Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: effects of a DHPR II–III loop peptide

Esther M. Gallant, James Hart, Kevin Eager, Suzanne Curtis, and Angela F. Dulhunty

Muscle Research Group, John Curtin School of Medical Research, Canberra, ACT 2601, Australia

Submitted 21 July 2003 ; accepted in final form 17 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca2+ release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca2+-release channels (RyRs) is not affected by the MH mutation (Arg615Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1,000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high-affinity activation by a peptide (CS) that corresponds to a part of the II–III loop of the skeletal dihydropyridine receptor (DHPR). Furthermore, subactivating concentrations of peptide CS enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine sensitivity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles 1) the caffeine sensitivity of native RyRs was enhanced, 2) the sensitivity of RyRs to a skeletal II–III loop peptide was depressed, and 3) an interaction between the caffeine and peptide CS activation mechanisms seen in normal RyRs was lost.

calcium ion homeostasis; excitation-contraction coupling; ryanodine receptor polymorphisms; muscle contraction


SUSCEPTIBILITY TO malignant hyperthermia (MH) is conferred in humans by a group of autosomal dominant mutations that lead to an anesthetic-induced increase in free cytoplasmic Ca2+, contractures, acidosis, high temperature, and death if treatment is not immediate. One of these mutations (Arg615Cys) produces MH in pigs with an autosomal recessive expression (36). The skeletal muscle ryanodine receptor (RyR) is altered in ~50% of the human mutations (21, 29). The RyR is the Ca2+ release channel in the sarcoplasmic reticulum (SR) and is the conduit for stored Ca2+ release to initiate contraction. In humans, 42 MH mutations have been identified at 34 different RyR residues and 2 MH mutations at 1 residue in the skeletal dihydropyridine receptor (DHPR) (21, 40). Mutations in equivalent regions of the cardiac RyR lead to excess Ca2+ release under stress and to sudden cardiac death (32).

Muscles from MH-susceptible humans and pigs show an increased sensitivity to caffeine and halothane in vitro, and this enhanced sensitivity is widely used as part of the diagnosis of MH susceptibility in humans and experimentally in pigs (21). Many of the MH-linked RyR mutations similarly lead to an increased sensitivity to caffeine-induced Ca2+ release (48, 49). A single amino acid substitution of Arg614 to Cys is responsible in 1.3–9% of MH-susceptible humans (depending on study population), and the same substitution at residue 615 of the RyR is found in all MH-susceptible pigs. Two mutations have been studied in detail in muscle cells and/or muscle-derived preparations: Arg615Cys in pigs and Gly2434Arg in humans. These mutants demonstrate similar RyR abnormalities including enhanced SR Ca2+ release and ryanodine binding with both caffeine and Ca2+ (22, 37, 42). It is therefore somewhat puzzling that purified RyR1 channels isolated from MH-susceptible porcine skeletal muscle are reported not to exhibit an enhanced sensitivity to caffeine (45). Although it is possible that the enhanced caffeine response in intact MH muscle was due to a higher resting cytosolic Ca2+ concentration, this cannot explain enhanced caffeine-induced Ca2+ release from isolated SR (37). In the light of these observations we have investigated the caffeine sensitivity of RyR channels from normal and MH-susceptible pigs (RyRN and RyRMH, respectively) by using native RyR channels that are incorporated into lipid bilayers along with their associated proteins and lipids. The native RyR channels are therefore structurally and functionally closer to the RyR channel in SR vesicles and intact muscles than purified RyR channels.

Because the actions of several RyR1 antagonists/agonists are altered by the pig MH mutation (13, 27, 45), we have also examined the effects of a peptide probe (peptide CS), derived from the skeletal DHPR (L-type Ca channel), on RyRN and RyRMH. Peptide CS has the sequence of residues 724–760 of the skeletal DHPR II–III loop, which have been implicated in skeletal muscle excitation-contraction (EC) coupling (39). The peptide enhances Ca2+ release from SR at low cytoplasmic Ca2+ concentrations (55) and activates rabbit skeletal RyRs at low peptide concentrations (100 nM) (17, 47) or inhibits channels at higher concentrations (17). EC coupling in normal muscle is more sensitive to caffeine than passive tension (14), indicating that the drug is more active when the DHPR interacts with the RyR. Therefore, we have also examined the combined actions of caffeine and peptide CS on RyRN and RyRMH.

