releasing timesas long as 4 s were used 13.2 f 3.3 (5) pH 7 2.7, the pCa used to load the vesicles. The observation that SR calcium channels fused in lipid bilayers are closed at pCa values < 3 (Ma et ul., 1988) provides a rationale for this inhibition. In triadsfrom frog, decreasing calcium to pCa > 8.0 reduced the amount of calcium released to 40% (Fig. 7 B ) , yet the release rate constant retained its high value, 13.2 s-l (Fig. 7 A ) . This result indicates that there are two populations of vesicles: one that contains channels that are opened by ATP in very low calcium, and another one that contains channels that to open require not only ATP but also extravesicular calcium concentrations higher than pCa > 8.0. The possible connection between these observations and the properties of the two ryanodine receptor isoforms present in frog muscle will be presented under “Discussion.” Different results were obtained in triads from rabbit, where at pCa > 8 the rate constantof calcium release was decreased to half of that found at pCa 5 (Table I), but the amount of calcium released was the same as at pCa 5. The same effect was described by Moutin and Dupont (1988), who reported a decrease in the release rate constants in thepresence of ATP at pCa > 7 in triads from rabbit. Experiments ut pH 7.4-In triads from frog, at pCa 5.0, increasing the pH of the ATP-containing releasing solutions from pH 6.8 to 7.4 affected neither the release rate constants (Table I) nor the amount of calcium released (not shown). Consequently, ATP-induced release is not underthe same pH control as calcium-induced calcium release, since the latter was apparent at pH7.4 but was strongly inhibited at pH6.8.
0
200
1000
400 600 800 Releasing Time, m s
FIG. 6. Effwt of ATP on calcium release. Triads were equilibrated with calcium and release was induced at pH 6.8, 2 mM ATP, and different extravesicular calcium concentrations. A , release was done at pCa 5 (0)or at pCa > 8 (0).B , release was done at pCa 3.65 (B), at pCa 3.4 (01, or at pCa 2 (0).Experimental points were adjusted to a single exponential decay plus an offset, except for the data obtained at pCa 3.4 where the points were adjusted to a double exponential decay plus an offset: the fastest component was adjusted to the ruthenium red-insensitive release as described in the legend to Fig. 1. The equations given by the fits to the experimental points were: N ( t ) = 40.5 exp(-13.2t) 59.5, forpCa > 8; N ( t ) = 84.5 23.5, for exp(-16.6t) + 15.5, for pCa 5.0; N ( t ) = 76.5 exp(-10.6t) 44, for pCa 3.4; pCa 3.65; N ( t ) = 17 exp(-12t) + 34 exp(-2.2t) N ( t ) = 15 exp(-9.4t) + 84.9, for pCa 2.0.
+
+
+
Effect of Magnesium-The effect of free [ M e ] on ATPinduced calcium release was examined in triads from frog at pH 6.8, pCa 7.0 or 5.0. We found that addition of increasing [Mg2+]caused a progressive inhibition of ATP-induced release at both pCa values studied, to reach complete block, ix. basal release, at 0.55 mM free [ M e ] (Fig. 8). In these experimental conditions, the inhibitory effect of M$+ on calcium release may be caused, at least in part,by the decrease in free [ATP] produced by the formation of the ATP-Mg complex, provided only free ATP but not the complex activated release. We found that thevalues of release rate constantsobtained for a given free [ATP] in thepresence of M P were undistinguishable from those obtained for the same free [ATP] in the absence of M P (not shown). This agreement strongly supports the assumption that in our conditions M e inhibits release by decreasing free [ATP].
25436
pH-sensitive Calcium Release in Isolated Triads
looh I00 1412 c
-
80
IO-
60
0
c
8 -
40
6-
-
4-
0
\
2-
\
> 8.0
6.0
4.0
2 .o
pCa
B
.-3
0
,\"
IO
200
400
800
600
1000
FIG. 8. Effect of magnesium on ATP-induced calcium release at pH 6.8,pCa 7.0. Triads were equilibrated with calcium,
:I 20
0
Releasing Time, ms
eo t
"
0
and release was induced with a solution containing 2 mM ATP, pH 6.8, and different extravesicular concentrations ofMgC12. Release was done at free [ M e ] : 0 mM (o), 0.08 mM (O), 0.35 mM (m), or 0.55 mM (0).Experimental points were adjusted to a double exponential decay plus an offset as described in the legend to Fig. 1, except for the data obtained a t 0 free [ M e ] and at0.55 mM free [ M e ] , where only one component was apparent and the points were adjusted to a single exponential decay plus an offset. The equations given by the 32.1, fits to theexperimental points were: N ( t )= 67.8 exp(-15.6t) 50.2 exp(-3.2t) + 35, for 0 mM free [ M e ] ; N ( t ) = 17 exp(-l2t) 52.3 exp(-1.3t) for 0.08 mM free [ M P ] ; N ( t ) = 17 exp(-12t) 35, for 0.35 mM free [M$+]; N ( t ) = 23.2 exp(-12.0t) 76.5, for 0.55 mM free [ M e ] .
