Effects of dihydropyridine receptor II-III loop peptides on Ca2+ release in skinned skeletal muscle fibers

Graham D. Lamb1, Roque El-Hayek2, Noriaki Ikemoto2, and D. George Stephenson1

1 School of Zoology, La Trobe University, Bundoora, Victoria 3083, Australia; and 2 Boston Biomedical Research Institute, Boston, Massachusetts 02114


    ABSTRACT
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In skeletal muscle fibers, the intracellular loop between domains II and III of the alpha 1-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca2+ release channel in the sarcoplasmic reticulum. We examined the effects of synthetic peptide segments of this loop on Ca2+ release in mechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg2+ concentration ([Mg2+]; 1 mM), a 20-residue skeletal muscle DHPR peptide [AS(20); Thr671-Leu690; 30 µM], shown previously to induce Ca2+ release in a triad preparation, caused only small spontaneous force responses in ~40% of fibers, although it potentiated responses to depolarization and caffeine in all fibers. The COOH-terminal half of AS(20) [AS(10)] induced much larger spontaneous responses but also caused substantial inhibition of Ca2+ release to both depolarization and caffeine. Both peptides induced or potentiated Ca2+ release even when the voltage sensors were inactivated, indicating direct action on the Ca2+ release channels. The corresponding 20-residue cardiac DHPR peptide [AC(20); Thr793-Ala812] was ineffective, but its COOH-terminal half [AC(10)] had effects similar to AS(20). In the presence of lower [Mg2+] (0.2 mM), exposure to either AS(20) or AC(10) (30 µM) induced substantial Ca2+ release. Peptide CS (100 µM), a loop segment reported to inhibit Ca2+ release in triads, caused partial inhibition of depolarization-induced Ca2+ release. In toad fibers, each of the A peptides had effects similar to or greater than those in rat fibers. These findings suggest that the A and C regions of the skeletal DHPR II-III loop may have important roles in vivo.

excitation-contraction coupling; ryanodine receptor


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IN VERTEBRATE SKELETAL MUSCLE, depolarization of the transverse-tubular (T-) system triggers Ca2+ release from the sarcoplasmic reticulum (SR) and subsequent contraction, without the need for inflow of extracellular Ca2+. It is currently thought that the dihydropyridine receptors (DHPRs) in the T-system membrane act as voltage sensors, sensing T-system depolarization and then by some protein-protein interaction activating adjacent ryanodine receptor (RyR)/Ca2+ release channels in the apposing SR membrane (21). The skeletal muscle DHPR is an oligomeric complex of five subunits. The alpha 1-subunit, which contains the dihydropyridine binding site, evidently plays a crucial role in the coupling with the RyR, as skeletal muscle-type coupling is absent in dysgenic mouse muscle, which lacks the alpha 1-subunit, but can be restored by expression of that protein (34). Additionally, it appears that the intracellular loop between repeats II and III of the skeletal DHPR is essential for skeletal-type excitation-contraction (E-C) coupling, because such coupling is lost if the skeletal loop is replaced with the homologous segment of the cardiac DHPR (33).

Lu et al. (16, 17) showed that the skeletal and cardiac II-III loop peptides could each activate isolated skeletal RyRs and increase ryanodine binding to skeletal but not cardiac RyRs. It was subsequently found (3) that the stimulatory region of the skeletal DHPR loop could be further localized to the sequence Thr671-Leu690, called peptide A. It has been since shown that peptide A (at 1-30 µM) increases the open probability of isolated RyRs and that it can also cause voltage-dependent block of the channels (2). Recently, it has been found that the COOH-terminal half of peptide A [called AS(10)] is considerably more potent than peptide A at inducing Ca2+ release and stimulating ryanodine binding in skeletal muscle SR vesicles, with maximal stimulation at ~20 µM and inhibition at higher concentrations (4). It should be noted, however, that these experiments showing the activating effect of the peptide A region were performed at a free Mg2+ concentration ([Mg2+]) of 0.2-0.3 mM rather than at the physiological level of ~1 mM, and such a lowered [Mg2+] may have augmented the stimulatory effect of the peptide (12, 14, 19, 31). Furthermore, experiments with chimeric DHPRs in dysgenic muscle indicate that a quite different region of the skeletal II-III loop (Phe725-Pro742) is crucial for skeletal-type E-C coupling (22). Nevertheless, when a peptide corresponding to this latter region was applied to isolated triads, it did not induce any Ca2+ release, and it was found that the region Glu724-Pro760 (called peptide C) actually inhibited both peptide A-induced and depolarization-induced Ca2+ release (3, 28). Thus, from the studies to date, it is unclear which region(s) of the DHPR II-III loop actually activate the RyRs in vivo.

Here we examine the effects of the skeletal and cardiac DHPRs II-III loop peptides in mechanically skinned fibers, where voltage-sensor control of Ca2+ release is retained (11, 13), and with important factors such as myoplasmic [Mg2+], [ATP], and the SR Ca2+ load all maintained at physiological levels. In this way we could investigate which peptides are capable of inducing Ca2+ release in mammalian muscle fibers under conditions close to those in vivo, which is an important test of their proposed role in normal E-C coupling. Because only every alternate RyR is associated with a DHPR tetrad in mammalian skeletal muscle (5, 21), we hypothesize that exogenously added loop peptides would have relatively free access to the key sites on at least one-half of the RyRs in a skinned fiber and consequently might activate Ca2+ release. (Here we assume that the absence of an adjacent DHPR would leave room for the exogenous loop peptide to bind to the normal coupling site on the RyR.) With this skinned fiber technique, we are also able to test whether any of the peptides interfere with normal E-C coupling, which is important for assessing whether a peptide has an inhibitory action in vivo or alternatively competes in some way with the normal coupling mechanism. Finally, we can also examine the effects of the peptides in toad skeletal muscle fibers, where there are two types of RyRs, alpha  and beta , homologous to the principal mammalian skeletal RyR (RyR1) and the smooth muscle brain RyR (RyR3), respectively, with the alpha -isoform thought to be linked to the DHPRs (21, 23). The DHPR II-III loop in amphibian skeletal muscle is very similar to that in mammalian muscle, and the arrangement of charged residues in both the A and C regions is in fact identical in mammals and amphibia (35). Thus it was of interest to see what effect the mammalian skeletal II-III loop peptides have in amphibian fibers.


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Skinned fibers. Mechanically skinned fibers were obtained and used as described previously (10, 13). Briefly, Long Evans hooded rats were anesthetized with halothane (2% vol/vol) in a bell jar and killed by asphyxiation, and then the extensor digitorum longus (EDL) or soleus muscles were removed. Cane toads (Bufo marinus) were stunned by a blow to the head and killed by pithing, and the iliofibularis muscles were removed. Single muscle fibers were mechanically skinned under paraffin oil, and a segment was attached to a force transducer (AME875; Horten, Norway) at 120% of resting length. The skinned fiber was then placed within a 2-ml Perspex bath containing a potassium hexamethylene-diamine-tetraacetate (K+-HDTA) solution (see below) for 2 min to allow the sealed T-system to become normally polarized, before being stimulated by rapid substitution of another bath with an appropriate solution. Fibers were used with their endogenous level of SR Ca2+ and were not additionally loaded, except where indicated. All experiments were performed at room temperature (23 ± 2°C).