The results show that native pig RyRMH channels are activated by caffeine at lower concentrations than native normal pig or rabbit RyRs. In addition, RyRMH was less sensitive to peptide CS than RyRN. The magnitude of the response of RyRN, but not RyRMH, to caffeine was enhanced by peptide CS. These novel observations suggest an interaction between the activation mechanisms for caffeine and peptide CS that is disrupted by the Arg615Cys mutation. The results further suggest that the response of RyRMH to caffeine either 1) depends on associated proteins or 2) is altered by purification.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Chemicals and biochemicals were from Sigma-Aldrich (Castle Hill, Australia). DHPR II–III loop peptide synthesis has been described previously (10). Peptides were synthesized with purification to 98–100% by using HPLC, mass spectroscopy, and NMR. Stock peptide solutions (~2 mM) were prepared in H2O and frozen in 20-µl aliquots. Precise stock solution concentrations were determined by Auspep. The peptide used in this study was peptide CS: 724Glu Phe Glu Ser Asn Val Asn Glu Val Lys Asp Pro Tyr Pro Ser Ala Asp Phe Pro Gly Asp Asp Glu Glu Asp Glu Pro Glu Ile Pro Val Ser Pro Arg Pro Arg Pro760.

Biological material and caffeine-halothane contracture test for MH susceptibility. The methods for genetic testing, anesthetic techniques, muscle dissection, caffeine-halothane contracture testing, preparation of SR vesicles, and single-channel recording have been described previously (27). Muscle and blood samples were obtained from three homozygous normal pigs (1 Belgium Landrace and 2 Landrace) pigs and three homozygous MH pigs (2 Belgium Landrace and 1 Landrace) ~4 mo old. Each animal was genetically tested for normal or MH RyR allele (containing either Arg615 or Cys615, respectively). The crude SR vesicle preparations were from the same animals as those used previously and were prepared in the same way (13, 18, 27). All fiber bundles from the three homozygous normal animals failed to respond to halothane or 2 mM caffeine, whereas all bundles from the three homozygous MH animals developed tension in response to both drugs. The experiments were carried out under a protocol approved by the Australian National University Animal Ethics Committee.

RyR solubilization and purification. The ryanodine receptor was isolated as described by Lai et al. (23a) using the method described in Shomer et al. (45) for caffeine-sensitivity studies of purified channels in bilayers. The SR vesicle membrane was solubilized in buffer A containing 1 M NaCl, 25 mM PIPES (pH 7.1), 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% phosphatidylcholine, and protease inhibitors (0.8 mM benzamidine, 0.1 mM PMSF, 0.6 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) for 1 h. Insoluble material was removed by centrifugation at 100,000 g for 30 min, and the supernatant was run on a linear sucrose gradient (5–20%, in buffer A) with centrifugation for 16 h at 4°C. Fractions were collected, and those containing the RyR were identified by SDS-PAGE, pooled, and concentrated.

Lipid bilayer techniques. The lipid bilayer and single-channel recording techniques were the same as those used previously (13, 18, 27). Bilayers were formed from phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (5:3:2 wt/wt/wt; Avanti Polar Lipids, Alabaster, AL) across an aperture with a diameter of 200–250 µm in the wall of a 1.0-ml Delrin cup (Cadillac Plastics). Terminal cisternae 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, with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). For experimental purposes, the cis chamber was held at ground and the voltage (V) of the trans chamber was controlled. Bilayer potential is expressed in the conventional way as VcisVtrans (i.e., VcytoplasmVlumen).

Bilayers were formed and vesicles were incorporated into the bilayer with cis solutions containing (mM) 230 Cs-methanesulfonate (CsMS), 20 CsCl, 1.0 CaCl2, and 10 N-tris(hydroxymethyl)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, 1) the cis solution was replaced (by perfusion) with an identical solution, except that Ca2+ concentration ([Ca2+]) was varied between 0.3 and 0.7 µM, and buffered by 2 mM BAPTA and 2) CsMS was added to the trans chamber to establish symmetrical ionic conditions (230 mM CsMS).

