cycle of the isometrically contracting state); bridges; and in both cases, there was an additional

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMlSTRY by The American Society for Biochemistq ' and Molecular Biology, Inc Vol. 262, No.28, Issue of October 5, PP ,1987 Printed in U. S. A. Effect of Rigor and Cycling Cross-bridges on the Structure of Troponin C and on the Ca2+ Affinity of the Ca2+-specific Regulatory Sites in Skinned Rabbit Psoas Fibers* (Received for publication, February 17,1987) Konrad GiithSB and James D. Pottergll With the technical assistance of Klara WinnikesS! From the!department of Pharmacology, University of Miami Schoot of Medicine (R-189), Miami, Florida and the $II Physwlogisches Institut, Universitat Heidelberg, Im Neuenheimer Feld, Heidelberg, West Germany Intrinsic troponin C (TnC) was extracted from small to regulatory proteins. A great deal has been learned about bundles of rabbit psoas fibers and replaced with TnC the biochemistry of this cycle (for review, see Refs. 1-3), and labeled with dansylaziridiae (6-dimethylamino- the relation of the biochemical cycle to the mechanical events naphthalene- 1-sulfonyl). The fluorescence of incorpo- is beginning to emerge. The essential features of the cycle rated dansylaziridine-labeled TnC was enhanced by which are generally accepted (1, 3) are that: 1) there are the binding of Ca2+ to the Ca2+-specific (regulatory) strongly attached states of the cross-bridge interaction with sites of TnC and was measured simultaneously with actin (actomyosin, actomyosin-adp); 2) there are weakly force (Zot, H. G., Guth, K., and Potter, J. D. (1986) J. attached states (actomyosin-atp, actomyosin-adp-pi); 3) Biol. Chem. 261, ). Various myosin the dissociation of rigor bridges by MgATP, the subsequent cross-bridge states also altered the fluorescence of dan- hydrolysis of MgATP, and the reattachment of cross-bridges sylaziridine-labeled TnC in the filament, with cycling to actin are rapid and not rate-limiting (in terms of the crosscross-bridges having a greater effect than rigor crossbridge cycle of the isometrically contracting state); bridges; and in both cases, there was an additional 4) the effect of Ca2+. The paired fluorescence and tension data association of weakly attached states with actin is probably were used to calculate the apparent Ca" affinity of the Ca2+-independent; 5) the association of strongly attached regulatory sites in the thin filament and were shown states with actin is Ca2+-dependent and cooperative; 6) force to increase at least 10-fold during muscle activation development occurs between actomyosin-adp-pi association presumably due to the interaction of cycling cross- with actin and Pi release (5); and 7) troponin control of this bridges with the thin filament. The cross-bridge state cycle probably occurs at this point in the cycle. Much of the responsible for this enhanced Caa+ affinity was shown above has been recently supported by mechanical studies to be the myosin-adp state present only when cross- using skinned fibers employing caged ATP (3,4, 6). bridges are cycling. The steepness of the pca force The early results of Bremel and Weber (7) indicated that curves (where pca represents the -log of the free CaZ+ rigor cross-bridges (in the absence of ATP) increased the concentration) obtained in the presence of ATP at short affinity of all four Ca2+-binding sites on troponin, whereas and long sarcomere lengths was the same, suggesting work by Fuchs (8) suggested that actively cycling cross- that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament. To understand the regulatory properties of the thin filament, one must consider the interactions of myosin with the thin filament as they relate to the sliding filament model. The essential features of the sliding filament model of muscle contraction include the cyclical interaction of myosin crossbridges with actin, the coupling of ATP hydrolysis to force production, and the regulation of these events by Ca2+ binding * This work was supported by National Institutes of Health Grants AR and HL A. The costa of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (To whom correspondence should be addressed Dept. of Pharmacology, University of Miami School of Medicine (R-189), P. 0. Box , Miami, FL bridges, unlike rigor bridges, do not alter troponin-ca2+ affinity. In this paper, evidence is given that cycling cross-bridges increase the affinity of the Ca2+-specific sites by at least 10- fold when muscle changes from the relaxed to the fully contracted state in the presence of MgATP. Evidence that also supports the idea that cycling cross-bridges alter the affinity of STnC' for Ca" comes from the recent experiments of Kerrick and Hoar (9) where, in skinned fibers, it was found that as the ratio of ADP to ATP was increased, force increased, whereas the ATPase rate decreased. These changes were accompanied by a shift toward lower free Ca2+ concentration (higher affinity) in the Ca2+ dependence of the force development. The opposite effect was seen when the Pi/ATP ratio was increased. All these changes have been interpreted as being due to a change in the population of strongly attached (actomyosin-adp) bridges and are supported by the results presented here. MATERIALS AND METHODS Preparation-Skinned rabbit psoas fibers were prepared and stored as described by Kawai and Schulman (10). These preparations were The abbreviations used are: STnC, skeletal troponic C; dansyl, 5- dimethylaminonaphthalene-1-sulfonyl; pca, -logof the free Ca2+ concentration.