Solutions. All chemicals were obtained from Sigma, unless specified otherwise. The K+-HDTA solutions used for rat and toad fibers contained (in mM): 117 (toad) or 126 (rat) K+; 37 Na+; 50 HDTA2- (Fluka, Buchs, Switzerland); 8 total ATP; 8.6 total magnesium; 10 creatine phosphate; 0.05 total EGTA; 60 (toad) or 90 (rat) HEPES; 1 N3-; pH 7.10 ± 0.01 and pCa (= -log10 [Ca2+]) 7.0. Skinned fibers were depolarized by substitution of an Na+-HDTA solution, which was identical to the corresponding K+-HDTA solution, except that all K+ was replaced with Na+. The above solutions all contained 1 mM free Mg2+ and had an osmolality of 295 ± 5 (rat) or 255 ± 5 (toad) mosmol/kgH2O. Solutions with a free [Mg2+] of 0.05 mM (toad) or 0.015 mM (rat) that were used to directly activate the Ca2+ release channel were similar to the standard K+-HDTA solution, except that they contained only 2.15 or 1.0 mM total Mg2+, respectively. The solution with 0.2 mM free Mg2+ was made by appropriate mixture of the low and normal [Mg2+] solutions. The loading solution used during sequences of depolarization-induced responses was made by adding Ca2+ to the standard K+-HDTA solution (50 µM total EGTA) to give a pCa of 6.0; such a solution could be washed out within 1 or 2 s, unlike loading solutions with heavier Ca2+ buffering (e.g., 1 mM total EGTA; see below) that require longer washout. Maximum Ca2+-activated force was determined using a solution ("max") similar to standard K+-HDTA solution, but with 50 mM Ca2+-EGTA (20 µM free Ca2+) replacing all HDTA and the total Mg2+ reduced to 8.12 mM to keep the free [Mg2+] at 1 mM (32). The Ca2+ sensitivity of the contractile apparatus was determined by exposing the fiber to a sequence of solutions of progressively higher [Ca2+] made by appropriate mixture of the 50 mM Ca2+-EGTA solution and a similar solution with 50 mM free EGTA (9, 32). Free [Ca2+] of solutions was measured with a Ca2+-sensitive electrode (Orion Research, Boston, MA).

Repeated Ca2+ load-release cycles for low [Mg2+]- and caffeine-induced release. The Ca2+ content of the SR could be estimated from the size of the force response when the SR was fully and rapidly depleted by exposing the fiber to a low [Mg2+] K+-HDTA solution (0.015 mM Mg2+) with 30 mM caffeine and 0.5 mM free EGTA (pCa 8) present to chelate the released Ca2+ (25). (As the caffeine-low [Mg2+] solution potently triggers Ca2+ release, the fiber was first preequilibrated for 10 s in the standard K+-HDTA solution with 0.5 mM EGTA to ensure EGTA was present within the fiber.) Because fibers were skinned under paraffin oil, the SR initially contained its normal endogenous level of Ca2+. After depletion, the SR was reloaded with Ca2+ by exposure to the standard K+-HDTA solution with 1 mM total EGTA at pCa 6.7, and then it could be subsequently depleted again. As found previously (25), the force response obtained from such load/release cycles was highly reproducible, and the time integral (i.e., "area") of the force response was approximately linearly related to loading time (until saturating with prolonged loads), indicating that it was indicative of the amount of Ca2+ loaded into the SR. In the experiments here, the EDL fibers were reloaded with Ca2+ to approximately the original endogenous level by 20 s loading under the above conditions.

To examine the ability of the peptides to trigger Ca2+ release at 0.2 mM Mg2+ (see Fig. 2), each EDL fiber was fully depleted of Ca2+ and reloaded as above (with the loading terminated by a 2-s exposure to a K+-HDTA solution with 0.5 mM free EGTA) and then 1) equilibrated for 20 s in the standard K+-HDTA solution (1 mM Mg2+, 50 µM EGTA, pCa 7.0) with or without peptide, 2) exposed for 15 s to the 0.2 mM Mg2+ solution (50 µM EGTA, pCa 7.0) with or without peptide, and 3) once again fully depleted of Ca2+ with the 30 mM caffeine-low [Mg2+] solution ("full release" in Fig. 2). When examining the effect of the peptides on caffeine-induced Ca2+ release at set SR loading levels, the sequence was the same as above, except that the fiber was exposed to a solution with 7 mM caffeine (at 1 mM Mg2+) instead of the 0.2 mM Mg2+ solution. (In some experiments, the effect of caffeine was examined without depleting and loading the SR to a set level, e.g., as in Fig. 3B.) Where noted, similar experiments were also carried out with all solutions made from Na+-HDTA instead of K+-HDTA, to ensure the T-system was chronically depolarized and hence the voltage sensors were kept inactivated.

Peptides. Peptides were synthesized on an Applied Biosystems model 431A synthesizer employing N-(9-fluorenyl)methoxycarbonyl as the alpha -amino protecting group. Peptides were cleaved and deprotected with 95% trifluoroacetic acid. Purification was carried out by reverse-phase high-pressure liquid chromatography using a Rainin Instruments preparative C8 column; all peptides were purified to homogeneity. It is unlikely that there was any substantial heterogeneity in the secondary structure of a given peptide type, because each peptide was only 10 or 20 residues long. The peptide regions of the DHPR alpha 1-subunit II-III loop (Fig. 1) are classified as A or C in conformity with previous usage (3), with subscripts s and c denoting the rabbit skeletal and cardiac muscle DHPR, respectively, and the number of amino acids indicating the full length A peptide [i.e., AS(20) or AC(20)] or the 10-amino acid segment at the COOH-terminal end [i.e., AS(10) or AC(10)]. Peptides were dissolved in double-distilled water at 100-1,000 times the highest final concentration. Tests with a Ca2+-sensitive electrode showed no detectable Ca2+ contamination associated with the peptides.


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Fig. 1.   Amino acid sequence of the various synthetic peptides encompassing different regions of the II-III loop of the alpha 1-subunit of the skeletal and cardiac dihydropyridine receptors (DHPRs; denoted with subscripts S and C, respectively), as well as decalysine (see METHODS). The charges on the amino acids at neutral pH are indicated.

Force traces and analysis. In force traces, unless otherwise indicated, the skinned muscle fiber was bathed in the standard K+-HDTA solution (1 mM Mg2+, 50 µM total EGTA, pCa 7.0). In the text, mean values are given ± SE. Statistical probability (P) was determined with Student's t-test or the nonparametric Mann-Whitney test when the data were apparently not normally distributed, with P < 0.05 considered significant.