Recording and analysis of single-channel activity. Currents were filtered at 1 kHz and digitized at 5 kHz. Analysis of single-channel records (with Channel 2, developed by P. W. Gage and M. Smith, John Curtin School of Medical Research, Canberra, ACT, Australia) yielded channel open probability (Po), frequency of events (Fo), open times, closed times, and mean open (To) and closed (Tc) times, as well as mean current (I'). Po, To, and Tc 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 Po approaches 1.0. The mean current measurement becomes less accurate when the frequency of channel opening is very low, as it was in pig RyR channels when the cytoplasmic (cis) Ca2+ concentration was 300 nM. In this case, mean current It' was calculated from threshold analysis measurements: ,, where ii is the amplitude of a single channel opening, to,i is the duration of the opening, and T is the duration of the record from which the openings were measured (with a program kindly provided by Dr. C. S. Haarmann, Colorado State University, Fort Collins, CO).

Bilayers that appeared to contain one channel under reference conditions often showed multiple channel openings (i.e., a maximum conductance of 2 or 3 times the single-channel conductance) after addition of caffeine. I' provided an accurate measure of the current flowing through two or three channels after addition of peptide. Because bilayers containing more than one channel could not be used for measurements of single-channel parameters (i.e., Po, To, and Tc) and because most bilayers contained more than one channel, routine measurements of channel activity were done with mean current (I') analysis. Single-channel parameters were measured in the few records containing only one active channel, to assess how single-channel activity was affected by caffeine.

Statistics. Average data are given as means ±SE. The significance of the difference between reference and test values was tested with either 1) a Student's t-test, one- or two-sided and for either independent or paired data, as appropriate, or 2) the nonparametric "sign" test (38). Differences were considered to be significant when P <= 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The conductance of normal and MH channels was ~250 pS. This is the usual conductance for channels with symmetrical 250 mM Cs+ and 1 mM Ca2+ in the trans chamber. We used 1 mM trans Ca2+ because it is the physiological luminal Ca2+ concentration and because it maintains calsequestrin association with triadin and junctin (2). However, the Ca2+ competes with Cs+ for the pore and reduces the conductance (15, 51). Channels were identified as RyRs by their characteristic conductance and their sensitivity to block by ruthenium red, tested at the end of the experiment.

Effect of caffeine on RyR channels from normal and MHsusceptible pigs. RyRs from normal (RyRN) and MH-susceptible (RyRMH) pigs were exposed to caffeine with a subactivating cytoplasmic (cis) [Ca2+] of 300 nM. In contrast to previous channel experiments (45), but in agreement with Ca2+ release from SR vesicles (discussed in the introduction to this paper), we found that channels from the MH-susceptible pigs were activated by lower concentrations of caffeine than RyRs from normal pigs (Fig. 1). RyRN channels were unaffected by caffeine at <1–10 mM (Fig. 1A). In contrast, RyRMH channels were activated by >=10 µM caffeine (Fig. 1B). Channel opening to both maximal and submaximal conductance levels was prolonged by caffeine, particularly in the RyRMH channel.



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Fig. 1. Native ryanodine receptor (RyR) from malignant hyperthermia (MH)-susceptible pig (RyRMH) channels were activated by lower concentrations of caffeine than RyR from normal pig (RyRN) channels. The cytoplasmic (cis) Ca2+ concentration was 300 nM. Consecutive records from 1 RyRN channel are shown at left, and records from 1 RyRMH channel are shown at right. Records are shown at –40 mV in A and at +40 mV in B. The first trace in each panel was obtained under reference conditions; traces were then obtained after 1-min exposure to each of 10 µM, 100 µM, 1 mM, and 10 mM caffeine. Caffeine was added to the cis bilayer solution, i.e., to the cytoplasmic side of the channels. Note that the activity of the RyRN channel did not increase until the caffeine concentration was increased to 10 mM. In contrast, the RyRMH channel was activated by 10 µM caffeine. C, closed current level; O, maximum open current level.

 

There was little difference between the reference activity for RyRN and RyRMH at either 300 or 700 nM cis Ca2+, although there was a trend toward higher activity in RyRMH channels with 700 nM cis Ca2+ (Table 1). Po, estimated from the mean current (i.e., mean current/maximum single-channel current) was ~0.0005 for RyRN and RyRMH with 300 nM cis Ca2+ or ~0.011 and ~0.017 for RyRN and RyRMH, respectively, with 700 nM Ca2+.