2 13628 Effect of Cross-bridges on TnC in Skinned Muscle Fibers used for up to 3 weeks. Prior to th experiments, small fiber bundles of three or four fibers were teased from the preparation. Some of the fibers were treated with 1% Brij 35, although this did not influence the results reported here. Mechanical and Optical Setup-The optical and the mechanical apparatus and the procedure to mount small fiber bundles have been described in detail by Guth and Wojciechowski (11). The diameter of the focus field was approximately 1.2 mm. In our later experiments, we used a tweezer technique to attach the fibers, and this gave results identical to those using the hook and loop technique. For the determination of the sarcomere length, a 5-milliwatt helium/neon laser was used. The measurements of STnC fluorescence and sarcomere length were taken from the same area of the fiber bundle. Many of the experiments reported in this paper were performed with an illumination set and microscope configuration which gave a high background due to autofluorescence problems in the microscope. This has been improved with a new illumination set which gives a very low background fluorescence, and in this case, the dansylaziridinelabeled TnC signal in the absence of Ca2+ is 50% of that seen at high Ca2+. The results obtained with the latter configuration were not different than those obtained with the first configuration but were of higher quality. Solutions-The free Ca2+ and the free M$+ concentrations were calculated according to the procedure and the stability constants of Fabiato and Fabiato (12). Stock solutions were made for pca 8, pca 7, pca 6, and pca 5. The major anion was propionate rather than chloride. The ionic strength was adjusted with potassium propionate. To prepare solutions with lower MgATP concentrations (2, 0.5, and 0.1 mm MgATP), they were mixed from solutions B and I which contained the regenerating system (see Table I). To all solutions that contained creatine phosphate, 50 units/ml creatine kinase was added (Sigma), and the ph was adjusted to 7. The regeneration system was not included in the rigor experiments or in the solutions which contained ADP. Incorporation of Dansylaziridine-labeled STnC-The endogenous STnC was extracted by soaking the fiber bundle for min in a solution of 2 mm EDTA, ph 7.8. This solution was renewed every 20 s. After extraction, dansylaziridine-labeled STnC (for preparation of STnC, see Ref. 13; for the labeling procedure, see Ref. 14) was incorporated at low Caz+ concentration (pca 8) into the fibers by soaking it for 20 min in solution A or B (Table 11) which contained 20 pg/ml dansylaziridine-labeled STnC (see also Fig. 1B). Solution A or B was used depending on whether the ionic strength was 150 or mm, respectively. Experimental Protocol--An example of the experimental protocol is shown in Fig. 1 (A and B). In these experiments (results shown in Fig. 2), a pca force curve was determined before the STnC was extracted from the fiber bundle (Fig. la, upper). Measurements of force or dansylaziridine-labeled STnC fluorescence at intermediate Caz+ concentrations were always followed by a measurement at pca 4. Fluorescence and force values at the intermediate Ca2+ concentrations were normalized to the value determined before (i.e. at pca 8 to 0%) and after (ie. at pca 4 to 100%) the determination. In Fig. la (lower), the procedure of extraction of the intrinsic STnC from the fiber bundle and incorporation of the dansylaziridinelabeled STnC are shown. At arrow 1, the intrinsic STnC was extracted, and the force was then increased due to the absence of ATP and the development of rigor. After extraction for 30 min, the fiber was transferred into relaxing solution (solution A, Table I). A test contraction at pca 4 was performed to estimate the amount of STnC extracted. At the time marked by arrow 2, 20 pg/ml dansylaziridinelabeled STnC in relaxing solution A (Table I) was added to the incubation solution for 20 min. Fig. 1B shows the simultaneously measured force (lower trace) and fluorescence (upper trace) of the incorporated dansylaziridine-labeled STnC. The overall decrease of the fluorescence signal is probably caused by photobleaching of the dansylaziridine-labeled STnC. (The amplitude of the relative fluorescence change from the level at pca 8 and 4 is essentially unchanged because this relative change is small compared to the total fluorescence signal from the incorporated dansylaziridine-labeled STnC.) Regulation of the Muck by the Incorporated Dansylaziridine-la- beled STnC-Since it was possible that the fluorescence label on STnC might change the Ca" binding or the regulatory properties of STnC and thus the Ca2+ regulation of the muscle, we measured the pca force relationship before and after the exchange of the intrinsic STnC with dansylaziridine-labeled STnC. The result is shown in Fig. 2. The crosses correspond to the pca force relationship with intrinsic STnC; the filled squares were obtained after the incorporation of A 6 FIG. 1. A, upper: force development of a small fiber bundle (three fibers) at different Ca2+ concentrations. The Ca" concentration was changed as indicated by the arrows. The experiment was camed out with solution A (Table I). Lower: force transient continued. At the time marked by arrow 1, the intrinsic STnC was extracted from the fiber bundle at low ionic strength and low M$ concentration (2 mm EDTA, ph 7.8). After the extraction, the fiber bundle was relaxed (arrowpca 8), and then a test contraction was performed (arrow pca 4, followed by arrow pca 8). At the time marked by arrow 2, dansylaziridine-labeled TnC was added to the incubation solution (20 pg/ ml dansylaziridine-labeled STnC in relaxing solution; pca 8). B, the upper trace corresponds to the dansylaziridine-labeled STnC fluores- cence and the lower trace to the simultaneously measured force (force transient continued from A). " PC0 FIG. 2. The crosses show the pca force relationship of the contracting fiber before the intrinsic STnC was exchanged (four fiber bundles; four determinations). The doffed line interconnecting the measured points corresponds to the best fit of the data to the Hill equation with a Hill coefficient of The filled squares show the pca force relationship after the STnC exchange (four fiber bundles; eight determinations). The dotted line interconnecting the data points corresponds to the best fit to the Hill equation with a Hill coefficient of The open circles show the fluorescence of the dansylaziridinelabeled STnC detected simultaneously with the force (four fiber bundles; six total determinations). IS, ionic strength.