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Effect of A peptides in skinned fibers. Previous experiments (3, 4) showing that peptides from the A region of the skeletal muscle DHPR II-III loop induced rapid Ca2+ release in triad preparations were performed in the presence of ~0.2-0.3 mM free Mg2+. We first sought to test whether the peptides were effective in inducing Ca2+ release in mechanically skinned fibers under such conditions before examining their effect at physiological [Mg2+] (~1 mM). Because it was necessary to have the SR loaded with Ca2+ to the same extent when examining each of a number of successive treatments, each fiber was first emptied of its endogenous SR Ca2+ and then subjected to repeated cycles in which it was reloaded to a set level (close to the endogenous level), exposed to a test treatment, and then fully depleted of Ca2+ again (see METHODS). Lowering the [Mg2+] from the standard level of 1 mM to 0.2 mM never induced a force response in any rat EDL fiber before exposure to a peptide (at least 2 repetitions in each fiber). In contrast, lowering the [Mg2+] to 0.2 mM after 20 s preequilibration in 30 µM AS(20) [previously (3) called peptide A] induced a substantial force response in every fiber examined (mean 40.3% of maximum Ca2+-activated force; Table 1). This stimulatory effect of AS(20) was not readily reversed by washout of the peptide (e.g., Fig. 2A and Table 1), even over several load/release cycles, indicating that some peptide continued to act on the stimulatory site on the release channels. Similar results were found in two other fibers when the voltage sensors were kept inactivated by having all solutions Na+ based [response at 0.2 mM Mg2+ in AS(20): 77% and 25% of maximum force in those fibers]. Peptide AC(20), the matching segment of the cardiac DHPR II-III loop (Fig. 1), did not cause any response at 0.2 mM Mg2+ (Table 1), but its COOH-terminal end [AC(10), 30 µM] triggered substantial Ca2+ release, with its effect being more easily reversed by washout than found with AS(20) (e.g., Fig. 2B).

                              
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Table 1.   Mean force response of rat EDL fibers on exposure to II-III loop peptides at 0.2 mM Mg2+



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Fig. 2.   Peptides AS(20) and AC(10) induce Ca2+ release in mechanically skinned mammalian fibers at 0.2 mM Mg2+. A: skinned extensor digitorum longus (EDL) fiber of the rat was subjected to repeated load-release cycles in which the sarcoplasmic reticulum (SR) was loaded with Ca2+ to a set level (close to the endogenous level) and then the fiber exposed to a solution with 0.2 mM free Mg2+ with or without peptide AS(20), before inducing release of the remaining SR Ca2+ (full release) with a 30 mM caffeine-low [Mg2+]-0.5 mM EGTA solution (see METHODS). There was never any force response at 0.2 mM Mg2+ before exposure to AS(20) (e.g., left), but a large response was induced in presence of 30 µM AS(20) (e.g., middle), and the effect of the peptide could not be readily reversed by washout (e.g., right). B: exposure to 30 µM AC(10) had an effect similar to AS(20), but this was more readily reversed by washout. C: there was no force response at 0.2 mM Mg2+ in the presence of 30 µM AS(10), but importantly the response on subsequently depleting the SR with 30 mM caffeine (full release) was markedly reduced, showing that the peptide was directly blocking the release channels. After washout of AS(10), the responsiveness to low [Mg2+] was heightened, indicating persistence of the activating effect of the peptide. Different EDL fibers in A, B, and C. Time scale: 2 s throughout, except during wash period between 0.2 mM Mg2+ and full release, where it is 30 s. Maximum Ca2+-activated force was ascertained in each fiber with a heavily buffered Ca2+ solution with 20 µM free Ca2+ (max).

In contrast to the case with AS(20), there was no significant force response at 0.2 mM Mg2+ in the presence of 30 µM AS(10), the COOH-terminal end of peptide AS(20) (Fig. 1; e.g., Fig. 2C and Table 1). However, when trying to trigger full release of SR Ca2+ (with the 30 mM caffeine-low [Mg2+] solution) straight afterward, the response was invariably much smaller than would have been expected considering the lack of Ca2+ release in the peptide (e.g., Fig. 2C, n = 6), and this almost certainly indicates that the peptide was blocking the release channels to some extent. Furthermore, when the treatment cycle was subsequently repeated in the absence of the peptide, the response to the full release solution was invariably much larger, and in three of the six cases there was also an appreciable response in 0.2 mM Mg2+ (e.g., Fig. 2C). The most straightforward explanation of these data is that AS(10) stimulates Ca2+ release at 0.2 mM Mg2+ but that this effect is countered by an additional blocking action of the peptide on the release channels (see Effects of AC(20) and 10 amino acid peptides at 1 mM Mg2+); on washout, the blocking action is more easily reversed, revealing the persistent activating effect of the peptide. A similar, though much smaller, inhibitory effect may also occur with peptide AS(20), because in some fibers that rate of rise of the force response in 0.2 mM Mg2+ was even faster after washout of the peptide than it was in the presence of the peptide (e.g., Fig. 2A). In summary, peptides AS(20), AS(10), and AC(10) [but not AC(20)] all evidently stimulated Ca2+ release at 0.2 mM Mg2+, with AS(10) also having a potent blocking effect. Thus these data indicate that the A peptides have Ca2+-releasing effects in skinned muscle fibers, in accord with their reported effects in isolated triads (3, 4).

Effect of peptide AS(20) on depolarization-induced responses in skinned fibers at 1 mM Mg2+. As described previously in both rat EDL fibers and toad iliofibularis fibers (10-13), the E-C coupling mechanism is retained in mechanically skinned fibers and is quite functional at the physiological intracellular [Mg2+] of 1 mM. The skinned fibers retain the endogenous level of SR Ca2+ and require no additional loading. The T-system seals over on skinning and repolarizes if the fiber segment is bathed in a high [K+] solution, and can then be rapidly depolarized by substituting a solution in which all K+ is replaced with Na+ (see METHODS). Such stimulation elicits a rapidly rising force response, reaching close to the Ca2+-activated maximum, and lasting for only 1-3 s owing to inactivation of the voltage sensors and reuptake of released Ca2+ into the SR (10, 11, 13) (e.g., Fig. 3). If after each depolarization the fiber is repolarized in the K+ solution for >= 30 s to reprime the voltage sensors, many similar depolarization-induced responses (~15 in toad fibers and 20-30 in rat EDL fibers) can be elicited before the fiber shows substantial rundown. The time course, voltage dependence of activation and inactivation, and modulation by pharmacological agents (e.g., D600) (10-13) all strongly indicate that the responses in these skinned fibers are controlled by the same basic E-C coupling mechanism as in intact fibers.


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Fig. 3.   Effect of peptide AS(20) on Ca2+ release in skinned fibers of the rat. Addition of 30 µM peptide AS(20) potentiated the force responses to depolarization (A and B) and caffeine application (B) in EDL fibers. C: in a soleus fiber, AS(20) also induced substantial spontaneous force responses lasting >20 s. Time scale in A and B: 2 s during brief depolarizations (by Na+ substitution) and caffeine application and 30 s between stimulation and during the prolonged depolarization (dashed line). Depol, depolarized.

Under such conditions, addition of 30 µM peptide AS(20) to the K+ (polarizing) bathing solution induced small, spontaneous force responses (1-6% of maximum Ca2+-activated force) lasting ~1-20 s, within 30 s to 2 min of application, in ~40% of cases in skinned EDL (fast-twitch) fibers of the rat (Table 2). The response to depolarization in the presence of peptide AS(20) was on average potentiated relative to preceding control responses in every fiber examined (13/13) (Table 2), reaching approximately the maximum Ca2+-activated level in each case. Broadly similar responses were obtained on successive (typically 3-6) depolarizations in the presence of the peptide (e.g., Fig. 3A). A similar potentiating effect was also seen with 5 µM peptide AS(20) (not shown; 110% and 104% of control response to depolarization, n = 2). Peptide AS(20) (30 µM) also potentiated the response to caffeine application (5 or 7 mM; e.g., Fig. 3B), with the mean force response increasing from 55 ± 6% to 92 ± 4% of maximum Ca2+-activated force in the three fibers examined; the peptide also caused similar potentiation of caffeine-induced responses when the voltage sensors were kept inactivated using Na+-based solutions (not shown). Ca2+ release induced by lowering the free [Mg2+] to 0.05 mM or below (12, 13) was also potentiated by AS(20) (30 µM; not shown). The potentiation of the force responses reflected increased Ca2+ release and not an effect on the properties of the contractile apparatus, because additional experiments with heavily buffered Ca2+ solutions (see METHODS) showed that neither the maximum Ca2+-activated force nor the Ca2+ sensitivity of the contractile apparatus was significantly altered in any of the three fibers examined.