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Table 1. Average mean current for ryanodine receptors

 

The increase in mean current occurred at 50-fold lower caffeine concentrations in RyRMH channels than in RyRN channels at +40 mV (Fig. 2). There was a trend toward an increase in activity with only 10 µM caffeine in the RyRMH channels at –40 mV, indicating that the threshold for caffeine activation was 1,000-fold lower at negative potentials (where current flows from the luminal to the cytoplasmic side of the channel, as it does during Ca2+ release).



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Fig. 2. RyRMH channels were activated by caffeine at concentrations >=10 µM, in contrast to RyRN channels, which were activated by concentrations >=5 mM. All data were recorded with a cytoplasmic (cis) Ca2+ concentration of 300 nM. Data obtained at –40 mV are shown in A and data at +40 mV in B. {circ}, Average mean current for RyRN channels (n = 6 at –40 mV or 5 at +40 mV); , average mean current for RyRMH channels (n = 5 at –40 mV or 8 at +40 mV). The cytoplasmic (cis)Ca2+ concentration for these experiments was 300 nM.

 

The effects of adding caffeine to either the cis (cytoplasmic) or trans (luminal) side of RyRMH channels were compared. There was a trend toward a greater increase in activity after the cis addition of 10 mM caffeine at +40 mV and a significantly greater increase at –40 mV (Table 2). This result suggested that caffeine acted at a site that was most easily accessed from the cytoplasmic solution. We predicted that a site accessible from the membrane would show the same activation whether caffeine was added to the cis or trans solution, whereas a site on the luminal or cytoplasmic side would be preferentially activated by the trans or cis addition, respectively. The increased activation with cis addition supports other data indicating that the caffeine binding site is on the cytoplasmic domain of the RyR (43).


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Table 2. Effect of caffeine on RyRMH mean current

 

Caffeine sensitivity of purified RyRMH. Because previous experiments showing that the caffeine sensitivity of RyRMH channels in bilayers was not enhanced were performed on purified channels (45), we examined purified RyRMH. As previously reported, activity in these channels was not enhanced by 100 nM or 1 µM caffeine but was enhanced by 5 and 10 mM caffeine (Fig. 3). Thus the caffeine sensitivity of RyRMH is altered by the solubilization or purification procedures. In addition, the relative increase in purified RyRMH activity, of ~2-fold with 10 mM caffeine, was similar to the 2- to 3-fold increase seen previously with purified channels at pH 7.4 (45) and considerably smaller than the 10- to 30-fold increase in native RyRMH activity shown in Figs. 2 and 6.



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Fig. 3. Purification of RyRMH removes the channel's sensitivity to caffeine at concentrations <=1 mM. Relative mean current is plotted at +40 mV () and –40 mV ({circ}) as a function of caffeine concentration (n = 5 experiments). The cytoplasm (cis) Ca2+ concentration was 300 nM. Note that the failure of caffeine to activate the channels at concentrations <5 mM was observed in each of the 5 experiments.

 


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Fig. 6. Peptide CS increased the sensitivity of normal RyR channels, but not RyRMH channels, to caffeine in a species-independent manner. Results obtained at –40 and +40 mV have been combined in the average data. Relative mean current is shown for channels exposed to caffeine in the absence of peptide CS ({circ}) and in the presence of 10 nM peptide CS (). A: average data for RyRN channels (n = 4 at –40 mV or 5 at +40 mV) in both the absence and presence of peptide CS. B: average data for RyRMH channels (n = 6 at –40 and +40 mV in the absence of peptide CS and n = 6 at –40 mV or 5 at +40 mV with peptide CS). The cytoplasmic (cis) Ca2+ concentration for experiments in A and B was 700 nM. C: average data for native rabbit RyR channels (n = 4 at –40 and +40 mV) in the absence and presence of peptide CS. The cytoplasmic (cis) Ca2+ concentration for these experiments was 300 nM. *Significant difference between the data obtained in the presence and absence of peptide CS.