3 dansylaziridine-labeled STnC. The average maximum force developed at pca 4 after extraction of the intrinsic STnC was 41% of the force before the extraction. Incubation of the fiber bundle in dansylaziridine-labeled STnC restored the maximum force at pca 4 on average to 92% of the force before extraction. Some fibers had higher force after reincorporation (e.g. Fig. 1) than before extraction, suggesting that some TnC was already lost from the fiber. The Hill coefficients obtained from a least-squares fit of the data to the Hill equation (dotted lines in Fig. 2) are 3.8 before and 2.8 after the STnC extraction-incorporation procedure, respectively, and are not considered significantly different. This slight decrease in the Hill coefficient caused by the extraction-incorporation procedure may be caused by a slight loss of the net STnC that was also reflected by the 8% loss in the maximum force at pca 4. After extraction of small amounts of intrinsic STnC, a similar phenomenon has been reported (15). In addition, no significant shift in the pca force relationship was observed,indicating that the Ca" sensitivity of the muscle reconstituted with dansylaziridine-labeled STnC is the same in muscle regulated by intrinsic STnC. The open circles in Fig. 2 represent the fluorescence detected simultaneously with the force as described above and shown in Fig. 1B. RESULTS Simultaneous Measurement of Force ana' Fluorescence in Skinned Fibers-The crosses in Fig. 3 show the pca force relationship, and the open circles show the pca fluorescence relationship obtained during Ca2+ activation of the skinned fiber (solution B, Table I). The pca fluorescence curve is remarkably shifted to the left compared to the pca force curve. We have reported a similar shift between fluorescence and force (16) and have modeled the shift by assuming that Ca2+ binding to either Ca2+-specific site of troponin increased the fluorescence maximally and that two Ca2+ ions must be FIG. 3. The crosses show the pca force relationship of the contracting skinned fiber in the full overlap state (sarcomere length, -2.3 pm). The dotted line refers to the best fit of the data to the Hill equation. The Hill coefficient of the best fit was The open circles show the simultaneously measured dansylaziridine-labeled STnC fluorescence. Both fluorescence and force have been normalized to 100% at pca 4 and to 0% at pca 8. The error bars correspond to the standard error of the mean (three fiber bundles; six determinations). Solution A B C D F G H I Effect of Cross-bridges on TnC in Skinned Muscle Fibers TABLE I Solution cornmsitions Free concentrations Total concentrations Imid- Phos- Mg MgATP EGTA" ADP azo,e phate nhnte mm EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. Ionic strength bound to these sites to allow the formation of force-generating cross-bridges. The formalism given in Ref. 16 also assumed independent Ca2+ binding to different troponin-tropomyosin units along the actin filament. This simplification which fully accounted for the experimental data was made since the steepness of the curves reported in Ref. 16 was low; and thus, no cooperative binding of Ca2+ along the actin filament was apparent. However, the curves in this report, where we have used different conditions than those in Ref 16, are steeper. To account for this increased steepness, positive cooperativity between adjacent troponin-tropomyosin units was considered (17). Calculation of K,, as a Function of [Ca2+]-These steep responses indicate that the apparent binding constant for Ca2+ binding to the thin filament increases during muscle activation. The following is an attempt to get a quantitative measure for the apparent binding constant of the thin filament for Ca2+ at different states of muscle activation. It has been reported that Ca2+ binds independently and with the same affinity to the two low affinity Ca2+-specific regulatory sites of STnC (18), and this has been assumed in our calculations presented here. Assuming further that the different STnC molecules do not interact with each other, the fluorescence of dansylaziridine-labeled STnC is given by the equation of Grabarek et al. (19). F= BK[Cal + (K[CaI)' (1) (1 + K[Ca])' K is the stability constant for the binding of Ca2+ to the Ca2+specific sites. The parameter p describes the fluorescence properties of dansylaziridine-labeled STnC (p = 0: the fluorescence of the molecule is only enhanced when both sites are occupied by Ca2+;,6 = 1: (a) Ca2+ binding to one particular site increases the fluorescence of the molecule independent of whether the other site is already occupied by Ca2+, or (b) Ca2+ binding to both sites independently increases the fluorescence fractionally;,6 = 2: the binding of Ca2+ to either site increases the fluorescence maximally; see also Ref. 19). We also assume that Ca2+ binding to the two Ca2+-specific sites remains in- dependent when STnC is incorporated into the thin filament (recent experiments confirm this (25)). In our calculations, although Ca2+ binding to the two Ca2'-specific sites of a single troponin molecule is assumed to be independent, Ca2+ binding to adjacent troponin-tropomyosin units along the actin filament is assumed not to be. Consequently, during activation, different classes of troponin-tropomyosin units may coexist which have different Caz+-binding constants (Ki). If the minimum (at pca 8) and the maximum (at pca 4) fluorescence outputs from all of these species are the same, then the total normalized fluorescence of the incorporated dansylaziridinelabeled STnC is given by Equation 2 (see Equation 1 or Ref. 19 for the normalized fluorescence of a single class). or F'1 " where p = PI/& The ai values are the relative fractions of the troponin-tropomyosin units with the stability constants (Ki); F' is the fluorescence output which is made up of the regulated units having one Ca2+ ion bound to either site (F'l) and the units having two Ca2+ ions bound (F'2); and F is the normal- F'z

4 13630 Effect Cross-bridges of Skinned TnC on Muscle ized total fluorescence. The ai values are a function of muscle activation (ie. ea2+-dependent), and the relative number of units in the ith state vary with it. This Ca2+ dependence of the ai values can account for the measured high Hill coefficient without invoking cooperative Ca2+ binding to STnC. We also assume that the binding of ea2+ to both Ca2+- specific sites is required to allow the formation of forcegenerating cross-bridges (see also Ref. 16). The normalized force of the muscle is given by Equation 3. = 'iai [ Kj[CaJz 1 + Ki[Cal2] Equations 2 and 3 are not suitable to fit the measured pca fluorescence or the pca force relationships since neither the ai nor the Ki values are known. However, without the knowledge of these parameters, an apparent binding constant for ea2+ at different states of muscle activation can be derived. The fraction of bound Ca2+ normalized to 1 is given by Equation 4. From Equations 2-4, Equation 5 can be derived. B= F + (B - 1)T B An apparent binding constant (Kapp; defined by B = K.,,[Ca] /(1 + Kapp[Ca])) can then be calculated from Equation 6. From the pca fluorescence and the pca force curves shown in Figs. 2, 3, and 10, Kapp is calculated assuming that 6 = 2 (see Ref. 16, but also the justification below). The results are shown in Fig. 4. The filled triangles correspond to the data presented in Fig. 2, the filled squares to the.data from Fig. 3, and the filled circles to the data from Fig. 12. It is apparent fromfig. 