                              
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Table 2.   Effect of II-III loop peptides 1) in inducing spontaneous Ca2+ release and 2) on the response to depolarization in skinned EDL fibers of the rat at 1 mM Mg2+

It was also of interest to examine the effect of AS(20) in soleus (slow-twitch) muscle fibers, where the ratio of RyR Ca2+-release channels to DHPR voltage sensors is reportedly higher than in EDL fibers (1), so even more than 50% of the release channels should not be directly coupled to DHPR molecules (see Introduction). AS(20) (30 µM) caused spontaneous Ca2+ release in three of the eight soleus fibers examined (e.g., Fig. 3C), inducing force responses of between 2 and 16% of maximum force (mean 7 ± 5%). In many cases, the soleus fibers did not respond to depolarization by Na+ substitution, possibly because the T-system was not well sealed or polarized (note that the fibers always responded well to direct activation of Ca2+ release by caffeine or low [Mg2+]). In the soleus fibers responding to depolarization, the first depolarization-induced response in 30 µM AS(20) was on average 156% of the preceding control response (50 ± 16% and 32 ± 13% of maximum Ca2+-activated force, with and without peptide, respectively, n = 4). Thus AS(20) caused comparatively greater potentiation of depolarization-induced responses in soleus fibers than in EDL fibers, although this predominantly reflects the relatively poor control response in soleus fibers rather than the effectiveness of the peptide per se. In every soleus fiber examined, peptide AS(20) (30 µM) potentiated the response to lowering [Mg2+] (to 0.015 mM) or applying 2 or 4 mM caffeine [mean response as a percentage of maximum force increasing from 61 ± 1% to 85 ± 2% (n = 3) and from 21 ± 15% to 35 ± 19% (n = 3) for low [Mg2+] and caffeine, respectively], showing that the peptide augmented direct stimulation of the Ca2+-release channels.

Effects of AC(20) and 10-amino acid peptides at 1 mM Mg2+. The effects of other A-region peptides were also examined in EDL fibers in the presence of physiological [Mg2+] (i.e., 1 mM). AS(10) induced much larger spontaneous force responses at 30 µM than did AS(20) (21% vs. 2% of maximum force; Table 2; e.g., Fig. 4), and the response to depolarization decreased greatly over the first few repetitions (e.g., Figs. 4 and 5A and Table 2), indicative of depletion of releasable Ca2+ and/or inhibition of release. AC(20), the cardiac DHPR loop peptide, had little if any effect, with only a single, brief spontaneous response being observed (after 3-min exposure) in one of six EDL fibers examined and with there being no potentiation of the depolarization-induced response (Table 2) or of the response to lowered [Mg2+] (not shown). In contrast, AC(10), the COOH-terminal end of AC(20), caused significant potentiation of depolarization-induced responses (e.g., Fig. 4), and its actions quantitatively resembled those of AS(20) (Table 2). Interestingly, the highly positive peptide, decalysine (30 µM; Fig. 1), did not cause any spontaneous responses or significantly alter the response to depolarization (Table 2; Fig. 5A).


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Fig. 4.   Effects of peptides AS(10) and AC(10) on depolarization-induced responses in a rat EDL fiber. Depolarization-induced responses were slightly potentiated in presence of 30 µM peptide AC(10) (top). Peptide AS(10) caused a progressive a decrease in the depolarization-induced responses in the same fiber (bottom). Washout of the peptide for 2 min and reloading (30 s) largely restored the response to depolarization and low [Mg2+]. Time scale: 2 s during depolarization (by Na+ substitution) and low [Mg2+] and 30 s elsewhere.



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Fig. 5.   Effects of decalysine and peptide AS(10) in a rat skinned fiber. A: depolarization-induced responses were little affected by decalysine (30 µM). In contrast, peptide AS(10) induced a spontaneous force response (shown on slow time scale) and a great reduction in the response to both depolarization and low [Mg2+]. B: trace from another EDL fiber showing that the response to low [Mg2+] was inhibited in the presence of AS(10) and could be largely restored simply by washing out the peptide. Time scale: 2 s during depolarization (by Na+ substitution) and low [Mg2+] (0.015 mM) and 30 s elsewhere.

We further examined the effects of AS(10) to identify the basis of its inhibitory action on depolarization-induced responses. Some of the progressive reduction in the response was probably due to SR depletion resulting from the Ca2+-releasing action of the peptide, because fibers showing a spontaneous response during the initial minute of exposure to 30 µM AS(10) (i.e., before the first depolarization) gave a significantly smaller response to the first depolarization (41 ± 9% of preceding control response, n = 4) than did fibers giving a spontaneous response afterward or not at all (78 ± 4% of control response, n = 6). It was also apparent that AS(10) had an inhibitory effect directly on the Ca2+-release channels, because the response to lowering [Mg2+] (3 fibers; e.g., Fig. 5B) or applying 10 mM caffeine (not shown, 2 fibers) was largely or completely suppressed in the presence of 30 µM AS(10) but could be restored simply by 30-60 s washout of the peptide without any reloading of the SR. Further experiments indicated that AS(10) also had a third type of effect. It was first shown that lower concentrations of AS(10) caused a less-marked decline in the depolarization-induced response: at 5 µM AS(10) there was very little spontaneous response (1% of control depolarization-induced response in 1 of 3 EDL fibers), and the response to depolarization declined to 94 ± 1% and 86 ± 4% of the control response on the first and third depolarizations, respectively (n = 3, P < 0.05; cf. Table 2). The falling phase of the depolarization-induced responses was unchanged in the presence of AS(10) (e.g., Fig. 6), which shows that the peptide did not cause any appreciable reduction in SR Ca2+ uptake. Experiments were then performed in which an EDL fiber was subjected to a second depolarization 10 s after each control response, to determine the extent to which the E-C coupling mechanism had reprimed in that time (e.g., Fig. 6). In the absence of any peptide, a fiber almost fully reprimed in 10 s, but in the presence 10 µM AS(10), the repriming was substantially slowed, with the response to the second depolarization 10 s later being only ~5% of the response to the first (e.g., Fig. 6). This effect could be reversed within 2 min by washout of the peptide. Similar results were found in all three fibers examined in this way with 5 or 10 µM AS(10). Thus part of the marked decline in the response to successive depolarizations (60 s apart) observed at 30 µM AS(10) (Table 2) is probably due to incomplete repriming of the voltage sensors.