 

Effects of peptide CS on RyR activity. Peptide CS, corresponding to the C region of the skeletal DHPR II–III loop, activates skeletal RyRs at 1–10 µM and inhibits at higher concentrations (17). The cis [Ca2+] was buffered to 700 nM to increase the probability of channel opening under reference conditions and to increase the likelihood that the peptide would enhance channel activity (17). We examined the effect of peptide CS (at 10 nM and 1 µM) on pig RyR channels. Peptide CS caused a 50% increase in RyRN activity at 10 nM and a larger twofold increase in relative mean current at 1 µM (Fig. 4). In contrast, RyRMH channels were not significantly affected by peptide CS at either 10 nM or 1 µM and had a reduced sensitivity to the peptide.



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Fig. 4. Peptide CS induced significant high-affinity activation of RyRN but not RyRMH. Filled bars show average relative mean current for 13 RyRN channels with 10 nM peptide CS and 4 RyRN channels with 1 µM peptide CS. Data were recorded with a cytoplasmic (cis) Ca2+ concentration of 700 nM. Open bars show average relative mean current for 9 RyRMH channels with 10 nM peptide CS and 3 channels with 1 µM peptide CS. *Significant increase in relative mean current. Note that there was a significant increase in relative mean current for RyRN with 10 nM and 1 µM peptide. There was no significant change in average relative activity of RyRMH.

 

Effects of peptide CS on caffeine sensitivity of RyRN and RyRMH. The ability of caffeine to activate RyRs in the presence of 10 nM peptide CS was examined with 700 nM cis Ca2+. Although the activity of the normal pig RyR was not obviously increased when 10 nM peptide CS was added to the cis solution, the response to caffeine was greater in the presence of peptide CS, with an increase in activity apparent with 10 µM caffeine at –40 mV (Fig. 5). Indeed, two RyRN channels became active in the bilayer with 10 mM caffeine. In contrast to the enhanced sensitivity of the RyRN channel to caffeine in the presence of peptide CS, the sensitivity of the RyRMH channel did not appear to be altered by peptide CS (Fig. 5), being similar to that of the channel shown in Fig. 1 (in the absence of peptide CS).



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Fig. 5. RyRN was activated by 10 µM caffeine in the presence of 10 nM peptide CS. Consecutive records obtained from 1 RyRN channel are shown at left, and records from 1 RyRMH channel are shown at right. The cytoplasmic (cis) Ca2+ concentration was 700 nM. Data are shown at –40 mV in A and at +40 mV in B. The first trace in each panel was obtained under reference conditions; traces were then obtained after 1-min exposure to 10 nM peptide CS and after 1-min exposure to each of 10 µM, 100 µM, 1 mM, and 10 mM caffeine in the presence of peptide CS. The peptide and caffeine were added to the cis bilayer solution, i.e., to the cytoplasmic side of the channels. Note that the activity of the channels was not much affected by the addition of peptide but that the activity of both the RyRN and RyRMH channels increased with 10 µM caffeine at –40 mV or 100 µM caffeine at +40 mV.

 

Because similar results were obtained at +40 mV and –40 mV, the data for the two potentials were combined to calculate average relative mean current. In the RyRN channels the increase in average mean current for caffeine plus peptide CS was significantly greater (with 1–10 mM caffeine) than with caffeine alone (Fig. 6). The interaction between peptide CS and caffeine in the RyRN channels was more than a simple additive effect. Peptide CS sensitized the channels to caffeine because, although the peptide enhanced activity by <50%, the caffeine response increased threefold. RyRMH channels did not share the synergistic actions of peptide CS and caffeine with RyRN channels (Fig. 6B).

Effects of peptide CS on caffeine sensitivity of rabbit RyRs. The caffeine sensitivity of rabbit skeletal RyRs and the effect of peptide CS were examined to ensure that the properties of the normal pig RyR were not species specific. The rabbit RyR channels, with 300 nM cis Ca2+, showed a small increase in activity with 1–10 mM caffeine (Fig. 6C). In agreement with previous reports (17), there was no significant change in the activity of the rabbit RyRs with 1 µM peptide CS at the subactivating cis [Ca2+]. However, as with normal pig RyRs, there was a dramatic effect of the peptide on the sensitivity of the rabbit RyRs to caffeine.

Changes in channel gating with caffeine are not altered by either MH or peptide CS. Single-channel parameters were measured in a subset of experiments in which only one channel was open in the bilayer under all conditions. The results show an increase in Po and To, with a decrease in Tc, which were significant under most conditions and tended to be greater in the presence of peptide CS (Fig. 7). In general, there was a trend toward larger changes in the single-channel parameters in RyRMH with caffeine in the absence of peptide, but the trend was reversed in the presence of peptide CS. In summary, the changes in channel gating with caffeine and with peptide CS were the same in RyRN and RyRMH, although the relative sensitivity to caffeine was altered by the mutation and by the peptide.