4 that Kapp estimated from the above equations increases more than 10-fold during muscle activation. Fibers Effect of RigorCross-bridgeson the Structure and Ca2+ Affinity of STnC-The observed high steepness of the pca force and the pca fluorescence relationships may be caused by the cooperative binding of ea2+ to the thin filament irrespective of the formation of cross-bridges. On the other hand, it may be mediated by cross-bridge binding to the thin filament that results in an "apparent" cooperative binding of ea2+ (17). If the cooperativity in Ca2+ binding is cross-bridgemediated, it is likely that binding of cross-bridges changes the conformation of the troponin-tropomyosin units along the actin filament, thus causing a fluorescence change in the incorporated dansylaziridine-labeled STnC. This experiment is shown in Fig. 5 where the solution bathing the skinned fiber was changed (left-hand arrow, Fig. 5) from relaxing solution (pca 8, solution A, Table 11) to a rigor solution (pea 8, solution D, Table 11). The upper trace corresponds to the change in force and the lower trace to the fluorescence. Since very little MgATP is needed to keep muscle in the relaxed state, there was a delay of -15 s before rigor force began to develop. It can be seen that the rise in rigor force is paralleled by an increase in the fluorescence. When the rigor solution was changed back to the relaxing one, the fluorescence decreased to the original level (right-hand arrow, Fig. 5). Since no Caz+ was present in the relaxing or in the rigor solution, the fluorescence increase can only be caused by the formation of rigor cross-bridges or by the strain in the actin filament brought about by the rigor force. The rigor force generated by a fiber depends largely on the Ca2+ concentration present when ATP is withdrawn from it, with force increasing at higher [Ca"]. However, once the fiber goes into the rigor state, the force no longer depends on the ea2+ concentration. Thus, it is possible to generate different rigor force levels that are independent of the Ca2+ concentration once the rigor state is entered and therefore makes it possible to measure dansylaziridine-labeled STnC fluorescence at the same ea2+ concentration but at different force levels. We studied the ea2+ dependence of the dansylaziridine- *TP pcoao FIG. 4. K,,(ordinate) was calculated from Equation 6 with B = 2 and pairs of relative fluorescence and force measurements determined at the same state of activation, i.e. the same free Ca2+ concentration (abscissa). The filled squares and the filled triangles show the pca force and pca fluorescence relationships in the contracting 5tate without phosphate (data from Figs. 2 and 3, respectively). The filled circles correspond to the contracted state in the presence of 10 mm inorganic phosphate (data from Fig. 12). The error bars correspond to the standard error of the mean. For the number of fibers and the number of determinations, see the corresponding pca force and pca fluorescence relationships (Figs. 2, 3, and 12). The dashed line corresponds to the data shown in Fig. 2 and to the filled squares in this figure; however, it was calculated from Equation 6 with 0 = Arrows a and b mark the affinity of the actin filament calculated from Equation 1 in the absence of ATP for the full and small overlap states, respectively (see Fig. 7). 15s ' FIG. 5. The upper trace shows the force, and the lower trace the fluorescence changes after ATP was withdrawn or added (see arrows) to the incubation solution. The Ca" Concentration was low (pca 8) in the presence and absence of ATP (solution D (Table I) when ATP was absent; solution A when it was present). The fiber bundle contained three fibers. TABLE I1 Effect of rigor and cycling cross-bridges on fluareseenee In all cases, the zero point was taken at pca 8 (+ATP) at the length used (full or small overlap). The value of 100% represents the value at pca 4 (+ATP) at low overlap. This value was corrected for the volume change which occurs upon stretch. Relative fluorescence % Full overlap, +ATP, pca Small overlap, +ATP, pca Full overlap, rigor, pca Full overlap, rigor, pca 8 83

5 labeled STnC fluorescence at the two force levels (Fig. 6) and found no apparent alteration in the Ca2+ affinity of the Ca2+specific sites. Here, the rigor state was entered in the presence or absence of Ca2+, i.e. the solution was changed from ATPcontaining pca 4 or 8 solutions into high (pca 4) or low Ca2+ (pca 8)-containing rigor (no ATP) solutions, respectively. Despite the considerable difference in the developedrigor force between the two states (18 and 73% of the force at full contraction), the Ca2+ sensitivity of the dansylaziridine-labeled STnC fluorescence was not significantly altered. Effect of Sarcomere Length on the Ca2+ Affinity of STnC in the Rigor State-Bremel and Weber (7) have reported that the apparent affinity of the regulated actin filament for Ca2+ is distinctly increased upon binding of rigor cross-bridges. To investigate this in our system, we studied the effect of rigor conditions at short (2.3 pm) and long (3.8 pm) sarcomere lengths on the Ca2+ dependence of dansylaziridine-labeled STnC fluorescence. The pca fluorescence relationship obtained in the absence of ATP (solution C, Table I) at 2.3-pm sarcomere length is shown in Fig. 7 (open squares). At this sarcomere length, the actin and myosin filaments overlap fully, and thus, a large number of cross-bridges are bound to the actin filament. In order to reduce the extent of crossbridge formation, we stretched the muscle prior to the experiment to a sarcomere length of -3.8 pm. At this sarcomere "I ty.p 0.a ;.".,, -,..Q...' pco FIG. 6. Dansylaziridine-labeled STnC fluorescence (open circles) is shown at full overlap (sarcomere length, 2.3 pm) in the rigor state entered at low [Ca2+](pCa 8). The average force obtained was 18% of that found in the fully contracted state (five fibers; five determinations). The open squares correspond to the values obtained when rigor was entered in the presence of ea2+ (pca 4). The average force was 72% of that obtained in the fully contracted state (six fibers; seven determinations). 50- Effect of Cross-bridges TnC on in Skinned Muscle Fibers length, there is reduced overlap between the thin and thick filaments (20), and thus, fewer numbers of cross-bridges should be bound to the actin filament. The corresponding pca fluorescence relationship is shown in Fig. 7 (open triangles). As can be seen, there is a 2-fold increase in the Ca" affinity of Ca2+-specific sites on transition from the long to the short sarcomere length. It is also clear from Fig. 7 that in the absence of ATP, the steepness of the pca fluorescence curve at both sarcomere lengths is much less than in the presence of ATP (compare Figs. 2,3, and 12 with Fig. 7), i.e. the cooperativity is lost in the absence of ATP. The data obtained at the 3.8-pm sarcomere length was fitted to Equation 1 = 2.38, which is similar to the B = 2 that was used for the calculation of the apparent binding constants shown in Fig. 4 (see also "Discussion"). A fit to the data at full overlap was only possible if fi was held constant since otherwise the fit routine diverged. The best fit was obtained with fi = 2.38 (taken from the small overlap case), allowing the stability constant (K) to vary, and is shown by the dashed line in Fig. 7. The observed shift in K between the curves is small and cannot account for the more than 10-fold increase of the apparent stability constant which occurs during muscle activation in the presence of ATP (Fig. 4). Since the number of rigor cross-bridges formed depends on the degree of filament overlap, the fluorescence increase upon rigor cross-bridge formation may depend also on the degree of overlap. Fig. 8 shows the fluorescence increase upon rigor cross-bridge formation in the absence of Ca2+ and the dependence on the sarcomere length. 100% corresponds to the value obtained under the same conditions and sarcomere length at pca 4. The solid line represents a linear regression to values obtained at sarcomere lengths larger than 2 pm. It is obvious from Fig. 8 that: 1) even at full overlap, the formation of cross-bridges in the absence of Ca2+ can only account for -80%of the signal obtained under the same conditions in the presence of saturating [Ca"] (compare also Table 11; note normalization is different); and 2) at longer sarcomere lengths, the lower number of recruited rigor links results in a smaller fluorescence signal. Effect of Cross-bridge Binding on Dansylaziridine-labeled STnC Fluorescence-The effect of cross-bridge binding to the thin filament on the dansylaziridine-labeled STnC fluorescence (summarized in Table 11) was determined by measuring the fluorescence intensity at saturating and at low Ca2+ concentrations at both full overlap (sarcomere length, 2.3 pm) and small overlap (fiber stretched out until less than 10% of the force observed at full overlap was detected). After correction for the volume change that occurs upon stretching the 0- i 6 S pco FIG. 7. The open trianglescorrespond to the pca fluorescence relationshipinstretchedfibersdeprivedof ATP. The sarcomere length (detected bylaser diffraction) averaged 3.8 pm (five fiber bundles; nine determinations). The dotted line corresponds to the best fit of the data to Equation 1 with fl = 2.38 and log(k) = The open squares correspond to the rigor state (absence of ATP, full overlap of thin and thick filaments). The sarcomere length averaged 2.3 pm (six fiber bundles; ten determinations). The dashed line corresponds to the best fit of Equation 1 to the data (fl held constant at 2.38). The best fit was achieved with log(k) = The error bars correspond to the standard error of the mean. The ionic strength was mm. FIG. 8. Dansylaziridine-labeled STnC fluorescence was obtained at different sarcomere lengths (SL) in the absence of ATP. Rigor was entered at pca 8. The values are normalized to 0% in the relaxed state (solution B) and to 100% in the absence of ATPat pca 4 (solution C).