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Fig. 6.   Peptide AS(10) slows repriming of depolarization-induced responses. This rat EDL fiber was subjected to pairs of depolarizing stimuli 10 s apart, with 60 s between successive pairs. Repolarizing the fiber in the K+ solution for 10 s resulted in almost full repriming of the response when no peptide was present. In the presence of 10 µM AS(10) there was little response to depolarization following 10 s repriming in K+, indicating that the peptide slowed repriming. Fiber depolarized in Na+ solution where "Depol" bar is shown and repolarized in K+ solution where bar is absent. Responses shown for fourth pair of stimuli in presence of peptide (4 min total exposure) and second after washout (2 min total). Time scale: 2 s throughout.

The effect of a very high concentration of AS(10) was also examined in an attempt to get the peptide to all sites within a fiber as quickly as possible. At 200 µM, peptide AS(10) elicited a single, comparatively large spontaneous force response (~28 ± 9% of maximum force) within 40 (±13) s in 6 of the 11 rat EDL fibers examined, after which the response to depolarization was virtually completely abolished (e.g., Fig. 7A). In the fibers not giving any spontaneous force response in the peptide (5 of 11), the response to depolarization was similarly abolished (mean response 2 ± 1% and 0% of maximum Ca2+-activated force on first and third depolarizations, n = 11). When the response to depolarization had been completely abolished by the presence of 200 µM peptide AS(10), lowering the [Mg2+] to 0.015 mM only induced a small force response (6 ± 5% of maximum force) in the seven fibers examined, and exposure to 10 mM caffeine similarly elicited little or no response (0 and 2%) in two other fibers (e.g., Fig. 7A). The total Ca2+ content of these fibers was then assayed (6, 24), revealing that there was a considerable amount of Ca2+ in stores within the fiber in every case. The assay involved briefly equilibrating the fiber in a solution with a particular concentration of a Ca2+ buffer (0.55 mM EGTA) and then lysing all the membranous compartments by moving the fiber into a Triton X-100/paraffin oil emulsion. As the force response in all six fibers examined reached the maximum possible in the Triton/oil environment (~50% of maximum in aqueous environment) (6, 24) (e.g., Fig. 7A), it was apparent that the SR Ca2+ content must have exceeded the capacity of the exogenous and nondiffusible endogenous Ca2+ buffers and hence was >= 0.7 mM (expressed as mmol/l fiber volume; see Refs. 6 and 24). [Note that the endogenous total Ca2+ content of rat EDL fibers is ~1 mM (6), and a content of 0.7 mM Ca2+ is normally sufficient for depolarization to induce at least 80% of the control depolarization-induced force response (24).] Thus it is apparent that 1) relatively little Ca2+ could have been lost from the SR in the period following the single spontaneous force response in 200 µM peptide AS(10), and 2) the Ca2+ release channels had become unresponsive to both T-system depolarization and direct stimulation with caffeine and low [Mg2+].


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Fig. 7.   Effects of high concentration of peptide AS(10) in primed and inactivated rat EDL fibers. A: addition of 200 µM peptide AS(10) to a fiber primed in the standard K+ solution induced a relatively large spontaneous force response after 30 s, following which neither depolarization nor 10 mM caffeine could elicit any response. Fiber was then equilibrated in a solution with 0.55 mM free EGTA (15 s) and lysed in an emulsion of Triton X-100 and paraffin oil (tx/oil) to release all Ca2+ in the fiber. The resulting force response indicated that there was at least 0.7 mM total Ca2+ in the fiber at the time of lysing, which implies that the peptide had blocked Ca2+ release and not simply depleted the SR of Ca2+. Time scale: 2 s during depolarization and exposure to caffeine, and 30 s elsewhere. B: when another EDL fiber was depolarized by Na+ substitution and then kept in that solution for 1 min to ensure the voltage sensors were inactivated, addition of 200 µM peptide AS(10) induced a spontaneous force response after ~30 s. When the fiber was returned to the K+ (polarizing) solution, a further force response was elicited, possibly indicating that the peptide had not exerted its full stimulatory effect during the period in Na+ solution. Time scale: 2 s during initial period of depolarization (continuous bar) and 30 s elsewhere (including dashed bar).

To determine whether the peptide was inducing spontaneous force responses by directly activating the RyRs or simply by depolarizing the T-system, we examined the effect of the peptide when the voltage sensors were inactivated. Each fiber was depolarized by Na+ substitution, generating a transient force response as usual, but then the fiber was kept in the Na+ solution for 1 min to ensure that the voltage sensors were inactivated (10, 11, 13), before substituting a similar solution with 200 µM peptide AS(10). In two of the five fibers examined (e.g., Fig. 7B), a spontaneous force response (~5% of maximum force) was elicited ~40 s after peptide application; this response was considerably smaller than that seen when the peptide was added to fibers normally polarized in the K+ solution (see above), although the response was substantially more prolonged (~20 s in Na+ vs. ~2-3 s in K+). In one case when the fiber was returned to the K+ solution, an additional spontaneous force response was elicited (e.g., Fig. 7B), probably indicating that the peptide was better able to elicit Ca2+ release when the fiber was in the K+ solution than in the Na+ solution (as has been observed in SR vesicle studies; Ref. 20). In summary, it was apparent that the peptide could elicit Ca2+ release even when the voltage sensors were inactivated, indicating that the peptide must have had a direct stimulatory effect on the RyRs.

Effect of peptide CS. El-Hayek et al. (3) have reported that peptide CS (Fig. 1), another segment of the II-III loop of the alpha 1-subunit of the skeletal muscle DHPR (previously called peptide C), did not elicit Ca2+ release or increase ryanodine binding in skeletal muscle triads, and in fact antagonized the ability of both AS(20) (3) and depolarization (28) to activate Ca2+ release (at 0.2 mM Mg2+). In seeming contrast, experiments with chimeras of cardiac and skeletal DHPRs expressed in dysgenic muscle indicated that this section of the skeletal II-III loop was essential for inducing normal skeletal-type activation of the Ca2+-release channels (22). In the experiments here, peptide CS (30 or 100 µM) did not induce any spontaneous response in any rat EDL fiber examined or cause any potentiation of Ca2+ release (Table 2) or evidence of SR Ca2+ depletion. At 100 µM, peptide CS did, however, cause significant inhibition of depolarization-induced responses (e.g., Fig. 8 and Table 2). This inhibitory effect could be reversed by washout in some fibers (e.g., Fig. 8B), although in most fibers it was not well reversed (e.g., Fig. 8A). The large response to low [Mg2+] at the end of the trace in Fig. 8A shows that the release channels were not noticeably blocked by the exposure to peptide CS (also see below) and that the SR was still loaded with Ca2+. Experiments in which fibers were subjected to pairs of depolarizations 10 s apart (as in Fig. 6) showed no significant difference in the repriming rate in the absence or presence of 100 µM CS (n = 7). The presence of 100 µM CS also resulted in a significantly smaller force response to 30 µM AS(20) at 0.2 mM Mg2+ (24.0.% vs. 40.3%, P < 0.05; Table 1). [Note that treatments were compared across different fibers because it was not possible to meaningfully compare the effects of the two treatments in the same fiber, as the effect of peptide AS(20) could not readily be reversed by washout.] The inhibitory effects of peptide CS on the response to depolarization and peptide AS(20) could not be due to some generalized blocking action on the release channels, because the response to 7 mM caffeine was unaffected by the presence of the peptide (32.8 ± 10.4% and 36.4 ± 7.7% of maximum force, with and without 100 µM CS in 3 EDL fibers).


