INTRODUCTION

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Contraction of striated muscle depends on Ca²⁺ release from the terminal cisternae (TC) of sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) calcium release channels. During excitation-contraction coupling (EC coupling) in skeletal muscle, an L-type Ca²⁺ channel (dihydropyridine receptor, DHPR) senses T-tubule depolarization and transmits an activating signal to the RyR. The signal is transmitted via a protein-protein interaction which requires the loop between the second and third repeats of the skeletal DHPR ₁ subunit (II-III loop, Tanabe et al., 1990). A region important for binding of the II-III loop is located on the skeletal RyR between Arg¹⁰⁷⁶ and Asp¹¹¹² (Leong and Maclennan, 1998). Functional interactions between the skeletal DHPR II-III loop and skeletal RyRs have been described. For example, the II-III loop increases [³H]ryanodine binding to SR vesicles, and activates purified RyR channels in lipid bilayers (Lu et al., 1994, 1995). A short peptide, corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ of the skeletal DHPR II-III loop (peptide A), activates Ca²⁺ release from SR, (El-Hayek et al., 1995; El-Hayek and Ikemoto, 1998; Dulhunty et al., 1999). At 40 mV, peptide A (10 nM) activates rabbit skeletal RyRs, but at +40 mV, peptide A (1 µM) inhibits channels because the positively charged peptide blocks the channel pore (Dulhunty et al., 1999). A different II-III loop peptide, peptide C (724-760) inhibits activation of Ca²⁺ release from SR by peptide A (El-Hayek et al., 1995; El-Hayek and Ikemoto, 1998).

Because of the ability of the skeletal II-III loop and peptide A to activate RyRs, it has been suggested that binding of the A region of the DHPR to the RyR is a step in EC coupling (El-Hayek et al., 1995; El-Hayek and Ikemoto, 1998). Although the A region peptide is undeniably a high affinity activator of the RyR, its precise role in EC coupling remains to be determined. Skeletal EC coupling occurs when the A region contains either a skeletal, cardiac, or scrambled sequence; skeletal EC coupling cannot proceed unless the II-III loop contains a skeletal sequence between residues 725 and 742 (Nakai et al., 1998; Proenza et al., 2000), which is encompassed in peptide C. Curiously, the cardiac DHPR II-III loop also increases the activity of skeletal RyRs (Lu et al., 1994, 1995). Therefore, perhaps not surprisingly, binding of the II-III loop, or the 20-amino acid peptide A, has less rigid isoform specificity than the full EC coupling process. The experiments with dysgenic myocytes (Nakai et al., 1998; Proenza et al., 2000) do not give quantitative or kinetic information about EC coupling. It is possible that signal transmission during normal skeletal EC coupling occurs through both the A and C regions, but that some Ca²⁺ release from the SR can be induced via the C region alone. The functional properties of the A peptide continue to be investigated (i) because it is a high affinity specific activator of the RyR and thus useful for probing RyR function and (ii) because the A region may play a role in EC coupling.

The skeletal RyR is altered in a potentially lethal manner by approximately half of the mutations that lead to susceptibility to malignant hyperthermia (MH; Loke and Maclennan, 1998; Jurkat-Rott et al., 2000). In humans, 22 MH mutations have been identified at 20 different amino acid residues in the RyR and two MH mutations at one residue in the DHPR (Jurkat-Rott et al., 2000). A single amino acid substitution of Arg⁶¹⁴ to Cys is responsible in 4% of MH-susceptible humans, and the same substitution at residue 615 of the RyR is found in all MH-susceptible pigs. Fifteen MH-linked RyR mutations are associated with an increased sensitivity to caffeine-induced Ca ²⁺ release when recombinant DNA for the mutant RyRs was expressed in heterologous systems (Tong et al., 1997). Only two identified mutants have been studied in detail in muscle cells and/or muscle-derived preparations: arg⁶¹⁵cys in pigs and gly²⁴³⁴arg in humans. A comparison of these mutants demonstrated remarkable similarities in RyR abnormalities, including enhanced sensitivity to both caffeine and calcium (Richter et al., 1997). Porcine MH SR vesicles are characterized by a greater than normal rate of Ca²⁺-induced Ca²⁺ release (Kim et al., 1984); MH RyR channels have reduced inhibition by millimolar concentrations of Ca²⁺ (Fill et al., 1991; Shomer et al., 1993) or Mg²⁺ (Laver et al., 1997). Both of these abnormalities would presumably lead to enhanced Ca²⁺ release from SR during EC coupling (Ohta et al., 1989) and increased force production (Gallant et al., 1980) in MH muscles. Whether or not resting cytoplasmic [Ca²⁺] is increased in MH muscles remains controversial (Mickelson and Louis, 1996), and resting [Ca²⁺] is not elevated when RyRs with MH mutations are expressed in heterologous systems (Otsu et al., 1994; Tong et al., 1997, 1999). Such an elevation could indicate greater than normal Ca²⁺ leak from the SR of MH-susceptible muscles.

It has been suggested that reduced Mg²⁺ inhibition could be responsible for a higher resting cytoplasmic [Ca²⁺] and enhanced EC coupling in muscle from MH-susceptible pigs (Laver et al., 1997). It is thought that RyRs are chronically inhibited by Mg²⁺ under resting conditions, and EC coupling proceeds after a decrease in the affinity of RyRs for Mg²⁺ (Lamb and Stephenson, 1991). Although the reduced Mg²⁺ inhibition explains many of the effects of the Arg⁶¹⁵Cys substitution on EC coupling, other aspects of RyR_MH activity relevant to EC coupling have not been examined. Therefore, we have compared the effects of peptide A on the activity of normal RyRs and on RyRs from MH-susceptible pig muscle.

We find that peptide A activation of Ca²⁺ release from SR of MH-susceptible pigs is eightfold greater than that from SR of normal pigs. The enhancement of single RyR channel activity, at 40 mV, by peptide A is also two- to fourfold greater in RyR_MH as compared to RyR_N. This activation is specific for the native sequence of peptide A. Activation is not seen with peptide NB, corresponding to a sequence in a different region of the skeletal II-III loop, whereas lesser activation is seen with a scrambled sequence of the peptide (peptide AS). Inhibition of RyR channels at +40 mV by peptide A and peptide AS is also enhanced in RyR_MH. The results show that the point mutation in MH can have multiple effects on RyR activity. It is possible that stronger binding of the peptide A region of the DHPR to the RyR_MH may contribute to greater Ca²⁺ release from the SR during EC coupling in MH-susceptible muscle.