6 13632 Effect of Cross-bridges Skinned TnC on Muscle Fibers fiber, the fluorescence signal at saturating [Ca"'] and at full overlap was -165% of the corresponding signal at small overlap (0% corresponds to the intensity obtained in the absence of Ca2+). This additional increase of 65% in the full compared to the low overlap state at pca 4 is probably brought about by the formation of actively cycling cross-bridges. The fluorescence signal obtained in the low overlap state is presumably due primarily to Ca2+ binding to STnC, with only a small contribution coming from any cycling cross-bridges. We also measured the fluorescence intensity brought about by the formation of rigor cross-bridges, i.e. the intensity increase obtained when the fiber was transferred from the relaxed state into the rigor state in the absence of Ca2+. The attachment of rigor cross-bridges to the thin filament at full overlap and pca 8 produced 83% of the change (Table 11) seen at small overlap at pca 4 (+ATP). Raising the [Ca"] to pca 4 gave a further increase of fluorescence to 119% (Table 11). Thus, both cycling and rigor cross-bridges can augment the change in fluorescence of dansylaziridine-labeled STnC brought about by the binding of Ca2+ (21). It is also clear that cycling cross-bridges have a greater effect on the fluorescence than do rigor cross-bridges (21). Effect of Sarcomere Length on the pca Fluorescence Relationship in the Presence of ATP-We next addressed the question of how many troponin-tropomyosin units along the actin filament might be cooperatively linked to produce the high steepness of both the pca force and pca fluorescence relationships. If the cooperative Ca" response is cross-bridgemediated, the steepness might depend on the degree of overlap between the filaments. Therefore, we measured the pca fluorescence relationship in the contracting state (presence of MgATP, solution B, Table I) at short and long sarcomere lengths. In Fig.9, the open circles correspond to the pca fluorescence relationship at full overlap (sarcomere length, -2.3 pm), and the open squares correspond to the low overlap condition (sarcomere length, -3.8 pm). As can be seen, there is no significant effect of sarcomere length on the pca fluorescence change, indicating that a small number of cycling cross-bridges at low overlap can have the same effect as a large number at full overlap. This would suggest that the entire thin filament can be activated by only a few crossbridges. Effect of ADP and pca on the pca Fluorescence Rehtwnship-As shown above, in the absence of ATP, the formation of rigor cross-bridges at high overlap (compared to low overlap) increased the Ca2+ affinity of the thin filament only 2- fold whereas the apparent affinity increases more than 10- fold during muscle activation as calculated from Equation 6. Moreover, it is obvious from Fig. 4 that the Ca2+ affinity, at short sarcomere lengths in the rigor state, is only slightly elevated compared to the Ca" affinity in the fully relaxed state where no cross-bridges are bound (compare in Fig. 4 the values obtained at low Ca2+ concentrations and the stability constant marked by arrow a which corresponds to the rigor state at full overlap). This may indicate that the apparent affinity of the thin filament for Ca2+ increases more when a cross-bridge species other than that found in rigor is bound. It is generally assumed that the rigor bridges, i.e. the nucleotide-free actomyosin complex, are formed at the end of the power stroke. Therefore, it is likely that the above-mentioned species of cross-bridge which increases the Ca" affinity more than the rigor bridge precedes it within the cross-bridge cycle. If it is the actomyosin-adp complex, it should be possible to increase the number of this species by increasing the ADP concentration in the incubation solution. However, it is furthermore generally assumed that in the contracting muscle, the nucleotide-free actomyosin complex (the rigor complex) is short-lived; and consequently, the number of this species is small. In order to increase the probability for the formation of the actomyosin-adp complex, we lowered the ATP con- centration to 0.5 mm and added 30 mm ADP to the incubation solution (solution F, Table I). Both the lowered ATP and the high ADP concentrations hinder the cross-bridge cycle, which might result in increased numbers of force-generating crossbridges (ie. S1-ADP bridges). Consistently, we observed that the force increased independent of the Ca2+ concentration to 121% of the force obtained at 7.5 mm ATP at pca 4 without ADP added to the solution. In Fig. 10, the open triangles correspond to the pca fluorescence curve obtained at 0.5 mm MgATP and 30 mm ADP. For easy comparison, the pca fluorescence curve obtained in the absence of MgATP in the stretched lowest affinity state is also included (sarcomere length, ~3.8 pm, open circles; same data as shown in Fig. 7). The open squares represent the pca fluorescence curve obtained during Ca2+ activation of the muscle in the presence of 7.5 mm ATP (same data as shown in Fig. 3). It is apparent from Fig. 10 that the pca fluorescence relationship obtained at 0.5 mm MgATP and 30 mm ADP is distinctly leftward of the other curves. Thus, the result is consistent with the postulate that the enhanced affinity of the thin filament for 1%) i M P 0' pco FIG. 9. The open circles correspond to the pca fluorescence relationship measured in the presence of ATP at short sarcomere length (average Z2.3 pm; six fiber bundles; nine determinations). The open squareshow the pca fluorescence relationship obtained from stretched fiber bundles where the sarcomere length averaged -3.8 pm (six fiber bundles; eight determinations). The error bars correspond to the standarderrorof the mean. The fluorescence signal was normalized to 100% at pca 4 and to 0% at pca 8. " pca FIG. 10. The open triangles correspond to the pca fluorescence relationship obtained at full overlap of the thick and thin filaments (sarcomere length, 2.3 pm) and in the presence of 0.5 mm ATP and 30 mm ADP (fourfibers;sevendeterminations). The open circles show the pca fluorescence relationship obtained at high sarcomere length (average, 3.8 pm) and in the absence of ATP. The dotted line corresponds to the best fit of the data to Equation 1. The data are also shown in Fig. 7. The open squares show the pca fluorescence dependence at full overlap of the thin and thick filaments (sarcomere length, 2.3 pm) and in the presence of ATP. The data are also shown in Fig. 3. The open hexugons show the pca fluorescence relationship at 1.5 mm MgATP and 30 mm ADP (one fiber; two determinations). The data are normalized to 100% at pca 4 and to 0% at pca 8.