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Fig. 7. Effects of caffeine on single-channel parameters were similar for RyRN and RyRMH in the presence or absence of peptide CS at +40 and–40 mV. Single-channel parameters were measured from a subset of experiments in which 1 channel remained active in the bilayer for the duration of the experiment. The numbers of experiments analyzed were 3 for RyRN and 5 for RyRMH in the absence of peptide CS and 5 for RyRN and 6 for RyRMH in the presence of peptide CS. The cytoplasmic (cis) Ca2+ concentration for these experiments was 700 nM. Data at –40 mV are shown at left and data at +40 mV at right. Filled bars, data for RyRN; open bars, data for RyRMH. A: open probability. B: mean open time. C: mean closed time. Top graph in each pair shows data obtained in the absence of peptide CS, and bottom graph shows data obtained in the presence of 10 nM peptide CS. Data plotted in each graph were obtained under reference conditions and in the presence of 10 mM caffeine. *Significant difference between a parameter under reference conditions and in the presence of peptide CS. Note that the trend toward an increase in open probability with 10 mM caffeine was accompanied by a tendency toward a 2- to 3-fold increase in mean open time and a 10- to 100-fold decline in mean closed time under all conditions.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We show here that the caffeine sensitivity of single native RyR channels isolated from MH pig muscle is significantly greater than the sensitivity of RyR channels isolated from either normal pig or rabbit muscle. Enhanced sensitivity to caffeine is predicted from the induction of contractures at lower caffeine concentrations in MH pig muscle than in normal muscle (14) and the enhanced caffeine-induced Ca2+ release from the MH SR (37). However, this is the first study to show that single-channel activity of pig RyRs is in fact more sensitive to caffeine in MH. Other novel findings include the following. 1) The sensitivity of RyRs to peptide CS is reduced in MH. 2) Peptide CS sensitizes normal RyRs to activation by caffeine. 3) The peptide CS enhancement of caffeine responses is abolished by the Arg615Cys mutation.

Native MH RyR channels are more sensitive to caffeine. Although it was predicted that the single-channel activity of RyRs from MH pigs would be more sensitive to caffeine than that of normal RyRs, previous studies with purified channels using conditions of ionic strength, pH, and [Ca2+] similar to ours failed to confirm this prediction (45). We also show that purified RyRMH channels do not demonstrate enhanced caffeine sensitivity. However, native channels from MH pigs (this study) and MH susceptible humans (12) do show enhanced caffeine sensitivity. Therefore, the fact that purified MH channels do not show enhanced caffeine sensitivity, whereas MH RyR activity in SR vesicles (inferred from Ca2+ release studies) is more sensitive to caffeine than normal RyR activity, is most likely explained by the purification process rather than by other differences between the experimental techniques such as ionic strength, which may influence the appearance of MH characteristics (44). Although studies showing enhanced caffeine sensitivity of Ca2+ release from SR and [3H]ryanodine binding were generally done with a salt concentration of 0.1 M (5, 44), experiments showing enhanced caffeine sensitivity in native MH RyRs (this study and Ref. 12) and a lack of enhanced caffeine sensitivity in purified MH RyRs (this study and Ref. 45) were all done under similar conditions of 0.2–0.25 M salt and pH 7.3–7.4.

The effects of purification on MH RyR activity could be due either to the absence of associated proteins or to the purification process per se. Native channels are incorporated into lipid bilayers with functionally active associated proteins. These include triadin, junctin, calsequestrin, FK506 binding proteins, Ca/calmodulin kinase II, and PKA (1, 2, 9, 33, 34). The enhanced sensitivity of RyRMH to caffeine, like the responses of the RyR to other ligands (2, 10, 24, 41), could require such a protein. The response to caffeine per se, seen in RyRs expressed in CHO cells (3, 4) and in the COOH-terminal 1,000 residues of RyR1 (50), does not require an associated protein. However, the possibility that associated proteins modify caffeine responses has not been explored. The hypothesis could be tested by examining caffeine sensitivity of normal and MH RyRs when associated proteins are added to purified RyRs, as has been done with calsequestrin, triadin, and junctin (16).