	METHODS

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Materials

Chemicals and biochemicals were from Sigma-Aldrich (Castle Hill, Australia). DHPR II-III loop peptide synthesis has been described previously (Dulhunty et al., 1999). Peptides were synthesized with purification to 98 to 100% using HPLC, mass spectroscopy, and NMR. Stock peptide solutions (~2 mM) were prepared in H₂O and frozen in aliquots of 20 µl. Precise stock solution concentrations were determined by Auspep Proprietry Ltd. (Melbourne, Australia). Peptides used in this study were:

Peptide A:

Peptide NB:

Peptide AS:

Biological material and caffeine-halothane contracture test for MH susceptibility

The methods for genetic testing, anaesthetic techniques, muscle dissection, caffeine-halothane contracture testing, preparation of SR vesicles, and single channel recording have been described previously (Otsu et al., 1992; Owen et al., 1997; Laver et al., 1997). Muscle and blood samples were obtained from 3 homozygous normal pigs (1 Belgium Landrace and 2 Landrace) and 3 homozygous MH pigs (2 Belgium Landrace and 1 Landrace) aged ~4 months. Each animal was genetically tested for normal or MH RyR allele containing either Arg⁶¹⁵ or Cys⁶¹⁵. The SR preparations were from the same animals as those used in Laver et al. (1997) and Haarmann et al. (1999). All fiber bundles from the 3 homozygous normal animals failed to respond to halothane or 2 mM caffeine, whereas all fiber bundles from the 3 homozygous MH animals developed tension in response to both drugs.

Isolation of SR vesicles

The preparation of crude SR vesicles was based on Meissner (1984) and Ma et al. (1995). Freshly dissected back and leg muscle was washed in cold phosphate-buffered saline containing 2 mM EGTA (pH 7.0), trimmed of fat and connective tissue, cut into cubes, and either frozen in liquid N₂ and stored at 70°C or freshly processed. The fresh or thawed muscle cubes were suspended in 5 mM Tris maleate, 100 mM NaCl, 2 mM EDTA, and 0.1 mM EGTA, pH 6.8 (5 ml/g of tissue). The muscle was homogenized in a Waring Blendor with four 15-s high-speed bursts. The homogenate was centrifuged at 2600 × g for 30 min and the supernatant filtered through cotton gauze and centrifuged at 10,000 × g for 30 min. The pellet (P2) was collected and the supernatant was centrifuged again at 35,000 × g and the pellet (P3) collected. Pellets P2 and P3 were resuspended in 5 mM Tris-2-(N-morpholino)ethanesulphonic acid (Tris-MES), 300 mM sucrose, 100 mM KCl, and 2 mM dithiothreitol, pH 6.8. Aliquots of the suspensions were frozen in liquid nitrogen and stored at 70°C. All buffers contained the protease inhibitors phenylmethylsulfonyl fluoride (0.7 mM), leupeptin (1 µg/ml), pepstatin A (1 µM), and benzamidine (1 mM).

Calcium release from SR vesicles

Extravesicular Ca²⁺ was monitored at 710 nm with the Ca²⁺ indicator, antipyrylazo III, using a Cary 3 Spectrophotometer (Varian, Sydney, Australia). Identical experiments at 790 nm showed that there were no changes in OD which would alter the rate of Ca²⁺ uptake measured at 710 nm. A step increase in OD upon addition of ruthenium red or caffeine, seen at both 710 nm and 790 nm, was subtracted from the data in Figs. 1 and 2 (Results). The temperature of the cuvette solution was thermostatically controlled at 25°C, and the solution was stirred with a magnetic stirrer. Ca²⁺ release was measured as described by Timerman et al. (1993). TC vesicles (100 µg of protein) were added to the cuvette, to a final volume of 2 ml of a solution containing 100 mM KH₂PO₄ (pH 7); 4 mM MgCl₂, 1 mM Na₂ATP, and 0.5 mM antipyrylazo III. Vesicles were partially loaded with Ca²⁺ by four sequential additions of CaCl₂, each initially increasing the extravesicular [Ca²⁺] by ~7.5 µM (~15 nmoles). It was necessary to suppress Ca²⁺, Mg²⁺-ATPase activity after loading (using 200 nM thapsigargin; Sagara and Inesi, 1991), to allow extravesicular [Ca²⁺] to increase after activation of the RyR. Peptide was then added, followed either by 10 mM caffeine (to determine RyR-releasable Ca²⁺ remaining in the SR vesicles) or by the RyR blocker ruthenium red (to a final concentration of 5 µM), to determine whether Ca²⁺ release from SR was via RyR channels. The Ca²⁺ ionophore A23187 (3 µg/ml) was finally added to release the Ca²⁺ remaining in the SR vesicles. A calibration curve for OD changes for a given increase in [Ca²⁺] was established at the start of each experiment, using 4 sequential additions of 12.5 µM (25 nmoles) of CaCl₂.

View larger version (11K): [in this window] [in a new window]   FIGURE 1   Increase in rate of Ca2+ release after addition of peptide A is greater in SRMH than in SRN. (A) and (B) show records of OD changes at 710 nm, with changes in extravesicular [Ca2+], measured using antipyrylazo III as the Ca2+ indicator. SR vesicles were loaded with Ca2+ by an initial uptake of ~16 nmoles of Ca2+ plus four additions of 15 nmoles of Ca2+ (not shown). The first arrow (t) indicates addition of 200 nM thapsigargin to block the SR Ca2+ ATPase. The second arrow (p) indicates addition of 36 µM peptide A. Caffeine (10 mM) was added at the third arrow (c) before the ionophore A23187 (last arrow (i), 3 µg/ml). The vertical calibration is in OD units: an increase of 0.1 OD unit occurred with an increase in extravesicular Ca2+ of 18 nmoles before adding caffeine, or 31 nmoles after adding caffeine (see Methods). Ca2+ is the non-RyR releasable Ca2+ remaining in the SR after exposure to caffeine.

Addition of H₂O alone to the cuvette after thapsigargin, produced a small decline in the rate of release of Ca²⁺. This was measured for each experiment and the rate of Ca²⁺ release in the presence of peptide was appropriately corrected. The increase in OD for a given increase in extravesicular [Ca²⁺] was ~40% less in the presence of 10 mM caffeine (see legend to Fig. 1). This factor was taken into account in calculations of releasable Ca²⁺.

Lipid bilayer techniques

The lipid bilayer and single channel recording technique are described in Laver et al. (1997) and Dulhunty et al. (1999). Bilayers were formed from phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (5:3:2 w:w) (Avanti Polar Lipids, Alabaster, AL) across an aperture with a diameter of 200 to 250 µm in the wall of a 1.0 ml Delrin cup (Cadillac Plastics, Australia). TC vesicles (final concentration, 10 µg/ml) were added to the cis chamber and stirred until vesicle incorporation was observed. The cytoplasmic side of channels incorporated into the bilayer faced the cis solution. The bilayer potential was controlled, and single channel activity was recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). For experimental purposes, the cis chamber was held at ground and the voltage of the trans chamber controlled. Bilayer potential is expressed in the conventional way as V_cis  V_trans, (i.e., V_cytoplasm  V_lumen).