7 Effect of Cross-bridges TnC on in Skinned Muscle Fibers Ca2+ is due to an active cycling cross-bridge state, a state which presumably precedes the rigor state within the cycle. In one particular experiment, the pca fluorescence curve at 30 mm ADP and 0.5 mm MgATP was considerably less shifted to the left than shown in Fig. 10. However, in the same experiment, the pca relationship could be shifted even further to the left by increasing the MgATP concentration from 0.5 to 1.5 mm (open hexagons, Fig. 10; solution G, Table I). The experiment shows that for a maximum shift, a certain amount of MgATP must be present. The finding that the optimum MgATP concentration for a maximum shift can be different from preparation to preparation is probably due to different diffusion conditions in different fiber bundles; since ADP is present in the solution, no ATP-regenerating system was used. Consequently, a diffusion-dependent concentration gradient of MgATP will be formed within the fiber bundle. This gradient, however, will depend strongly on the size of the fiber bundle. It is obvious from Fig. 10 that at 30 mm ADP and 0.5 mm MgATP (open triangles or hexagons), the steepness of the pca fluorescence curve is considerably smaller than the corresponding curve of the Ca2+-activated muscle in the presence of 7.5 mm ATP and in the absence of ADP (open squares). The steepness at 30 mm ADP and 0.5 mm MgATP is similar to the steepness of the pca fluorescence curve in the absence of ATP (Fig. 7; open circles, Fig. 10). It has been reported (22) that the pca force relationship is shifted to the left when the MgATP concentration is lowered. Therefore, the large shift to the left at 0.5 mm MgATP and 30 mm ADP could be a consequence of the lowered MgATP concentration in the solution. To test this, we varied the ATP concentration and measured the force and the dansylaziridine-labeled STnC fluorescence at an intermediate Ca2+ concentration (pca 6). The normalized force and fluorescence obtained at pca 6 are shown in Fig. 11 (100% corresponds to the value obtained under the same experimental conditions at pca 4; 0% to the value obtained at pca 8). It is apparent from Fig. 11 that lowering the MgATP concentration from 7.5 to 0.5 mm MgATP decreased rather than increased the relative fluorescence and force at pca 6, as would be expected if lowering the MgATP concentration produced a higher Ca2+ sensitivity (22). A slight increase was observed by further decreasing the MgATP concentration from 0.5 to 0.1 mm MgATP. Brandt et al. (22) also reported that the steepness of the pca force relationship depends on the MgATP concentration. Therefore, it is not clear whether the decrease in relative fluorescence and force at pca 6 indicates a decrease in the Ca2+ sensitivity or whether it is caused by a change in the steepness of the pca force and pca fluorescence relationships. In any event, the fluorescence increase with decreasing [MgATP] is not sufficient to explain the large shift in the pca fluorescence curve at 30 mm ADP and 0.5 mm MgATP. For comparison (Fig. 111, the fluorescence levels at pca 6 with 30 mm ADP and 0.5 mm MgATP and with 30 mm ADP and 1.5 mm MgATP are plotted (same data as shown in Fig. 10). The value obtained at 0.1 mm MgATP is close to the value obtained in the rigor state at short sarcomere lengths (rigor column in Fig. 11; extrapolated from the data shown in Fig. 7). Finally, to test the effect of Pi, we used conditions similar to those described for Fig. 3. In Fig. 12, the pca force and pca fluorescence relationships in the presence of 10 mm phosphate are shown (solution H, Table I). It is apparent from the comparison of Figs. 3 and 12 that the increased phosphate concentration shifts the midpoint of the pca force and pca fluorescence curves slightly, resulting in a greater separation between the pca force and pca fluorescence curves when Pi is present. DISCUSSION The data presented here yield valuable new insights into how the thick filaments interact with the Ca2+ regulatory system in the thin filaments. As shown in Fig. 5, the fluorescence intensity of dansylaziridine-labeled STnC incorporated into rabbit psoas fibers changes when rigor cross-bridges are formed in the absence of Ca2+ and is further enhanced upon Ca2+ binding. The fluorescence change is in the same direction as when Ca2+ binds to the Ca2+-specific sites of dansylaziridine-labeled STnC. The qualitative similarity of the fluorescence signal on Ca2+ binding to dansylaziridine-labeled STnC and on cross-bridge binding to the actin filament may indicate that the conformational changes in the troponin-tropomyosin units are similar upon Ca2+ and cross-bridge binding. Table I1 shows furthermore that the formation of cycling cross-bridges enhances the fluorescence considerably (65%) IS = mm FIG. 11. Comparison is shown of the relative force and fluorescence at an intermediate Ca2+ concentration (pca 6) and at different MgATP and/or MgADP concentrations in the incubation solutions. The concentrations are noted above the columns. On the left-hand side, the relative force and on the right, the corresponding relative fluorescence values are shown. Both fluorescence and force data have been normalized to 100% at pca 4 and to 0% at pca 8. The Rigor column is extrapolated from the data obtained at pca 5.9 and 6.2 from Fig. 7 (full overlap condition). The error bars correspond to the standard error of the mean. The number of determinations is noted above the columns J..=. I.' : PC0 FIG. 12. The crosses show the pca force relationship in the presence of 10 mm inorganic phosphate. The dotted line shows the best fit of the data to the Hill equation with a Hill coefficient of The open circles show the simultaneously measured dansylaziridine-labeled STnC fluorescence. Both the fluorescence and the developed tension have been normalized to 100% at pca 4 and to 0% at pca 8.