+
""""-""""
+
+
+
+
et al., 1993). In addition, the isolated triads exhibited significant [3H]ouabain binding, with a ratio of ouabain to nitrendipine binding close to two (Hidalgo et al., 1993), the same PC0 ratio displayed by isolated transverse tubulevesicles (JaimovFIG. 7. Effect of pCa on the rateconstants and the amounts ich et al., 1986). The isolated triads maintain an actual bond of ATP-induced calcium release at pH 6.8.A , the rates constants between transverse tubule andSR membranes, as verified by of ATP-induced calcium release, calculated from the data shown in comigration of the above SR and transverse tubule markers Fig. 6, are illustratedas a function of thepCa of the releasing solution. in sucrose density gradients (Hidalgo et al., 1993). B, the amount of calcium released, expressed as percentage of the The presence of a high concentration of leupeptin throughcalcium loaded and calculated as the difference between the 100% total calcium loaded - offset (see legend to Fig. 6), is plotted against out the preparation, aswell as in the solution utilized to store pCa. The dotted line represents the amount of calcium released that the preparations at -80 "C, may have helped to preserve the is insensitive to inhibition by magnesium and ruthenium red. Data native triadic structure. The use of protease inhibitors, such represent the mean f S.E. calculated from three to five different as leupeptin, is particularly important when studying calcium experiments. release, since the ryanodine receptor is highly susceptible to endogenous or exogenous neutral proteases, which are inhibDISCUSSION ited by leupeptin (Rardon et al., 1990; Iino et al., 1992). Also, digestion with trypsin or calpain results in stimulation of With the purpose of understanding the mechanisms that calcium efflux in heavy SR vesicles from rabbit muscle underly physiological calcium release, many experiments have (Trimm et al., 1988), and in activation of calcium-induced been done with in vitro systems. We studied calcium release calcium release in rabbit skinned muscle fibers (Iino et al., in triads, since vesicles that maintain the in vivo original 1992). triadic association present advantages relative to heavy SR Effect of pH on Calcium-induced Calcium Release-Calcium vesicles, where the connection with the transverse tubules has release in triads from frog skeletal muscle exhibited some been disrupted. The study of fluxes in triadsprovides comple- differences when compared to thewell studied calcium release mentary information to measurements of single channel ac- in SR vesicles isolated from mammalian muscle. The most tivity, where the channel protein fused in the lipid bilayer noticeable being the inhibition of calcium-induced calcium may lose potential contacts with other proteins that could release at pH6.8, and thehigh rates of partial release obtained exert important regulatory functions. at pCa > 8, pH 6.8, with ATP. The triadic origin of the vesicles used in these studies was In heavy SR vesicles from mammalian muscle, increasing routinely checked by measuring the binding of [3H]ryanodine, calcium to thepCa range 6-4 causes substantial release at pH as a marker for the SRcalcium release channels, and of [3H] 6.8 (Kim et al., 1983; Meissner, 1984; Moutin and Dupont, nitrendipine as a transverse tubular marker. The triadvesicles 1988), as confirmed in this work. Yet, as mentioned above, at from frog displayed a ryanodine receptor density of 10-15 pH 6.8 calcium failed to induce measurable release with the pmol/mg protein, and of 8-15 pmol/mg protein for nitrendi- fast filtration technique in triad vesicles isolated from frog pine, indicating 10% content of transverse tubules (Hidalgo skeletal muscle. This difference vanished at higher pH values.