Bilayers were formed and vesicles incorporated into the bilayer using cis solutions containing 230 mM Cs methanesulphonate (CsMS), 20 mM CsCl, 1.0 mM CaCl₂, and 10 mM N-tris[hyroxymethyl]methyl-2-aminoethanesulfonic acid (TES), pH 7.4, adjusted with CsOH. The trans solution had the same composition, except that CsMS was 30 mM. The cis solution sometimes contained 500 mM mannitol to aid SR vesicle fusion and RyR incorporation into the bilayer. After incorporation, the cis solution was replaced (by perfusion) with an identical solution, except that [Ca²⁺] was varied between 0.3, 10, and 100 µM, and buffered at 0.3 and 10 µM by 2 mM BAPTA, and 200 mM CsMS was added to the trans chamber to establish symmetrical conditions.

Recording and analysis of single channel activity

Currents were filtered at 1 kHz (10-pole low pass Bessel, 3dB) and digitized at 5 kHz. Analysis of single channel records (using Channel 2, developed by P. W. Gage and M. Smith, John Curtin School of Medical Research) yielded channel open probability (P_o), frequency of events (F_o), open times, closed times, and mean open (T_o) and closed (T_c) times, as well as mean current (I'). P_o, T_o, and T_c were measured by determining the number and duration of events in which the current exceeded a threshold level. An event discriminator set above the baseline noise at ~20% of the maximum current, rather than the usual 50%, was used so that openings to both subconductance and maximum conductance levels were included in the analysis. In contrast, I', the mean current, is the average of all data points in a record, in which the baseline is set to 0 pA. Ideally, in a channel lacking submaximal conductance activity, I' approaches the single channel conductance as P_o approaches 1.0. The threshold detection method is very accurate in measuring openings when the peak amplitude of openings exceeds the event discriminator. However, the technique fails when the channel openings are to low conductance levels that are close to the baseline noise. In this case, mean current provides the most accurate method of measuring channel activity.

Bilayers that appeared to contain one channel under control conditions often showed multiple channel openings (i.e., a maximum conductance of 2 or 3 times the single channel conductance) after addition of peptide A. I' provided an accurate measure of the current flowing through two or three channels after addition of peptide. The discriminator method was not used to measure activity when more than one channel was present in the bilayer because of the difficulty in deconvolution to meaningful single channel parameters. Since bilayers containing more than one channel could not be used for measurements of single channel parameters (i.e., P_o, T_o, and T_c) and since most bilayers eventually contained more than one channel, routine measurements of channel activity were done using mean current (I') analysis. P_o was measured in the few records containing only one channel to assess how single channel activity was affected by the peptide.

Statistics

Average data is given as mean ± SEM. The significance of the difference between control and test values was tested a using either Student's t-test, either 1- or 2-sided and either for independent or paired data, as appropriate, or the nonparametric sign test (Minium et al., 1993). Differences were considered to be significant when P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Calcium release from sarcoplasmic reticulum vesicles

Ca²⁺ release from skeletal SR of normal (SR_N) and MH-susceptible (SR_MH) pig muscle was examined in vesicles that were loaded with Ca²⁺ followed by block of the Ca²⁺, Mg²⁺-ATPase with 200 nM thapisgargin. A time-dependent increase in extravesicular [Ca²⁺] occurred after thapsigargin addition. The rate of calcium release increased further after adding peptide A (Fig. 1).

In the examples in Fig. 1, the rate of Ca²⁺ release from SR_N with thapsigargin was ~47 nmoles/mg/min and this increased to ~60 nmoles/mg/min after adding 36 µM peptide, showing a peptide-induced enhancement of 13 nmoles/mg/min (Fig. 1 A). Peptide A evoked stronger release from SR_MH; the rate increased from 66 nmoles/mg/min with thapsigargin to ~162 nmoles/mg/min after adding 36 µM peptide, with a peptide-induced enhancement of 96 nmoles/mg/min (Fig. 1 B).

The fraction of Ca²⁺ accumulated by SR_N and SR_MH that was not available for release through RyRs (presumably that not contained in terminal cisternae) was determined by exposing vesicles to 10 mM caffeine (~4 min after peptide addition), to release Ca²⁺ in TC vesicles through RyR channels, and then to the Ca²⁺ ionophore A23187 (3 µg/ml) to release all remaining Ca²⁺ (Fig. 1, A and B). The fraction of non-releasable Ca²⁺ was the difference between the Ca²⁺ released by caffeine and that released by the ionophore (Ca, Fig. 1; Ca is larger in SR_MH than in SR_N). Ruthenium red (10 µM) stopped Ca²⁺ release if it was added while extravesicular [Ca²⁺] was increasing (n = 8, data not shown), showing that Ca²⁺ release after addition of thapsigargin, and release activated by peptide A, was through RyR channels.

On average the rate of Ca²⁺ release from SR_MH with thapsigargin of 53 ± 5.2 nM/mg/min (n = 12) was significantly greater than the 41 ± 2.2 nM/mg/min (n = 13) for SR_N. The rate of Ca²⁺ release induced by peptide A is the rate after peptide addition, minus the rate with thapsigargin (inset, Fig. 2). A release rate of 76 ± 9.7 nmole/mg/min (n = 3) from SR_MH induced by 36 µM peptide A was significantly greater than the rate of 10 ± 3.2 nmole/mg/min from SR _N (n = 3, Fig. 2). Comparable results were obtained from three SR_N and three SR_MH preparations.

View larger version (30K): [in this window] [in a new window]   FIGURE 2   The initial rate of peptide A-enhanced Ca2+ release (nmoles/mg of TC vesicles/min) from SRN is less than from SRMH. The rate is the initial rate of release with peptide, minus the preceding rate with thapsigargin (n = 3 at each peptide concentration for each of SRN and SRMH). Filled bins show data from SRN and cross-hatched bins show data for SRMH. Data are averages ± SEM for TC vesicles from three different SR preparations. Asterisks show significant differences between data for SRN and SRMH. The insert shows average rates of Ca2+ release from SRN and SRMH in the presence of thapsigargin, just before addition of peptide (n = 12).