An alternative explanation is that the RyRs were modified by CHAPS solubilization and purification. The procedure does not alter FKBP12 or II–III loop binding to RyR1 (30, 31), although the Ca2+ sensitivity of RyRs can be altered (28, 52). If purification alone does alter the response of MH RyRs to caffeine, it would be necessary to speculate that the mechanism for enhanced caffeine activation in MH is particularly sensitive to purification, because purification does not appear to alter the caffeine sensitivity of normal RyRs. The threshold for caffeine activation is ~1 mM in purified (45) and native (this study) RyRN channels. Although we did not examine purified native RyRs, our results with purified MH channels were the same as those in Ref. 45 and it is likely that we would have obtained similar results with purified normal channels.

Sensitivity of RyR channels to peptide CS. Peptide CS corresponds to a sequence in the skeletal DHPR that is associated with skeletal EC coupling. The rabbit DHPR sequence was used in this study with both rabbit and pig RyRs. The CS region of the DHPR is highly conserved among other species, with 100% identity between rabbit and mouse and 97% identity between these rabbit/mouse and pig, human, or rat. Curiously, residue 754 is the only variable residue in the loop, being leucine in human, valine in rabbit and mouse, alanine in rat, and isoleucine in pig (Roberts M and Mickelson J, unpublished observations). Thus the hydrophobic nature of this residue is conserved across species. Residue 754 is not in the essential region for RyR activation, which is located in residues 725–742 (39) or 739–742 (23). The fact that we obtained the same result with normal pig and rabbit RyR suggests that the differences in residue 754 between pig and rabbit do not influence the effect of peptide CS on the RyR from these two species.

The functional changes in RyR activity caused by the peptide are indicative of its binding to the channel complex (17). The role of the interaction between the DHPR C region and the RyR during EC coupling and in resting muscle remains to be determined, as do the functional changes that occur with this binding in the presence of all the other modulatory factors in the intact fiber. Nevertheless, peptide CS is a useful probe of RyR activity and its activity is likely to be indicative of RyR-DHPR interactions in the muscle fiber. Curiously, the reduced sensitivity to peptide CS contrasts with the tendency for responses to be altered so as to increase RyR activity in MH (13, 27, 45). However, the reduced sensitivity to peptide CS could be related to enhanced EC coupling reported in porcine MH (6), because one postulated role for the C region of the II–III loop is to terminate the interaction with the RyR in EC coupling (55). A decreased ability to terminate EC coupling would facilitate Ca2+ release (6). This hypothesis could be tested by examining effects of conservative mutations in the C region of the DHPR on the onset and decay of activation during EC coupling in myocytes or muscle cell lines expressing normal RyRs or channels with MH mutations.

An alternative hypothesis is that binding of the C region of the II–III loop to the RyR removes Mg2+ inhibition and allows the RyR to open during EC coupling. It is well established that 1) depression of Mg2+ inhibition is an important step in EC coupling (26) and 2) Mg2+ inhibition is substantially reduced in MH, contributing to enhanced channel activity in vivo at rest (27). In addition, we have preliminary evidence that the CS peptide reduces Mg2+ inhibition in normal RyRs (Haarmann CS, Dulhunty AF, and Laver DR, unpublished data). Therefore, the interaction between the C region and the RyR might facilitate the reduced Mg2+ inhibition during EC coupling. An extension of this hypothesis is that the binding site for the CS peptide is intimately related to the Mg2+ inhibition mechanism and that the structural changes that disrupt Mg2+ inhibition in MH also disrupt the interaction between the C region and the RyR. Verification of this hypothesis will require a more detailed understanding of both the Mg2+ inhibition mechanism and the binding site on the RyR for peptide CS and the C region in the full II–III loop and their relationship to residue 615 on the RyR.

The fact that the sensitivity of the RyR to the peptide is reduced in MH provides another example of the multiple effects of the mutation. The result supports the hypothesis that RyR residue 615 is in a region of the protein that is involved in the interdomain interactions required for normal channel function (20, 25, 46, 53, 54).