0
> 8.0
6 .O
4.O
2 .o
pH-sensitive Calcium Release in Isolated Triads Significant calcium-induced calcium release took place in triads from frog at pCa 5.0, pH 7.4. Increasing pH caused a steep increase in release rate constants at pCa 5.0, from a value of 0.18 s” at pH7.1 to 1.80 s-l at pH8.6. The opening probability of the SR calcium channel from mammalian skeletal muscle incorporated in lipid bilayers increases with increasing pH (Ma et al., 1988; Rousseau and Pinkos, 1990), and ryanodine binding increases markedly at pH 9.0 relative to pH 7.0 (Valdivia et al., 1990). Since ryanodine supposedly binds to the open channel, these previous observations indicate that theincrease in rate constantscaused by raising pH reflects an increase in theopening frequency of the channels. The stimulation caused by increasing pH in triads from frog was less effective, though, than that produced by ATP. Even the highest rate constantsfound at pH8.2-9.0, pCa 5.0, were 5-6-fold lower than the release rate constants induced by addition of 2 mM ATP at pH 6.8. In both conditions the vesicles released essentially all their accumulated calcium, indicating that channels that responded to ATP or alkaline pH were present in every vesicle. Since ATP (Smith et al., 1986, Bull et al., 1989) or increasing pH (Ma et al., 1988) produce maximum channel opening when measured in steadystate conditions in thepCarange 5-4, the observed differences in release rate constants are rather unexpected. They may indicate that ATP acts fasterthan alkaline pH in opening the channels; such differences would not be detected in steadystate single channel experiments butwould contribute significantly to release rates in the millisecond time range. Ryanodine, for instance, takes some time in locking the channel in a low conductance state with an open probability close to unity (Rousseau et al., 1987; Imagawa et al., 1987). ATP-induced Release-In the pCa range 7-4, calcium release in triads from frog was stimulated maximally by ATP at pH 6.8, as judged by the observed increase in rateconstants, and in the amount of calcium released caused by ATP addition. No further increase was found after raising the pH to 7.4. This observation suggests different mechanisms of activation of release by ATP and calcium, since the latter was inhibited markedly at pH 6.8. The effect of ATP was completely inhibited by further addition of 0.55 m~ [M?], a concentration somewhat lower than its physiological resting concentration in amphibian and mammalian skeletal muscle (0.80-1.1 mM, Alvarez-Leefmans et al., 1986; Blatter, 1990: Konishi et al., 1993). Consequently, it seems unlikely that in physiological conditions ATP stimulates release, unless some additional factors contributeto channel opening (see below). A novel finding of this study is the observation that in triads from frog ATP activated calcium release at pCa > 8.0 with values of rate constants ashigh as thoseobserved at pCa 7-5. At pCa > 8, however, after subtraction of basal release only 20-25%of the vesicles released calcium. Thus, frog skeletal muscle may contain a subset of channels that are opened by ATP even at pCa > 8. This finding differs with previous reports of fast release kinetics in SR vesicles from rabbit (Meissner et al., 1986; Moutin and Dupont, 1988). In the presence of ATP a 50% decrease in rate constants was reported after decreasing calcium to pCa > 7.0 or beyond (Moutin and Dupont,1988),a finding confirmed in this work, whereas with the non-hydrolyzable ATP analog AMP-PCP a 75% decrease inrateconstants was reported on lowering calcium from pCa 5.4 to 8 (Meissner et al., 1986). Intrinsic dissimilarities in the nature of the channels present in amphibian muscle relative to those of mammalian species may contribute to theabove differences (Airey et ai,1990; Olivares et al., 1991; Lai et al., 1992; Murayama and Ogawa, 1992). It is interesting to note in this regard that the two isolated
25437
isoforms of the ryanodine receptor, purified from frog skeletal muscle, display different calcium sensitivities to ryanodine binding (Murayama and Ogawa,1992);while one of them binds ryanodine at pCa > 8, the other requires pCa < 7 to display some ryanodine binding. Furthermore, recent observations (Bull and Marengo, 1993)have described the presence, with equal frequency, of single calcium channels in frog muscle with different calcium sensitivities; one is activated by calcium in the pCa range > 6 whereas the other requires higher calcium concentrations, with half-maximal activation at pCa 5.3. These combined results fit well with our findings in triad vesicles, since they all indicate that frog skeIetal muscle possesses two types of calcium release channels that present different calcium sensitivities. Physiological Implications-Acidic pH affects contraction in whole muscle, and it may be involved in causing muscle fatigue (Westerblad et al., 1991). It has been shown that contraction of amphibian skeletal muscle persists at pH 6.0 (Lamb et al., 1992), indicating that thephysiological coupling mechanisms stilloperate at thispH.Incontrast, calcium release in SR vesicles from rabbit muscle is almost totally inhibited at pH 6.3 (Meissner, 1990), and single channels in bilayers cannot be activated by calcium and ATP below pH 6.5 (Rousseau and Pinkos, 1990). Our