Average nonreleasable Ca²⁺ was 16.5 ± 0.57 nmoles in SR_N and 20.3 ± 0.55 nmoles in SR_MH. Since ~60 nmoles of Ca²⁺ were loaded, non-RyR releasable Ca²⁺ fraction was 0.28 ± 0.01 in RyR_N and 0.34 ± 0.01 in RyR_MH. The significantly smaller nonreleasable Ca²⁺ in SR_N suggests that SR_N was more enriched in terminal cisternae than the SR_MH. The different fraction of nonreleasable Ca²⁺ indicated that the difference between peptide A-induced release from terminal cisternae was about eightfold greater in SR_MH than in SR_N (rather than the ~7.6-fold indicated by the uncorrected rates).

Control peptides NB and AS did not mimic the effects of peptide A. The rates of Ca²⁺ release from SR_N and SR_MH immediately after adding 36 µM NB (minus the rate with thapsigargin) were 8.2 ± 2.6 nmoles/mg/min (n = 4) and 1.1 ± 3.4 nmoles/mg/min (n = 4), respectively. Addition of AS resulted in excess release at rates of 6.3 ± 4.2 nmoles/mg/min (n = 4) and 29 ± 4.9 nmoles/mg/min (n = 4) for SR_N and SR_MH, respectively. The increase in Ca²⁺ release from SR_MH with AS was significant, but remained significantly less than that induced by peptide A.

RyR channel activity

Peptide A (0.6 nM to 30 µM) was added to the cis side of the bilayer, with 250/250 mM Cs⁺ (cis/trans), at [Ca²⁺]s of 3× 10⁷ M, 10⁵ M, or 10⁴ M (cis) and 10³ M (trans). Initial experiments with 2 mM Na₂ATP in the cis solution failed to show convincing peptide A-induced activation of normal pig skeletal RyRs (RyR_N, n = 23) or RyRs from MH-susceptible pigs (RyR_MH, n = 25), although the usual peptide A-induced inhibition (Fig. 6 below) was seen in the presence cis Ca²⁺ = 100 µM. Thus experiments examining activation by peptide A were performed in the absence of ATP. Low levels of activity were recorded under these conditions. Table 1 shows that average mean current in bilayers containing 1 to 3 RyR_N or RyR_MH channels was between 0.2 and 2.5 pA before addition of peptide. This was 0.01 to 0.05 of the maximum current (+15 pA or 15 pA at +40 mV or 40 mV, respectively). There were no significant differences in control mean current between RyR_N and RyR_MH.

Activating effects of peptide A on channels at 40 mV

The strongest activating effects of peptide A in rabbit are seen at negative potentials where there is less peptide-induced inhibition (Dulhunty et al., 1999). Peptide A (cis, 0.6 µM and 30 µM) activated RyR_N and RyR_MH channels at 40 mV (Fig. 3). The increase in activity was not associated with an increase in the single channel conductance. The conductance of the RyR_N channel was in fact reduced with 30 µM peptide, due to the peptide's inhibitory action. One RyR_MH channel is apparent in Fig. 3 under control conditions and a second and third channel are seen with peptide A. Washout of peptide with 10 volumes of cis solution caused an increase in activity (discussed below). Activity of RyR_N and RyR_MH was abolished by 30 µM cis ruthenium red added at the end of the experiment (Fig. 3 G).

View larger version (34K): [in this window] [in a new window]   FIGURE 3   Single channel activity at 40 mV is enhanced after adding 0.6 µM or 30 µM peptide A and activation is greater in RyRMH (right panel) than RyRN (left panel). Records were obtained with 10 µM cis Ca2+. (A) Control; (B) after adding 0.6 µM peptide A to the cis chamber; (C) after increasing cis (peptide A concentration) to 30 µM; (D-F), 1, 8, and 12 min, respectively, after perfusion of the cis chamber with peptide-free solution; (G) 1 min after adding 30 µM cis ruthenium red. The solid line shows the zero current (closed, C); the broken line shows the maximum open channel conductance for one channel (O1), or two channels (O2) in RyRMH.

A history plot of mean current during similar experiments with 10 or 100 µM cis Ca²⁺ are shown in Fig. 4. I' progressively increased during exposure to increasing concentrations of peptide and there was a further increase after washing out 30 µM peptide. A greater increase in activity with peptide addition is seen in RyR_MH than in RyR_N. The average normalized mean current (I'p/I'c) also increased more in RyR_MH than in RyR_N with peptide A at each cis [Ca²⁺] (Fig. 5, A-C). Maximum activation of both RyR_N and RyR_MH at was achieved with 0.6 to 6 µM peptide A. 

View larger version (19K): [in this window] [in a new window]   FIGURE 4   Continuous time course of channel activity during exposure to peptide A, washout of peptide A and exposure to ruthenium red. The mean current (I') in 30-s recordings at 40 mV (each separated by a 30-s recording at +40 mV). A and IB, with 100 µM cis Ca2+; data from one bilayer with 2 RyRN (open circles) and a second bilayer with 2 RyRMH channels (filled circles). C and D, with 10 µM cis Ca2+; data from one bilayer with 2 low activity RyRN channels (open circles) and from a second bilayer with 3 relatively high activity RyRMH (filled circles), to compare analysis techniques. Each data point is from a 30-s recording at 40 mV. Successive points are separated by 30 s at +40 mV (not shown). The initial two points in each graph show control activity. Peptide A was then added to progressively increase concentration from 0.6 nM to 6 nM, 60 nM, 600 nM, 6 µM, and 306 µM (indicated by short horizontal lines), with 120 s at each peptide concentration. Peptide was then perfused out of the cis chamber and washout effects recorded during the period labeled "wash". Finally, ruthenium red was added (horizontal line labeled r).

View larger version (20K): [in this window] [in a new window]   FIGURE 5   Peptide A causes a greater increase in mean current at 40 mV in RyRMH (right) than in RyRN (left). Relative mean current (I'p/I'c, where I'p is I' in the presence of peptide and I'c is I' under control conditions) is plotted against the logarithm of peptide A concentration in nM. Symbols show average I'p/I'c and vertical bars show ±1 SEM where this is greater than the dimensions of the symbol. Results were obtained with: (A) 300 nM cis Ca2+ (n = 11 for RyRN; n = 8 for RyRMH); (B) 10 µM cis Ca2+ (n = 19 for RyRN; n = 15 for RyRMH); (C) 100 µM cis Ca2+ (n = 9 for RyRN; n = 10 for RyRMH).). On average, peptide A caused up to a twofold increase in I'p/I'c for RyRN at each cis [Ca2+]. RyRMH showed greater increases in the normalized mean current at 40 mV with peptide A at each cis [Ca2+], with 4.0- to 7.5-fold increases in I'p/I'c.

Single channel parameters were measured from records with only one channel active in the bilayer before and during exposure to the peptide (Fig. 6). Such experiments were rare because summed openings of multiple (two or three) channels usually became obvious after addition of activating concentrations of peptide. Therefore, the numbers of channels analyzed in this way were too few to establish the Ca²⁺ dependence of single channel parameters or the effects of Ca²⁺ on peptide-induced changes in the parameters. The effects of cytoplasmic Ca²⁺ concentration on peptide A-induced activation are best seen in Fig. 5.