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Fig. 8.   Peptide CS partially inhibits depolarization-induced responses in rat EDL fibers. A: on initial exposure to 100 µM peptide CS, depolarization-induced responses decreased substantially and did not recover on washout of the peptide. A further small but reversible degree of inhibition was seen on reapplication of the peptide. Fiber broke on maximum activation. B: example of fiber showing reversible partial inhibition of responses on first exposure to peptide CS. Time scale: 2 s during depolarization and low [Mg2+] exposure and 30 s elsewhere.

Effects of peptides in toad fibers. When 30 µM peptide AS(20) was applied to skinned iliofibularis fibers of the toad at 1 mM Mg2+, in 3 of 12 cases it elicited spontaneous force responses that were 2-16% of maximum force and lasted up to 20 s (e.g., Fig. 9A, note slow time scale), showing that the peptide must have triggered appreciable Ca2+ release (24). The force response to depolarization decreased rapidly over the first few depolarizations in AS(20), dropping to 79 ± 9% and 19 ± 4% of the preceding control level on the first and third depolarizations, respectively (n = 12). In general, the actions of AS(20) in toad fibers more closely resembled those of AS(10) in rat EDL fibers, rather than those of AS(20) (cf. Table 2). AS(20) caused evident depletion of SR Ca2+ in some fibers (e.g., Fig. 9B). AS(10) (at 30 µM) caused more inhibition of depolarization-induced responses than did AS(20) (27 ± 15% and 0% of control on first and third depolarizations, n = 5), and AC(10) and decalysine had qualitatively similar, although smaller effects. In contrast to the case with rat EDL fibers, decalysine (30 µM) induced spontaneous Ca2+ release in all toad fibers examined (7 ± 2% of maximum force, n = 4).


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Fig. 9.   Effect of peptide AS(20) on Ca2+ release in toad skinned fibers. A: addition of 30 µM peptide AS(20) in this iliofibularis fiber evoked substantial spontaneous Ca2+ release lasting over 20 s, caused a progressive decrease in the response to depolarization, and reduced responses to low (0.05mM) [Mg2+] and to 30 mM caffeine. B: in a different fiber, the force response to depolarization was progressively lost after addition of peptide AS(20) (30 µM) but was restored over 3-6 min by washing out the peptide and repeatedly reloading the SR with Ca2+ (1-min periods in pCa 6.0; see METHODS). The progressive decrease in the depolarization-induced force responses evident after the second load period indicates that the SR was leaking an appreciable amount of Ca2+ at that time. Time scale in A and B: 2 s during depolarization and exposure to low [Mg2+] and caffeine and 30 s between stimulation including periods of spontaneous response (spont. resp.).

Finally, peptide CS at 30 µM did not elicit any spontaneous Ca2+ release in any of the 7 toad fibers examined or cause any apparent SR depletion or inhibition of depolarization-induced responses (response to first and third depolarizations in peptide: 97 ± 4% and 105 ± 4% of preceding control in 7 fibers). These results are similar to the findings with rat fibers at such a concentration. The effects of higher concentrations of peptide CS were not examined.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of A peptides in skinned fibers. It is presently not fully understood how the DHPRs in the T-system regulate the Ca2+-release channels in the SR. Studies in myotubes with DHPR chimeras have indicated that the II-III loop of the alpha 1-subunit of skeletal muscle DHPR plays an essential role in the coupling process (33, 34). Other recent studies have shown that II-III loop peptides of skeletal or cardiac DHPR can directly activate the Ca2+-release channel in isolated SR and bilayer preparations (2-4, 16, 17). Here we examined the effects of particular peptides on Ca2+ release in a skinned fiber preparation that retains E-C coupling and in which important factors, such as myoplasmic [Mg2+], [ATP], and SR Ca2+ loading, were kept close to physiological levels. In this way we could test which, if any, of the peptides were capable of eliciting (or interfering with) Ca2+ release under conditions approximating those in vivo, with the assumption that the key activation site(s) would be accessible in at least one-half of the release channels (see Introduction). It was found that 30 µM peptide AS(20), which has been previously shown to activate Ca2+ release channels in isolated triads and bilayers (2-4), induced only a small degree of Ca2+ release in skinned fibers with functional coupling in the presence of physiological [Mg2+] (1 mM) (Figs. 3 and 9 and Table 2), although it potentiated the response to both voltage sensor activation and caffeine application (Fig. 3). AS(20) apparently had access to the activation site(s) on many RyRs, because it potently induced Ca2+ release in the presence of a lower [Mg2+] (0.2 mM; Table 1). The inhibitory effect of 1 mM Mg2+ observed in this study is not in conflict with the finding that As(20) could activate isolated RyRs in bilayers in the presence of 2 mM Mg2+ (2), because the latter experiments were performed in the presence of a high [Cl-] that reduced the affinity of the Ca2+/Mg2+ inhibitory site for Mg2+ by fourfold or more compared with the conditions used here (15, 20).

Peptide AS(10), the COOH-terminal end of peptide AS(20) (Fig. 1), had considerably greater effects on Ca2+ release than did AS(20) at the same concentration (30 µM), inducing substantial spontaneous force responses (~20% of maximum force) in the majority of cases (Fig. 5A and Table 2). The spontaneous force responses were only slightly larger with 200 µM AS(10), but very much smaller at 5 µM. The ability of 200 µM AS(10) to elicit a single, comparatively large spontaneous force response, and yet not cause radical depletion of SR Ca2+ after application for several minutes (Fig. 7A), indicates that at later times the peptide no longer potently activated the RyR. This must have been due at least in part to a separate inhibitory action of the peptide on the RyR (see later), because the peptide clearly interfered with Ca2+ release to caffeine and lowered [Mg2+] (e.g., Figs. 2C, 5B, and 7A). Peptide AS(10) evidently also interfered with repriming of the E-C coupling mechanism, possibly because it affected the T-system potential (see later). Such an action, however, could not account for the ability of AS(10) to induce spontaneous Ca2+ release when the voltage sensors were inactivated (Fig. 7B), showing that the peptide must have had some relatively direct stimulatory effect on the RyR itself. The fact that the spontaneous force responses were larger when a fiber was kept polarized in the K+ solution than when inactivated in the Na+ solution (e.g., Fig. 7) is probably due in part to the apparently greater level of RyR activation that can be achieved in the presence of K+ compared with Na+ (Fig. 2B in Ref. 20), although the peptide's possible depolarizing action may have also contributed to the difference.

In contrast to AS(20) and AS(10), peptide AC(20), which is the segment of the cardiac DHPR II-III loop corresponding to AS(20) (Fig. 1), had little effect on Ca2+ release, but its COOH-terminal end, AC(10), had somewhat greater effect, potentiating Ca2+ release in a manner comparable with AS(20) (Table 2). AC(10), like AS(20), induced considerable Ca2+ release at 0.2 mM Mg2+ (Fig. 2, Table 1), and AS(10) was also evidently stimulatory, although this effect was confounded by the peptide's additional inhibitory action (Fig. 2C). Truncating AS(20) to AS(10), or AC(20) to AC(10), removes negatively charged amino acids closest to the region of greatest density of positively charged amino acids (Fig. 1). It is also noteworthy that the ability of the peptides to induce Ca2+ release in rat fibers is in the same rank order as the net positive charge of consecutive stretches of charged amino acids [AS(10) (5+) > AS(20) (3+) approx  AC(10) (3+) > AC(20) (1-)]. This could mean that the activity of the peptides is determined in large part by the presence of a region of positively charged amino acids, as suggested by Zhu et al. (36). This could not be the only important factor, however, because decalysine, which has an even higher positive charge than peptide AS(10), had little if any effect in rat fibers (Fig. 5A and Table 2), just as the peptide RKRRK had little effect in isolated triads (4). Thus it seems that other factors in addition to charge distribution influence the ability of the peptides to induce Ca2+ release (4).