8 13634 Effect of Cross-bridges on TnC in Skinned Muscle Fibers in addition to the signal brought about by Ca2+ binding to the filament alone. Thus, the formation of either rigor crossbridges or cycling cross-bridges causes a conformational change within the regulatory proteins on the thin filament which might be qualitatively similar to that evoked by the binding of Ca2+. This finding is consistent with our previous findings (23) which showed that the binding of Ca2+ to the Ca2+-specific sites is tightly coupled to cross-bridge interactions with the thin filament. The steepness of the pca force and pca fluorescence relationships in the presence of ATP that we report here is larger than can be explained by the binding of Ca2+ to the Ca2+specific sites of STnC alone (24). Therefore, it must be assumed that several troponin-tropomyosin units along the actin filament interact with each other. Since the binding of Ca2+ in vitro changes the affinity of the regulated actin filament slightly for strongly bound cross-bridges (for review, see Ref. 24), the formation of strongly bound cross-bridges may then change the affinity of the filament for Ca2+. Therefore, we calculated under "Results" an apparent affinity of the thin filament for Caz+ during muscle activation, i.e. when force-generating cross-bridges are formed. For the calculation using Equation 6, the value of the parameter /3 must be known. It was concluded previously (16) that the change in fluorescence intensity of dansylaziridine-labeled STnC incorporated into skinned rabbit psoas fibers was maximal when Ca2+ bound to either Ca2+-specific site, which corresponds to /3 = 2. The fit of Equation 1 to the pca fluorescence curve (obtained in the absence of ATP and at low overlap) yields /3 = 2.38 or 2.08 as obtained from the data shown in Figs. 7 and 6, respectively. At full overlap (Fig. 7), the scatter of the data is large; and thus, the fit procedure diverged. But at slightly lower ionic strength (150 mm; data not shown), we found at full overlap that (I = 2.32, which is also close to p = 2. All of the calculations above assumed that the fluorescence was only affected by Ca2+ binding to dansylaziridine-labeled STnC. However, as shown in Table 11, cycling cross-bridges further enhance the fluorescence over that produced by Ca2+ alone. In order to account for this, we have calculated a new value of /3. Since we postulated under "Results" that during muscle activation, two Ca2+ must be bound before forcegenerating cross-bridges can form (Equation 3), the enhanced fluorescence would only arise from that species (F12 term in Equation 2). Thus, in Equation 2, the weighting factor p2 would be 1.65 times higher when active cross-bridges are formed compared to the rigor state when the number of attached cross-bridges is constant. Thus, the p value (Equation 2) suitable for the activated state would be the value determined in the rigor state (p = 2.38) divided by 1.65, i.e. /3 = This value is considerably lower than the /3 = 2 which was used to calculate the Kapp from Equation 6 shown in Fig. 4. However, the Kspp calculated from Equation 6 is not terribly sensitive to variations in the parameter 6. This is shown in Fig. 4 where we plotted for one set of fluorescence-force pairs not only the Kapp values obtained with (3 = 2 (filled squares), but also the values obtained from /3 = 1.44 (dashed line). It can be seen that the curve corresponding to /3 = 1.44 is slightly shifted to higher affinities with respect to the curve obtained with p = 2, but the overall increase of KaPp during muscle activation is essentially the same. The values for the apparent affinity of the thin filament for Caz+ (Kapp) calculated from the data presented in Figs. 2, 3, and 12 increase steeply and with a roughly constant slope when Ca2+ is increased from intermediate to high concentra- tions (see Fig. 4). Since the curves do not level off to a maximum value of Kapp, it might be concluded that under the experimental conditions, the highest possible affinity of the thin filament had not been reached even when the force was maximal. At low degrees of activation, the curves in Fig. 4 show a tendency to level off. However, the curves do not bend distinctly enough to allow for an extrapolation to a minimum K., at low degrees of activation. This would correspond to the affinity of the thin filament if no force-generating, strongly bound cross-bridges are formed. Nevertheless, it is interesting to note that the lowest values of Kapp coincide with the affinity obtained in the absence of ATP and at low overlap of the actin and myosin filaments (arrow b, Fig. 4). Two curves in Fig. 4 correspond to slightly different ionic strengths (150 and mm, respectively), and these curves are very close. This is different from the finding of Brandt et al. (22) who reported a more substantial shift of the pca force relationship on changing the ionic strength within this range. Since they did not measure the dansylaziridine-labeled STnC fluorescence, no apparent binding constant could be derived; but nevertheless, a large change in the affinity wouldbe anticipated from the large reported shift in the pca force curve. One possible explanation for this is that the authors worked at extremely low free magnesium concentrations, and thus, the data may not be comparable. The KaPp values obtained in the presence of 10 mm Pi are different from those obtained in the absence of Pi (see Fig. 4). In the presence of Pi and at higher Ca2+ concentrations, the slope of the curve is less than in the absence of Pi. On the other hand, the maximum and minimum values for KaPp are not significantly different in the presence and absence of Pi. It is tempting to conclude that during muscle activation, the total affinity increment of the actin filament for Ca2+ does not depend largely on the Pi concentration; but that in the presence of Pi, a slightly higher Ca2+ concentration may be needed to enhance the affinity to the maximum level. This interpretation is consistent with the cross-bridge cycle in which the transition from the weakly bound actomyosin complex (ADP and Pi bound) to the strongly bound form (ADP bound with Pi released) is Ca2+-regulated (l), whereas Pi might increase the backward rate of the transition, and Ca2+ would decrease it or increase the forward rate (5), i.e. in the direction of the cycle. Thus, the relative number of strongly and weakly bound cross-bridges may depend contrarily on Ca2+ and Pi. Since Pi impedes the formation of strongly bound crossbridges, more Ca2+ would be required for the formation of strongly bound cross-bridges than in the absence of Pi. Thus, in the presence of Pi, more Ca2+ would be required to reach the maximum affinity of the thin filament for Ca2+, if it is assumed that it is this formation of strongly bound