View larger version (27K): [in this window] [in a new window]   FIGURE 6   The effects of peptide A on single channel characteristics of RyRN and RyRMH. Analysis was performed on 120 s of channel activity before addition of peptide and 120 s of activity at the peptide concentration giving greatest activation, i.e., between 0.6 and 30 µM. Channel activity was measured at 40 mV (A-D) and at +40 mV (E-H). Data is shown for open probability (Po, A and E), mean open time (To, B and F), opening frequency (Fo, C and G) and mean closed time (Tc, D and H). The symbols show data for individual channels with cis Ca2+ concentrations of 300 nM (), 10 µM (), and 100 µM (). Solid lines connect data for control conditions (con) with data obtained in the presence of peptide A (A).

In the set of experiments used for single channel analysis, activity tended to be low (P_o < 0.1) under all control conditions. Peptide A caused an increase in P_o, T_o, and F_o and a decrease in T_c in all 7 RyR_N channels and in 8 of 9 RyR_MH channels at 40 mV (Fig. 6, A-D). There was no consistent difference between parameter values for RyR_N and RyR_MH under control conditions at 100 µM cis Ca²⁺ (see also Fill et al., 1990; Shomer et al., 1993; Laver et al., 1997). However, in the presence of the peptide at 40 mV, P_o, and T_o tended to be greater in RyR_MH than in RyR_N (Fig. 6, A and B).

Overall, the results suggest that peptide A had similar actions on the gating of RyR_N and RyR_MH channels, reducing the mean closed time and increasing the duration and frequency of channel openings. The effect of the Arg⁶¹⁵Cys substitution in RyR_MH was to amplify these actions of the peptide, particularly the increase in the duration of channel opening.

Activating and inhibiting effects of peptide A on RyR channels at +40 mV

At +40 mV, RyR_N and RyR_MH channels were activated by peptide A at 0.6 µM, but inhibited by peptide at 30 µM (Fig. 7, A-C). Inhibition resulted in channel openings to lower conductance levels, which are apparent in RyR_N with 0.6 µM peptide (Fig. 7 B). Activation by 0.6 µM peptide at +40 mV was greatest in the RyR_MH channel. Perfusion of peptide A out of the cis chamber at +40 mV was followed by a slow increase, and then a decline in channel opening (Fig. 7, D-F). The increase in activity after perfusion was associated with recovery of the maximum open conductance, confirming that the reduced conductance with 0.6 µM peptide was due to the inhibitory effect of the peptide.

View larger version (24K): [in this window] [in a new window]   FIGURE 7   Single channel activity at +40 mV is enhanced after adding 0.6 µM peptide A, but is inhibited with 30 µM peptide A. Activation is greater in RyRMH (right panel) than RyRN (left panel). Records are from the same channels shown in Fig. 3 with 10 µM cis Ca2+. (A) control; (B) after adding 0.6 µM cis peptide A; (C) after increasing cis [peptide A] to 36 µM; (D-F), 1, 8, and 12 min, respectively, after perfusion of the cis chamber with peptide-free solution; (G) 1 min after adding 30 µM ruthenium red to the cis chamber. The solid line shows the zero current (closed, C); the broken line shows the maximum open single channel conductance (O).

Normalized mean current at +40 mV increased with peptide A between 0.6nM and 0.6 µM and then, in contrast to effects at 40 mV, declined with inhibition at higher [peptide] under most conditions (Fig. 8). Both the increase in average I'p/I'c and the subsequent decline with increasing [peptide] at +40 mV were more pronounced in RyR_MH than in RyR_N. As in rabbit (Dulhunty et al., 1999) the strongest inhibition by peptide A was seen in both RyR_N and RyR_MH when cis Ca²⁺ was 100 µM.

View larger version (21K): [in this window] [in a new window]   FIGURE 8   Peptide A causes a greater decrease in average mean current at +40 mV in RyRMH (right column) than in RyRN (left column). Relative mean current is plotted as described for Fig. 5. Symbols show average I'p/I'c and vertical bars show ±1 SEM where this is greater than the dimensions of the symbol. Results were obtained with: (A) 300 nM cis Ca2+ (n = 11 for RyRN; n = 8 for RyRMH); (B) 10 µM cis Ca2+ (n = 19 for RyRN; n = 15 for RyRMH); (C) 100 µM cis Ca2+ (n = 9 for RyRN; n = 10 for RyRMH). There were 3.4-fold and 5.0-fold declines in average I'p/I'c, from the maximum peptide-activated levels, in RyRMH with 30 µM peptide and 10 µM or 100 µM cis Ca2+ respectively, compared with 2.1-fold and 1.6-fold falls in I'p/I'c for RyRN. I'p/I'c did not fall significantly in RyRN with 300 nM Ca2+.

The peptide-induced changes in single channel parameters were smaller and less consistent at +40 mV than at 40 mV (Fig. 6, E-H). For example, an increase in T_o was seen in 5 of 7 RyR_N channels and 3 of 9 RyR_MH channels. This was presumably due to the competing activating and inhibiting effects of the peptide at +40 mV, since inhibition decreases T_o (Dulhunty et al., 1999).

Peptide A washout

The increases in channel activity after washout of peptide (Figs. 3, 4, and 7) were also seen in rabbit RyRs and were thought to reflect exposure of a high-affinity activating effect after washout of a lower affinity peptide A-induced inhibition (Dulhunty et al., 1999). The slower removal of peptide from its activating site after washout is consistent with a higher affinity binding to the activation site in RyR_N and RyR_MH, whereas faster removal from the inhibition site is consistent with lower affinity binding to the inhibition site in RyR_N and RyR_MH. The washout activation suggests that channel activity in the presence of the peptide is a sum of simultaneous activation and inhibition. The time to peak activity after washout reflects the time course of removal of inhibition. The lack of full reversal of activation after 7 to 12 min suggests that the peptide remained bound to its activation site for this time, as might be expected with high affinity binding. The slower washout of activation in RyR_MH suggests stronger binding of peptide A to the activation site on RyR_MH.