results, then, indicate that the release channels present in frog skeletal muscle are even more sensitive to inhibition by acidic pHthanthe channels of SR from rabbit skeletal muscle. These results, plus our findings in triads from frog demonstrating inhibition of calcium-induced calcium release at pH 6.8, make unlikely a role for calcium as thephysiological agonist responsible for channel opening, unless a mechanism exists in the cell to overcome the inhibition of calcium-induced calcium release by acidic pH observed in uitro. It could be argued that the ATP present in muscle might overcome the pH inhibition of calcium-induced calcium release, since we found that in the presence of 2 mM ATP release took place at pH6.8. However, addition ofMg2+ at concentrations in the range normally present in resting skeletal muscle (Alvarez-Leefmans et at., 1986; Blatter, 1990; Konishi et al., 1993) completely inhibited release. It has been suggested recently that M F binding to the calcium release channels, that strongly inhibitstheir opening, may be under voltage control; thus depolarization would removethe inhibition by inducing dissociation of Mg2’ from the channels (Lamb and Stephenson, 1992). Furthermore, phosphorylation has been reported to overcome the inhibitory effect of M e in SR from rabbit muscle at the single channel level (Hain et al., 1993), but no information exists regarding the effect of phosphorylation on calcium release in vesicles or muscle cells. It is too early, then, to ascertainwhether voltage-induced MgZ+ dissociation from inhibitory sites, or channel phosphorylation, has physiological relevance as modulators of calcium release. The physiological significance of the stimulation of calcium release by alkaline pH observed in vesicles is not clear, It is known that depolarization elicits large force responses in amphibian skeletal muscle fibers, even at intracellular pH values as high as 8.0 (Lamb et al., 1992), indicating that E-C coupling operates adequately at this pH. Furthermore, after heavily loading the SR with calcium, changing intracellular pH to pH8.0 produced a large force response in the absence of depolarization (Lamb et al., 1992).This resultis compatible with the pH-induced activation of calcium channels (Ma et al., 1988; Rousseau and Pinkos, 1990), and of calcium release in vesicles (Meissner, 1990; Dettbarn and Palade, 1991). It has been pointed out, however, that while alkaline pH suffices to trigger calcium release in heavily loaded fibers, this mech-
pH-sensitive Calcium Release in Isolated Triads anism does not play a major role in normal E-C coupling, where calcium release stays under the control of the voltage sensors (Lambet al., 1992). To summarize, our findings show that triads isolated from frog skeletal muscle release calcium at rates that, in the presence of ATP and atphysiological resting calcium concentrations, are fastenough to be compatible with the physiological release rates. In fact, after subtraction of basal release and offset, the triad vesicles from frog on average contained 100 nmol/mg of protein of calcium amenable to specific release. From the rate constantvalues of 10-13 s-’, initial rates of 5-6 nmol of calcium released/mg of protein in 5 ms canbe calculated, giving release rates of 1to 1.2 pmollmg of protein/ s. The same calculation for triads from rabbit, that on average contained 60 nmol/mg of protein of calcium amenable to specific release, gives initial rates of release of 0.6-0.8 pmol/ mgof protein/s. Thus, the release rates in rabbit skeletal muscleseem to be lower than in frog, in agreement with previous reports (Volpe et al., 1988). In frog skeletal muscle, peak values for the in vivo calcium release fluxes of 30 pM/s have been calculated (Rios and Pizarro, 1991). Assuming 5 mgof SR protein/g of muscle (Meissner et al., 1973) and a volume of 0.58 ml/g of wet weight (Baylor et al., 1983), these fluxes can be converted to peak values of release rates of 3.5 pmollmg of protein/s. The in vitro rates determined in the present work are in thesame range. This release, however, is completely inhibited by resting concentrations of M$+, indicating that an additional mechanism should exist in vivo to achieve calcium release following muscle depolarization. Acknowledgments-The help of Humberto Prieto in some of the experiments is gratefully acknowledged. The authors thank Drs. M. T. N S e z and E. Jaimovich for their critical reading of the manuscript. REFERENCES Airey, J. A., Beck, C. F., Murakami K., Tanksley S. J., Deerinck T. J., Ellisman, M. H., and Sutko, J. L. (1b90)J. Bwl. CLm. 266, 14187-i4194 Alvarez-Leefmans,F. J., Gamiiio, S. M., Glraldez, F., and Gonzalez-Serratos, H. (1986)J. Physiol (Lond.) 378,461-483 Baylor., S.M., Chandler, W. K., and Marshall, M. W.(1983)J. Physiol. ( L a n d . ) 344,625-666 Blatter, L. A. (1990)Pfliigers Arch 416,238-246 Bull, R., and Marengo, J. J. (1993)FEBS Lett., in press Bull, R., Marengo, J. J., Suarez-Isla, B. A., Donoso,P., Sutko, J. L., and Hidalgo, C. (1989)Blophys. J. 66,749-756 Dettharn, C., and Palade, P. (1991)J . BioL Chem. 266,8993-9001 Donoso, P., Ronjat, M., and Hidalgo, C. (1992)Biophys. J. 61, A423 Dulhunty, A. F. (1992)Frog. Biophys. Mol. Bid. 67,181-223 Endo, M. (1977)PhysioL Reu. 67,71-I08
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