Inhibitory effects of AS(10). In addition to its stimulatory effect, AS(10) had a direct inhibitory effect on Ca2+ release (Figs. 2C, 5B, and 7A). This dual action is similar to that observed when applying AS(20) to Ca2+ release channels in bilayers (2). The inhibitory effect in that study was greatest for potentials that induced movement of the positively charged peptide into the channel, and the results were consistent with the peptide blocking the channel pore (2), as has been found with other positive peptides (18). We also note that Dulhunty et al. (2) did not detect any such inhibitory effect of AS(20) when inducing Ca2+ release from SR vesicles, which is consistent with the lack of any marked inhibition with 30 µM AS(20) in this study. Thus it seems that the release channels are not as susceptible to block by the peptides under physiological conditions where Ca2+ is flowing out of the SR down its electrochemical gradient. Nevertheless, it was evident that, when the more strongly charged peptide AS(10) was present at high enough concentrations (>= 30 µM), Ca2+ release did become inhibited. This may have been due to blockage of the channel pore (and possibly only occurs on channel opening). Alternatively, the peptide may have inhibited the release channel by acting at a modulatory site on its cytoplasmic face. Many divalent and polyvalent cations have been shown to act at a low-affinity Ca2+/Mg2+ inhibitory site, with the half-inhibitory concentration being ~1 µM for trivalent metal cations (and ruthenium red) and ~100 µM for divalent metal cations (e.g., Ca2+ or Mg2+) in skeletal RyRs, and ~10-fold higher in cardiac RyRs (14, 15, 19, 20, 31). The peptides may also have such an action as ryanodine binding and Ca2+ release in skeletal muscle triad decrease with >30 µM AS(20) or AS(10) and >10 µM polylysine, with there being little or no inhibition in cardiac RyRs, even with 100 µM polylysine (4). Certainly, a pore-blocking effect alone would not seem to account for the much greater inhibitory effect of the peptides on skeletal compared with cardiac RyRs (4). A peptide corresponding to the COOH-terminal region of the DHPR also inhibits RyR activation (30), and, given that this region also has concentrations of positively charged residues, its actions may be similar to those of AS(10).

Irrespective of exactly how the peptides inhibit the RyRs, it is noteworthy that this effect of AS(10) could be reversed within ~30 s by washout of the peptide (e.g., Fig. 5B), revealing a persistent activating effect of the peptide (Fig. 2C). This too is in good agreement with the finding that isolated RyRs were actually more active after washout of AS(20) than in its presence, which was interpreted as meaning that the activating effect of the peptide was less easily reversed by washout than the inhibitory effect of the peptide (2).

It also appeared that AS(10) had an additional inhibitory action, in which it slowed repriming of the E-C coupling mechanism (e.g., Fig. 6). The very small response after 10 s repriming compared with 60 s repriming is not readily explained by direct inhibition of the release channel. Such slow repriming would be expected if the T-system was depolarized to some extent by the presence of AS(10), and this might arise from the peptide-blocking K+ channels in the T- system membrane, given that AS(10) in many ways resembles K+ channel inactivation peptides, which act from the cytoplasmic side and have a strongly positively charged region and an adjacent hydrophobic region (18). Alternatively, it is possible that AS(10) slowed repriming of the voltage sensor/DHPRs by acting directly on those molecules or by interfering with their function via their coupling with the release channels.

Effects of peptide CS. Peptide CS, which corresponds to a part of the skeletal DHPR II-III loop thought to be critical for initiating normal skeletal-type coupling (22), never induced detectable Ca2+ release or evidence of SR depletion in any rat or toad fiber (e.g., Fig. 8 and Table 2). In fact, peptide CS (at 100 µM) caused partial inhibition of both depolarization-induced responses (e.g., Fig. 8 and Table 2) and peptide AS(20)-induced Ca2+ release (Table 1). These effects were not due to some generalized inhibitory action on the Ca2+ release channels, because the response to caffeine (7 mM) was not affected by the presence of peptide CS. Furthermore, the inhibition of depolarization-induced responses was not due to peptide CS in some way depolarizing the T-system, because the repriming rate was not noticeably altered. These results with peptide CS are in good agreement with the findings in triads (3, 28); the extent of the inhibition is smaller in the present study, but this is probably largely the result of quantifying responses in terms of peak force [which is indicative of total Ca2+ release (24)], rather than the rate of Ca2+ release (3, 28). Diffusional limitations would also have been greater in the skinned fibers than in the isolated triads. The fact that peptide CS interfered with activation by both the normal E-C coupling mechanism and the application of AS(20) is consistent with there being some commonality in the two methods of activation (3, 4) but does not mean that the two are identical. It is possible that exogenously added peptide CS occludes a common binding site on the release channel that is critical to both E-C coupling and peptide A activation. Given that even the first depolarization-induced response in the presence of peptide CS was reduced (Fig. 8 and Table 2), it seems that peptide CS exerted its inhibitory action either without the voltage sensors having to be activated or extremely quickly on such activation. The exact basis of the inhibitory effect of CS, however, is not apparent.

Relative effect of peptides in toad fibers. The A peptides, which correspond to segments of the mammalian DHPR II-III loop, also induced Ca2+ release in toad muscle fibers, with the extent of spontaneous release being, if anything, somewhat larger than in EDL fibers (e.g., Fig. 9A). This could reflect greater potency of the peptides on either the alpha - or the beta -isoform of the RyR present in amphibian muscle (23) or that the peptides activate the alpha -isoform, which in turn triggers substantial Ca2+-induced Ca2+ release through the beta -isoform. It is also likely that the toad fibers show greater net release and depletion of SR Ca2+ than rat fibers because the Ca2+ pump in the SR is less able to recover released Ca2+, given that we find that the rat fibers can refill the SR about twice as fast as can the toad fibers at the same [Ca2+] (e.g., at pCa 6.7 or 6.0) (unpublished observations). It is pertinent that the mammalian A peptides have stimulatory effects in toad fibers, because the amphibian skeletal DHPR II-III loop, which presumably physically activates the alpha -RyR in vivo, has A and C regions with high homology and identical charge distribution compared with the mammalian skeletal counterparts (35). In fact, the A region in frog (TSAQKAKAEERKRKKLARAN) closely resembles a chimera of AS(20) with the COOH-terminal end of AC(20) (cf. Fig. 1). The fact that the A region of the mammalian II-III loop triggers some Ca2+ release in toad fibers much as it does in mammalian fibers under the same conditions suggests that there is a binding site for the A peptide on the RyRs in amphibia (presumably on the alpha -isoform) similar to that on RyR1.