The increase in RyR activity after removal of peptide A may provide a more accurate indication of the ability of the peptide to activate RyR_N and RyR_MH than channel activity in the presence of the peptide, which is the sum of activation and inhibition. Long-term washout experiments were performed with 10 µM and 100 µM cis Ca²⁺. I'p/I'c increased significantly after washout and was significantly greater at 40 mV than at +40 mV under all conditions (Fig. 9). I'p/I'c after washout at 40 mV was significantly greater in RyR_MH than in RyR_N with both 10 µM and 100 µM cis Ca²⁺ (note the different scales on the y axes). Curiously, there was no difference between RyR_N and RyR_MH in I'p/I'c after washout at +40 mV. This may have been due to slower removal of the stronger inhibition of RyR_MH

View larger version (26K): [in this window] [in a new window]   FIGURE 9   Mean current increases after peptide A removal from the cis chamber. The increase is significantly greater at 40 mV in RyRMH (right panel) than in RyRN (left panel) at each cis [Ca2+] (note the different scales on the y axes; see text). The results are from a subset of data in which RyRN or RyRMH channels were held for 6 to 10 min after washout of peptide A, with 10 µM cis Ca2+ (n = 6 for RyRN; n = 5 for RyRMH) (A) or 100 µM cis Ca2+ (n = 6 for RyRN; n = 8 for RyRMH) (B). The [Ca2+] was the same in the peptide-containing solution and the perfusion solution. Filled bins, I'p/I'c (see legends to Figs. 5 and 6, for definition) during exposure to 30 µM peptide A. Cross-hatched bins, I'p/I'c for 60 s of maximum activity, 6 to 8 min after washout of peptide A. The asterisks indicate that the difference between I'p/I'c before and after peptide removal is significant according to the Student's t-test. The hatch marks (#) indicate the difference is significant according to the nonparametric sign test. Results are shown for 40 mV and +40 mV as indicated.

Washout activation provides minimum values for the effectiveness of the peptide in activating RyRs, since maximum I'p/I'c occurred when some inhibition remained and activation was declining. Slower washout of inhibition in RyR_MH would further reduce the observable activation for these channels. The greatest average activation was a sevenfold increase in I'p/I'c in RyR_N and an 11-fold increase in RyR_MH (with 10 µM cis Ca²⁺). Highest individual values were a 12-fold increase in one RyR_N channel and a 15-fold increase in one RyR_MH.

Control experiments

Three types of control experiment for the effects of peptide A and its removal on RyR activity are shown in Fig. 10. In the first type of experiment (Fig. 10, A and B), bilayers were exposed to one concentration of peptide (30 µM) for 20 min. There was a slow increase in activity during the 20 min of exposure to the peptide in RyR_N and RyR_MH, with a greater increase in RyR_MH. The small increase in activity during exposure to peptide was followed by a massive increase upon washout. Similar results were obtained in 5 bilayers containing RyR_MH and 3 bilayers containing RyR_N channels. Fig. 10, A and B, show examples of washout effects with 300 nM cis Ca²⁺ (not shown in Fig. 9 above) and also show that the washout-induced increase in activity after 20 min exposure to 30 µM peptide was similar to that after 2 min exposure to 30 µM peptide, with a total exposure to increasing peptide concentration of 12 min (Fig. 4 above). Thus, the increase in activity after 12 min was a specific effect of washout and not a function of time after exposure to peptide.

View larger version (19K): [in this window] [in a new window]   FIGURE 10   A large increase in channel activity upon washout of peptide A occurs after 20 min exposure to 30 µM peptide and is not seen unless channels have been exposed to peptide. Analysis of continuous channel activity in 30-s segments at 40 mV (each separated by a 30-s recording at +40 mV), recorded with 300 nM cis Ca2+. The data in A and B were from a single RyRN (A) and a single RyRMH channel (B), under control conditions (first 3 points), during exposure to cis peptide for 20 min (30 µM, A1), and after perfusion of the cis chamber (wash) and then exposure to ruthenium red (r). The data in C were obtained from a bilayer containing two RyRN channels, under control conditions (first 4 points) during exposure to increasing volumes of water (0.5, 4.5, 4.5, 4.5, 4.5, and 20 µl, equivalent to that added with stock solutions to give 0.6 nM, 6 nM, 60 nM, 0.6 µM, 6 µM, and 30 µM peptide, respectively), and then after perfusion of the cis chamber. The data in D were from three different bilayers containing RyRMH channels. The open circles show data from one bilayer containing three channels and the filled circles show data from a second bilayer containing one channel. The first two or three data points were obtained under control conditions; data were then obtained with water additions (as above) and after perfusion of the cis chamber, and finally after addition of ruthenium red (r). In the third experiment (D, ), data are shown from one channel under control conditions (first 3 points), and then during a 12-min period during which the cis solution was stirred every 2 min, but no additions were made to the solution. Perfusion was performed in the usual way after 12 min, and ruthenium red was added at the end of the experiment.

In the second type of experiment the volumes of water normally added with peptide were added at the usual intervals, but in the absence of peptide, and then the cis chamber perfused (Fig. 10, C and D, circles). Finally, in the third type of experiment, the cis solution was stirred at 2min intervals, with no additions, and then perfused after 12 min (Fig. 10 D, triangles). There was no significant increase in RyR_N activity with water or with stirring alone. Neither did activity increase in RyR_N after perfusion. There was a consistent approximately twofold increase in I' in RyR_MH after the second perfusion of the cis solution in RyR_MH, after either water addition or stirring alone (all experiments were preceded by a perfusion step to replace the incorporation solution with the recording solution; see Methods). The increase after the second perfusion was small compared with the increase in activity after washout of peptide. Similar results were obtained for the water control in 4 bilayers containing RyR_N and 10 bilayers containing RyR_MH, and for the stirring controls in 3 bilayers containing RyR_MH. Fig. 10, C and D, shows that the increase in activity during exposure to the peptide and upon its washout are specific effects of peptide A on channel activity, since they do not occur in the absence of peptide.

Long-lasting substate openings induced by peptide A

Channel opening to lower conductance levels as a result of the inhibitory action of peptide A was seen in all channels. Very long lasting substate openings (Tripathy et al., 1998; Gurrola et al., 1999) were seen only in a subpopulation of RyR_N and RyR_MH and usually in channels that showed strong substate activity under control conditions (Fig. 11, A and B). There was no consistent difference between RyR_N and RyR_MH in substate levels or in the prolongation of substates in the presence of the peptide. Thus an example from one type of channel only is shown in Fig. 11. The predominant substate level in the RyR_MH channel in Fig. 11 was at ~33% of the maximum conductance under control conditions and the prolonged substate opening in the presence of 0.6 µM peptide A was to the same level. This level (arrow) is clearly seen in the all points histograms in Fig. 11, C and D. Twelve of 27 RyR_N and 15 of 23 RyR_MH showed substate activity that was prolonged in the presence of the peptide.