Location of caffeine and peptide CS binding sites. Our results show that the caffeine binding site is most accessible to the cytoplasmic solution. The binding site may encompass more than one domain of RyR1. Sensitivity to caffeine is diminished by mutations in residues 2370, 2373, or 2375 (8), which are in the putative cytoplasmic region of the channel. However, the caffeine binding site may also involve the pore region because channels formed by the last 1,000 residues of the RyR retain caffeine sensitivity (50). MH mutations in all three MH-sensitive regions increase caffeine sensitivity (7). The finding that peptide CS enhanced the response of normal RyRs to caffeine, and that this was abolished by the MH mutation, suggests that 1) the peptide CS and caffeine binding sites are physically close or are functionally related and 2) interactions between the binding sites are disrupted in MH. Assessment of these suggestions awaits the discovery of the caffeine and peptide CS binding sites on the RyR and the solution of the three-dimensional structure of the protein.

Lack of difference between activity of normal and MH RyR channels at subactivating cytoplasmic [Ca2+]. We find that there is little difference in the activity of normal and MH channels with 0.1–0.7 µM cis [Ca2+]. This is consistent with previous reports that the channels in bilayers have similar activity at subactivating cis [Ca2+] but that RyRMH activity is enhanced at activating [Ca2+] (45). It is useful to compare these results (obtained with 0.2–0.25 M salt solutions) with those of [3H]ryanodine binding and Ca2+ release from SR vesicles. In the presence of 0.5–1 M NaCl there is little difference between [3H]ryanodine binding to normal and MH SR at [Ca2+] of 50 µM (5) or Ca2+ between 1 nM and 1 mM (44). In contrast, [3H]ryanodine binding to MH vesicles is higher at all [Ca2+] (1 nM to 10 mM) with 0.1 M salt (19, 35, 44). Similarly, the initial rates of Ca2+ release from SR vesicles are higher in MH than in normal preparations with [Ca2+] between 10 nM and 1 mM in the presence of 0.1 M salt (5, 11). The fact that MH RyR channels in bilayers do not show enhanced activity in the presence of subactivating cis [Ca2+] may be due to the use of intermediate salt concentrations in these studies (0.20.25 mM) that could tend to reduce the differences between the normal and MH channels, which are maximal with 0.1 mM salt and abolished with 1 M salt.

It could be argued that the subconductance activity in native (this study) and purified (45) normal and MH RyR channels might be a source of variation between measurement of Po in single channels and [3H]ryanodine binding to, or Ca2+ efflux from, SR vesicles. For example, an increase in openings to low subconductance levels at subactivating cis [Ca2+] in RyRMH channels could cause an increase in Ca2+ efflux through the RyR. This may not be seen as an increase in Po if the threshold for channel opening is set above the subconductance level. However, this explanation does not hold up, because similar results were obtained when either Po (45) or Po and mean current (this study) were used as an index of channel activity. In contrast to Po, mean current includes both full and subconductance opening and is a better approximation to the total cation flux through the channel than Po.

In conclusion, the results in this study show that native RyR channels from MH pigs have enhanced sensitivity to caffeine and suggest that this sensitivity may be lost with channel purification. In addition, we show that normal rabbit and pig RyRs were activated by low concentrations of peptide CS and that addition of 10 nM peptide CS could dramatically enhance the response of normal RyRs to caffeine. In contrast, the RyR from MH pigs had a reduced response to peptide CS, and its caffeine sensitivity was unaffected by the peptide. Single-channel analysis suggests that the nature of changes in single-channel gating caused by caffeine was not altered by either MH or peptide CS. However, the magnitude of these changes in gating was enhanced in both cases. Finally, the results support the concept that the Arg615Cys mutation in MH causes a broad disruption of ryanodine receptor ion channel function that is likely to contribute to the changes in Ca2+ release and EC coupling seen in intact muscle studies.


    ACKNOWLEDGMENTS
 
We are grateful to Suzy Pace and Joan Stivala for assistance with the preparation of SR vesicles and purification of RyRMH and thank Monica Roberts and James Mickelson for permission to cite their unpublished sequence data for the pig DHPR II–III loop.

GRANTS

This project was supported by US National Science Foundation Grant INT9907846 (to E. M. Gallant and A. F. Dulhunty).


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. F. Dulhunty, John Curtin School of Medical Research, Australian National Univ., PO Box 334, Canberra, ACT 2601, Australia (E-mail: angela.dulhunty{at}anu.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.


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