Relevance to E-C coupling. The ability of the A region to trigger Ca2+ release under some circumstances and the fact that it, like normal E-C coupling, is partially inhibited by peptide CS could suggest that the A region plays a role in normal E-C coupling. Movement of the A region of the II-III loop would be consistent with a number of other findings. It has been found that heparin, a strongly negatively charged molecule that binds to the A region of the II-III loop (8), interferes with E-C coupling in toad skinned fibers if and only if the DHPR/voltage sensors are activated in its presence (9). This could mean that that region of the loop only moves into the triadic gap when the DHPRs are activated, and this would also be consistent with other studies indicating that the associated serine residue (Ser687) is more readily phosphorylated if the DHPRs are repetitively activated (26, 29). In addition, it has also been shown that E-C coupling involves a nonlinear surface potential change associated with the DHPRs, and one possible cause of this could be the exposure of positive charges on the intracellular face of the DHPR that were occluded in the resting conformation (7). Thus there are a number of different lines of evidence that are consistent with DHPR/voltage sensor activation involving the movement of the A region of the II-III loop. Nevertheless, we point out that this does not mean that the A region itself initiates Ca2+ release.

It is possible that the ability of the A peptide to activate the release channels is unrelated to the normal E-C coupling mechanism, perhaps resulting from peptide binding to a region of the RyR that is normally inaccessible to the intracellular loop of the DHPR. Importantly, it was found here that the A peptides induced only a very small degree of Ca2+ release in the skinned fibers at physiological [Mg2+] (1 mM), with appreciable release only occurring at a substantially lower [Mg2+] (Table 1). The rate of Ca2+ release in the fibers here at 1 mM Mg2+ was probably underestimated owing to the occurrence of simultaneous Ca2+ reuptake into the SR, but nevertheless it must still have been far lower than during normal E-C coupling. As the skinned fibers evidently have functional E-C coupling, it cannot be argued that peptides are comparatively ineffective simply because some cofactor normally present in vivo has been lost. This lack of effectiveness of AS(20) at physiological [Mg2+] may imply that normal E-C coupling involves some different or additional action to the binding of AS(20) to the release channel. Alternatively, it is possible that the applied peptides are acting at the same site on the RyR as the II-III loop does in vivo, but that the exact interaction is not the same because the peptide is not held in its normal orientation or conformation by structural connections with the rest of the DHPR. It may be a relatively rare occurrence for loop peptides to be simultaneously bound to each of the four subunits of a RyR tetramer, and, if this is a prerequisite for channel opening, it could explain why the peptides only rarely induced substantial Ca2+ efflux. Here it is noteworthy that single cyclic nucleotide-gated (CNG) channels, which also have a tetrameric structure, are activated in a highly nonlinear manner with increasing levels of bound ligand, having an open probability of 0, 0.01, 0.33, and 1.0 with 1, 2, 3, and 4 bound nucleotides, respectively (27). Furthermore, when fewer than four ligands were bound, the CNG channels showed substate activity (27) that seems highly comparable to that observed with AS(20) stimulation of isolated RyRs (2). Thus the poor ability of A peptides to induce Ca2+ release in the skinned fibers at physiological [Mg2+] could simply be due to such highly nonlinear activation behavior. Another possibility is that lateral interactions between adjacent RyRs result in the array of RyRs acting in concert, which could make exogenously applied peptides a less effective stimulus than synchronously activating all the DHPRs facing the RyR array, as presumably happens in vivo. Finally, we cannot exclude the possibility that the applied A peptides fail to reach the activating site on even the uncoupled RyRs, although it is evident that AS(20) must have reached stimulatory sites on some RyRs because it was effective at inducing Ca2+ in 0.2 mM Mg2+ (Fig. 2A and Table 1).

The finding that peptide CS did not induce any detectable Ca2+ release in the fibers here does not readily fit with that region of the II-III loop having a direct stimulatory effect on the release channels, as would have been expected from the simplest interpretation of the results of experiments with DHPR chimeras (22). Because peptide CS actually interfered with the coupling mechanism, it is apparent that this peptide must have had access to at least some regions of the DHPR-RyR complex within the triad junction, presumably including the putative coupling site(s) on at least half of the RyRs (5). It is possible that the ability of CS to interfere with the coupling mechanism was due to the exogenous peptide competing with the in situ C-region peptide for the activation site, but with the exogenous peptide being poorly stimulatory. Alternatively, binding of the CS region may not be sufficient by itself to fully stimulate the Ca2+ release channel, even though it may be a prerequisite for proper coupling.

One possibility is that normal E-C coupling involves some coordinated movement or interaction of both the C and A regions of the II-III loop, as suggested previously (28). This could be consistent with the finding that a chimera of the cardiac DHPR with the skeletal CS region was able to give skeletal-type E-C coupling (22), because it was found that AC(10) could also induce Ca2+ release under some circumstances (Tables 1 and 2 and see Ref. 4). Another perhaps related possibility is that the peptide C region has a repulsive action on the RyR that destabilizes the closed state of the channel, with this interaction only being possible because the DHPR and RyR are held in fixed apposition in situ (5).

In conclusion, this study has shown that the A and C regions of the DHPR II-III loop have activating and inhibitory actions, respectively, on Ca2+ release in functioning muscle fibers, showing that these regions may be important in normal E-C coupling.


    ACKNOWLEDGEMENTS

We are grateful to Maria Cellini and Aida Yousef for technical assistance.


    FOOTNOTES

This work was supported by the National Health and Medical Research Council of Australia (Grant 9936582) and by grants from the National Institutes of Health.

Address for reprint requests and other correspondence: G. D. Lamb, School of Zoology, La Trobe Univ., Bundoora, Victoria, 3083 Australia (E-mail: zoogl{at}zoo.latrobe.edu.au).

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

Received 28 December 1999; accepted in final form 11 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Delbono, O, and Meissner G. Sarcoplasmic reticulum Ca2+ release in rat slow- and fast-twitch muscles. J Membr Biol 151: 123-130, 1996[ISI][Medline].

2.   Dulhunty, AF, Laver DR, Gallant EM, Casarotto MG, Pace SM, and Curtis S. Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12. Biophys J 77: 189-203, 1998[Abstract/Free Full Text].

3.   El-Hayek, R, Antoniu B, Wang J, Hamilton SL, and Ikemoto N. Identification of calcium release-triggering and blocking regions of the II-III loop of the skeletal muscle dihydropyridine receptor. J Biol Chem 270: 22116-22118, 1995[Abstract/Free Full Text].

4.   El-Hayek, R, and Ikemoto N. Identification of the minimum essential region in the II-III loop of the dihydropyridine receptor alpha 1 subunit required for activation of skeletal muscle-type excitation-contraction coupling. Biochemistry 37: 7015-7020, 1998[ISI][Medline].

5.   Franzini-Armstrong, C, and Jorgensen AO. Structure and development of E-C coupling units in skeletal muscle. Annu Rev Physiol 56: 509-534, 1994[ISI][Medline].

6.   Fryer, MW, and Stephenson DG. Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle. J Physiol (Lond) 493: 357-370, 1996[Abstract].

7.   Jong, D-S, Stroffekova K, and Heiny JA. A surface potential change in the membranes of frog skeletal muscle is associated with excitation-contraction coupling. J Physiol (Lond) 499: 787-808, 1997[Abstract].

8.   Knaus, HG, Scheffauer F, Romanin C, Schindler H-G, and Glossmann H. Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. J Biol Chem 265: 11156-11166, 1990[Abstract/Free Full Text].

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