View larger version (33K): [in this window] [in a new window]   FIGURE 11   Openings to submaximal conductance levels are prolonged by peptide A. The recordings in (A) and (B) show 4 s of continuous recording from an RyRMH channel at 40 mV with 10 µM cis Ca2+, before (A) and after (B) addition of 0.6 µM peptide A to the cis chamber. The solid line (C) shows the zero current, closed level; the dotted line (S) shows the dominant submaximal conductance level which is the same in control activity and after peptide addition. The broken line (O) shows the maximum open single channel conductance. (C) and (D) are all-points histograms showing the probability (P) of current levels (I(pA)) for the data in (A) and (B), respectively. The closed level at 0 pA is apparent in both histograms. The submaximal level at ~3.8 pA is indicated by the arrow and is clearly enhanced after peptide A addition (D).

Effects of control peptides on single channel activity

The specificity of the native sequence of peptide A was examined by adding peptide NB or peptide AS to the cis solution while recording activity from RyR_N and RyR_MH in the presence of 10 µM cis Ca²⁺, where peptide A induced the strongest activation. Peptide NB had no effect on RyR_N or RyR_MH at either 40 mV or +40 mV (Fig. 12, A and B). Peptide AS did not alter RyR_N activity at +40 mV or 40 mV or RyR_MH at +40 mV. Mean current increased by 1.8-fold with 6 µM AS at 40 mV, but this was significantly less than the 7.5-fold increase seen with peptide A. 

View larger version (28K): [in this window] [in a new window]   FIGURE 12   Control peptides NB and AS do not reproduce the effects of peptide A on RyRN or RyRMH with 10 µM cis [Ca2+]. (A and B) Relative mean current (I'p/I'c) for RyRN (left) and RyRMH (right) at 40 mV (A) and +40 mV (B). Open circles show average data for peptide AS (n = 3, RyRN; n = 4, RyRMH) and closed triangles show average data for peptide NB (n = 4, RyRN; n = 5, RyRMH). The broken lines, included for comparison, connect data for peptide A (from Figs. 5 and 6). Asterisks indicate significant differences between either peptide NB or peptide AS and peptide A at each concentration. (C) Effects on relative mean current through RyRN (left) or RyRMH (right) of removing 30 µM of peptides AS or NB from the cis chamber. Average I'p/I'c is shown in the presence of 30 µM peptide AS or NB (filled bins) and 3 to 5 min after removal of AS or NB (cross-hatched bins). For comparison, the open bins show average I'p/I'c before and after removal of 30 µM peptide A (data from Fig. 7). Asterisks indicate significant differences between control peptide (NB or AS, data bins) and peptide A (open bins), under each condition.

No significant washout activation was seen with NB or with AS in RyR_N or RyR_MH at either 40 or +40 mV (Fig. 12 C). An increase in activity was, however, seen in 3 out of 3 RyR_MH channels at 40 mV after removal of AS, but the average relative mean current of 4.6 ± 1.4 was significantly less than the average of 11.2 ± 1.2 seen after washout of peptide A. When comparing AS or NB data with peptide A data, the relative mean current at 40 mV was significantly less both before and after washout of AS or NB than before and after washout of peptide A.

Thus, the degree of activation of RyR_N and RyR_MH by peptide A was a specific effect of the peptide and depended on the sequence of amino acids in the peptide. The small activation of single channels by the scrambled sequence AS at high peptide concentrations is consistent with the small increase in Ca²⁺ release from SR vesicles evoked by the peptide.

The experiments in Fig. 12 do not provide information on the ability of the control peptides to inhibit RyR activity, since inhibition is minimal when cis [Ca²⁺] is 10 µM (Fig. 8 and Dulhunty et al., 1999), but is significant with 100 µM cis Ca²⁺. Therefore, inhibition by the control peptides was examined in a separate series of experiments with 100 µM cis Ca²⁺ and also 2 mM cis Na₂ATP (which suppresses activation, but not inhibition, by peptide A; see Results above). Under these conditions, peptide NB failed to inhibit either RyR_N or RyR_MH at either 40 mV or +40 mV (Table 2). On the other hand, 6 µM peptide AS inhibited RyR_MH at 40 mV, whereas 0.6 µM and 6 µM AS inhibited RyR_MH at +40 mV. Peptide AS did not alter activity in RyR_N. These results are consistent with a previous report that AS can inhibit RyR activity (Dulhunty et al., 1999) and confirm the observation that RyR_MH is more susceptible than RyR_N to inhibition by positively charged peptides.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We show here that Ca²⁺ release from the SR of MH-susceptible pig skeletal muscle is more strongly activated by the peptide A segment of the skeletal DHPR II-III loop than Ca²⁺ release from SR of normal pig muscle. In addition, the peptide A-induced increase in RyR channel activity in lipid bilayers was greater in RyR_MH. These results suggest that enhanced tension production (Gallant et al., 1980; Ohta et al., 1989) in MH could, in part, be due to stronger activation of RyRs by the A region of the DHPR II-III loop. This would be consistent with observations that MH muscles are more sensitive than normal to some, but not all, RyR agonists, e.g., caffeine, halothane, and ryanodine (Mickelson and Louis, 1996), but not sulfydryl oxidation (Haarmann et al., 1999).

Activation by peptide A

MH mutations might influence relatively distant sites within the RyR. Identified RyR-linked MH mutations occur in two widely separated clusters: between N-terminal residues 35 and 615 (MH₁ domain) and between residues 2163-2458 (MH₂ domain; Tong et al., 1997). The only direct comparison of mutations from these two domains found that the physiological abnormalities were nearly identical (Richter et al., 1997). It is possible that the MH domains of the RyR are structurally important and/or have allosteric effects over widespread areas of the RyR. The Arg⁶¹⁵Cys substitution could alter the conformation of the MH₁ domain and its interaction with other regions of the RyR or allosterically alter the II-III loop binding site or the structure of regions critical for transmission of information from the DHPR binding region to the RyR ion channel pore.

It is not possible, from the present experiments, to distinguish among several likely mechanisms for stronger activation of RyRs by peptide A. The MH mutation may alter the peptide binding site on the RyR so that the peptide binds more effectively and causes greater activation. A second possibility is that the MH mutation facilitates transmission of a signal from the remote cytoplasmic DHPR-binding domain, presumably adjacent to the T-tubule membrane, to the channel gating mechanism. Alternatively, the response of the RyR gating mechanism to a signal from the binding domain might be facilitated. In the last case, any activating signal should induce a stronger response in RyR_MH than in RyR_N. This is not the case, because the increase in activity upon sulfhydryl oxidation is not greater in RyR_MH (Haarmann et al., 1999).

We have suggested that the A region of the DHPR is bound to the RyR in resting muscle (Dulhunty et al., 1999). Increased resting activation of RyR_MH by the A region in vivo could contribute to enhanced Ca²⁺-activated Ca²⁺ release (Kim et al., 1984; Ohta et al., 1989; Mickelson et al., 1988) and increased force (Gallant et al., 1980). If mechanisms that lower myoplasmic [Ca²⁺] are unable to cope with the high Ca²⁺ leak, resting cytoplasmic [Ca²⁺] would also be higher than normal (Lopez et al., 1986).