cycling cross-bridges which increases the affinity of the filament. In addition to the anticipated increase in the affinity of the thin filament for Ca2+ mediated by the formation of strongly bound cross-bridges, it is possible that Ca2+ binding to the thin filament itself is cooperative irrespective of the formation of cross-bridges. Fig. 7 shows that in the absence of ATP, no cooperative Ca2+ binding to the thin filament is apparent since the measured pca fluorescence curve could be fitted to Equation 1, which is derived assuming no cooperative binding of Ca2+ to STnC. No cooperative binding was also indicated by the low steepness of the pca fluorescence relationship obtained at 30 mm ADP and 0.5 mm MgATP (Fig. 10). Under these conditions, the forcewashigh and the same in the presence and absence of Ca", indicating that the number of force-generating, strongly bound cross-bridges was independent of Ca". Also in the absence of ATP, i.e. in the rigor state, it is generally assumed that the number of attached cross-

9 Effect of Cross-bridges on TnC in Skinned Muscle Fibers bridges is independent of the Ca2+ concentration. If, on the other hand, different Ca2+ concentrations result in different numbers of force-generating, strongly bound cross-bridges, the resulting pca fluorescence relationship will be steep (as seen in the presence of ATP; Figs. 2, 3, and 12), and cooperative binding of Ca2+ to different troponin-tropomyosin units along the actin filament must be assumed. We conclude that the formation of force-generating cross-bridges that are formed concomitantly when Ca2+ is bound to troponin affects the apparent cooperativity of Ca2+ binding to the actin filament and that Ca" binding to the filament itself, i.e. with no or a constant number of strongly bound cross-bridges formed, is not cooperative. It is apparent from Fig. 7 that in the absence of ATP, the shift between the pca fluorescence curve obtained at high and at low overlap is comparatively small (approximately 0.3 pca unit). Thus, in our measurements, the affinity of the thin filament for Ca2+ increases only 2-fold on formation of rigor cross-bridges. On the other hand, it was difficult in our experiments to stretch skinned psoas fibers to true nonoverlap conditions. Furthermore, the control of the sarcomere length in our experiments stems from the laser diffraction pattern detected during the experiment. The laser diffraction pattern, however, only provides information about the most prominent sarcomere length within the fiber, and it does not give information about the organization of the actin and myosin filaments within the sarcomere. Thus, even at the measured sarcomere length of 3.8 pm which is thought to correspond to the non-overlap condition (ZO), it is possible that a considerable number of cross-bridges are formed between the actin and myosin filaments. This is likely since these fibers, at long sarcomere lengths, developed 5-15% of the force seen at short sarcomere lengths. Also, the fluorescence signal brought about by the formation of rigor bonds in the absence of Ca2+ is not zero at 3.8-pm sarcomere lengths, as indicated in Fig. 8. If the formation of these cross-bridges transfers the thin filament into the state where it binds Ca2+ with high affinity, the formation of further cross-bridges in the full overlap state might not have an additional effect. Since in the stretched state the formation of the cross-bridges probably only takes place at the ends of the filaments, this interpretation includes the postulate that the cooperative interaction between the troponin-tropomyosin units spreads along the entire actin filament. This idea is supported by the results shown in Fig. 9, where in the presence of ATP, the pca fluorescence curve is steep irrespective of the degree of overlap of the actin and myosin filaments (see also Ref. 20). The measured fluorescence change of the incorporated dansylaziridine-labeled STnC is probably caused by Ca2+ binding to all parts of the thin filament and not only to its ends where the cross-bridges are formed. Thus, the formation of strongly bound cross-bridges at the ends of the filament may be able to switch the entire filament from, a low to a high affinity for Ca2+, i.e. all troponin-tropomyosin units along the actin filaments are related cooperatively. It is interesting that although we observed no change in the pca fluorescence curve in the presence of ATP at long and short sarcomere lengths, we did observe a change in the magnitude of the fluorescence intensity obtained under these conditions. It is possible that only a small number of cycling cross-bridges are required to alter the pca force relationship, whereas greater numbers are needed to alter the extent of the fluorescence enhancement analogous to that seen with rigor cross-bridges (Fig. 8). Fig. 10 shows that in the presence of 30 mm ADP and 0.5 mm MgATP, the pca fluorescence curve is distinctly shifted to the left compared to the curve obtained in the rigor state at full overlap. It must be concluded that different species of strongly bound cross-bridges cause a different affinity of the thin filament for Ca2+. It is obvious from Fig. 4 that the affinity of the thin filament in the rigor state (see arrow a in Fig. 4) is also much lower than the maximum affinity achieved during Ca2+ activation in the presence of MgATP (Fig. 4). At low MgATP concentration and at 30 mm ADP, a large number of cross-bridges probably have ADP bound; and thus, it is likely that the highest affinity of the actin filament for Ca2+ might be caused by an actomyosin-adp complex. It was also demonstrated under "Results" that some MgATP must be present to observe the large leftward shift of the pca fluorescence curve seen in the presence of 30 mm ADP. Although it is difficult to have ATP-free ADP solutions, in one experiment in the presence of 30 mm ADP, we saw the same Ca2+ dependence of fluorescence as seen in the rigor state. These results may indicate that only the actomyosin-adp complex which is formed in the active cycle of the cross-bridge is responsible for the increased affinity of the actin filament. REFERENCES 1. Eisenberg, E., and Hill, T. L. (1985) Science 227, Rosenfeld, S. S., and Taylor, E. W. (1984) J. Biol. Chem. 269, Hibberd, M. G., and Trentham, D. R. (1986) Annu. Reu. Biophys. Chem. 16, Ferenczi, M. A., Homsher, E., and Trentham, D. R. (1984) J. Physiol. (Lond.) 362, Chalovich, J. M., Greene, L. E., and Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, Goldman, Y. E., Hibberd, M. G., and Trentham, D. R. (1984) J. Physiol. (Lond.) 364, Bremel, R. D., and Weber, A. (1972) Nat. New Bwl. 238, Fuchs, F. (1985) J. Muscle Res. Cell Motil. 6, Kerrick, W.G. L., and Hoar, P. E. (1987) Pfluegers Arch., in press 10. 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