The scrambled peptide AS released Ca²⁺ from SR_MH, and from rabbit skeletal SR, at a lower rate than the native peptide A (Dulhunty et al., 1999) and enhanced RyR_MH activity. Since peptide AS contains the same number of positively charged residues as peptide A, it is likely that it interacts weakly with the A-binding region on the RyR, but cannot interact strongly because it lacks the appropriate structure (Casarotto et al., 2000).

Inhibition

Inhibition by high concentrations of the peptide A at +40 mV was enhanced by the MH mutation. The voltage and current direction dependence of inhibition suggest that the peptide blocks the channel pore (Dulhunty et al., 1999) as a result of strong interactions between its numerous positively charged residues and negative sites within the pore (Mead et al., 1998). In contrast, activation is likely to depend on peptide binding to a II-III loop binding-site on the RyR. The enhanced inhibition of RyR_MH could be due to a greater negative charge density in the channel vestibule as result of the Arg⁶¹⁵Cys substitution. Alternatively, the geometry of the vestibule might change to allow the peptide greater accessibility. In either case, the results show that the MH mutation changes more than one property of the RyR and underlines the importance of the MH domains in influencing the structure of the entire RyR. The MH mutation in the N-terminal part of the RyR alters the properties of C-terminal residues, which form the channel pore (Bhat et al., 1997).

Inhibition by peptide A was not apparent in Ca²⁺ release from SR_N and SR_MH and is not apparent in Ca²⁺ release from rabbit skeletal SR (Dulhunty et al., 1999). Inhibition is minimal in these experiments where ion flow is from the SR lumen into the cytoplasm. Inhibition is strongest when ions flow from the cytoplasm to the lumen (i.e., +40 mV). It is unlikely that inhibition occurs in vivo where the II-III loop is tethered to the DHPR in the T-tubule membrane well away from the RyR pore.

Multiple effects of the MH mutation

In addition to effects of the MH mutation on activation and inhibition by peptide A, the Arg⁶¹⁵Cys substitution is associated with a reduced affinity of RyR_MH for Ca²⁺ (Fill et al., 1991; Shomer et al., 1993; Richter et al., 1997) or Mg²⁺ (Laver et al., 1997) at a low affinity inhibitory site. We did not use [Ca²⁺] in the inhibitory range (>100 µM), so did not observe this difference between RyR_MH and RyR_N. Mg²⁺ inhibition is thought to play a critical role in EC coupling in that the RyR is chronically inhibited at myoplasmic [Mg²⁺]. Ca²⁺ release during EC coupling proceeds because the EC coupling signal from the DHPR reduces the affinity of the RyR for Mg²⁺ (Lamb and Stephenson, 1991). The lower than normal affinity of RyR_MH for Mg²⁺ means that RyR activity is higher than normal and may be enhanced more than normal during EC coupling (Laver et al., 1997). The Ca²⁺/Mg²⁺ inhibition site has been located on the C-terminal portion of the RyR (3692 to 4969, Lynch et al., 1999), and changes to this site might be related to changes in the channel region that lead to enhanced inhibition by peptide A. Involvement of the C-terminal Mg²⁺-inhibition region further emphasizes the wide-spread influence of the MH domain on RyR structure and function.

As mentioned previously, it is not clear whether the MH mutation increases the ability of all activating agents to enhance channel activity by altering a common site in the activation process or whether the mutation causes multiple changes in ligand binding sites or links between the binding sites and the channel pore. Both activation by relief of Mg²⁺ inhibition (with reduced [Mg²⁺]) and by caffeine are enhanced in MH, suggesting that both physiological and nonphysiological activators may share a common activation mechanism. On the other hand, physiological activation by sulfhydryl oxidation is not enhanced in MH (Haarmann et al., 1999), so that the common mechanism is not shared by all activating influences. Thus, the question remains open whether peptide A is a physiological or a nonphysiological activator of the RyR channel, since the A region is not essential for skeletal EC coupling (Nakai et al., 1998, 2000). The peptide, however, has several merits as a probe for studying normal and disordered RyR activity, including its high affinity and specific binding to the calcium release channel.

Peptide A-evoked Ca²⁺ release from normal pig SR

The low Ca²⁺ release from SR_N induced by peptide A was unexpected, since EC coupling and contraction in pig muscle proceed as effectively as in rabbit muscle (Gallant et al., 1980). In contrast to Ca²⁺ release, activation of single pig RyR_N channels by peptide A was similar to activation of rabbit RyRs (Dulhunty et al., 1999). Ca²⁺ release from the SR was, of necessity, measured in the presence of ATP, which prevented peptide A activation of single pig RyR channels (Results). Thus ATP may have suppressed Ca²⁺ release from pig SR. We are further investigating this effect of ATP on peptide A activation of pig RyRs.

There are several possible reasons for the difference between peptide A-induced activation of normal pig and rabbit RyRs. Rabbit and pig RyRs differ by one amino acid in the RyR sequence required for DHPR II-III loop binding with Thr¹⁰⁸²Ala substitution in the pig (Fujii et al., 1991; Takeshima et al., 1989). First, this difference may destabilize peptide A binding and reduce in vitro peptide A activation of normal pig RyRs (in vivo, the RyR-DHPR loop interaction could be stabilized by the spatial constraints imposed by insertion of the two large proteins in their respective membranes). Second, the sequence differences between normal pig and rabbit RyRs may mean that additional cytosolic factors are necessary for effective peptide A, or A region of the II-III loop, binding to the RyR in normal pig muscle. Finally, there maybe be changes in the porcine II-III loop sequence that accommodate and stabilize binding to the porcine RyR_N. A number of species-specific sequence differences occur in the DHPR II-III loop, although the pig sequence is not yet reported (Tanabe et al., 1987; Hogan et al., 1994; Chaudhari, 1992). In any case, the lack of peptide A sensitivity of normal pig RyRs in vitro in the presence of ATP is partially reversed by the MH mutation.

In conclusion, we find that RyR channels from MH-susceptible pig muscle are more easily activated by peptide A than RyR channels from normal pigs, in the absence of Mg²⁺ or inhibiting concentrations of cytoplasmic Ca²⁺. The channels from MH-susceptible muscle are also inhibited more by high concentrations of the positively charged peptide. The results suggest that activation by the DHPR II-III loop might be enhanced during EC coupling and contribute to an increase in Ca²⁺ release from the SR of MH-susceptible muscle. In addition, the results show that the MH point mutation alters the response of the RyR to more than one ligand, and thus has multiple actions on the RyR.