Modulation of Ca2+-gated cardiac muscle Ca2+-release channel (ryanodine receptor) by mono- and divalent ions

Wei Liu, Daniel A. Pasek, and Gerhard Meissner

Departments of Physiology and Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The effects of mono- and divalent ions on Ca2+-gated cardiac muscle Ca2+-release channel (ryanodine receptor) activity were examined in [3H]ryanodine-binding measurements. Ca2+ bound with the highest apparent affinity to Ca2+ activation sites in choline chloride medium, followed by KCl, CsCl, NaCl, and LiCl media. The apparent Ca2+ binding affinities of Ca2+ inactivation sites were lower in choline chloride and CsCl media than in LiCl, NaCl, and KCl media. Sr2+ activated the ryanodine receptor with a lower efficacy than Ca2+. Competition studies indicated that Li+, K+, Mg2+, and Ba2+ compete with Ca2+ for Ca2+ activation sites. In 0.125 M KCl medium, the Ca2+ dependence of [3H]ryanodine binding was modified by 5 mM Mg2+ and 5 mM beta ,gamma -methyleneadenosine 5'-triphosphate (a nonhydrolyzable ATP analog). The addition of 5 mM glutathione was without appreciable effect. Substitution of Cl- by 2-(N-morpholino)ethanesulfonic acid ion caused an increase in the apparent Ca2+ affinity of the Ca2+ inactivation sites, whereas an increase in KCl concentration had the opposite effect. These results suggest that cardiac muscle ryanodine receptor activity may be regulated by 1) competitive binding of mono- and divalent cations to Ca2+ activation sites, 2) binding of monovalent cations to Ca2+ inactivation sites, and 3) binding of anions to anion regulatory sites.

sarcoplasmic reticulum

    INTRODUCTION
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Abstract
Introduction
Procedures
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Discussion
References

IN MUSCLE, AN ACTION potential activates intracellular Ca2+-release channels to cause the release of Ca2+ from an intracellular Ca2+ compartment, the sarcoplasmic reticulum (SR), into the myofibrillar space (for review, see Refs. 5, 26). The SR Ca2+-release channels are also known as ryanodine receptors (RyRs) because they can bind the plant alkaloid ryanodine with high affinity and specificity (for review, see Refs. 3, 15). The cardiac muscle (RyR2) and skeletal muscle (RyR1) RyRs have been purified as 30S protein complexes composed of four large (relative molecular weight of ~560,000) (3, 15) and four small (FK-506-binding protein, relative molecular weight of ~12,000) (24) subunits. Physiological and biochemical evidence suggests that RyR1 and RyR2 are ligand-gated channels with Ca2+ as a major regulator. Activation by micromolar Ca2+ and inhibition by millimolar Ca2+ suggest at least two classes of Ca2+ binding sites, high-affinity activation sites and low-affinity inactivation sites. Various other endogenous and exogenous effectors have been identified, including Mg2+, ATP, calmodulin, caffeine, and ryanodine (3, 15, 30).

The neutral plant alkaloid ryanodine binds with nanomolar affinity and high specificity to RyRs and has been used as a sensitive probe in assessing the activity of RyRs (1, 16, 29). As a general rule, conditions that open the channel, such as the presence of micromolar Ca2+ or millimolar adenine nucleotide increase the affinity of [3H]ryanodine binding.

RyR activity is sensitive to ionic strength and composition, as assessed in [3H]ryanodine binding, vesicle Ca2+ efflux, and single-channel measurements. This phenomenon has been especially observed for RyR1. Inorganic phosphate and anions often classified as chaotropic ions (ClO<SUP>−</SUP><SUB>4</SUB>, SCN-, I-, NO<SUP>−</SUP><SUB>3</SUB>) stimulated RyR1 activity, whereas replacement of Cl- by gluconate, propionate, and buffer anions such as 2-(N-morpholino)ethanesulfonic acid (MES) anion and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) anion was inhibitory (6, 7, 13, 18). An increase in KCl or NaCl concentration increased RyR1 activity (1, 9, 16, 18, 19, 21). In contrast, RyR2 activity has been reported to be only little affected by ionic composition (6, 19).

It is well established that there exist major differences in the regulation of the skeletal muscle and cardiac muscle receptor isoforms by Ca2+, Mg2+, ATP, and other endogenous and exogenous effectors (3, 15). Here, we show that the two receptors are regulated in a similar, but not identical, manner by mono- and divalent ions. Our results indicate that cations regulate Ca2+-gated RyR2 activity by at least two different mechanisms: 1) competitive binding to high-affinity Ca2+ activation sites and 2) binding to low-affinity Ca2+ inactivation sites. Cl- had an activating effect.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
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Preparation of SR vesicles. Cardiac SR vesicles enriched in [3H]ryanodine binding activity were prepared from canine hearts in the presence of protease inhibitors (100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin, 1 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride) using a discontinuous sucrose gradient of 20, 30, and 40% sucrose (17). Membranes at the 20%/30% sucrose interface were recovered, quick-frozen in small aliquots, and stored at -135°C before use. "Heavy" rabbit skeletal muscle SR vesicles were prepared in the presence of the above protease inhibitors as described previously (14). The maximum number of high-affinity [3H]ryanodine binding sites determined under optimal binding conditions (11) ranged from 2.5 to 8 pmol/mg protein for cardiac SR vesicles and from 11 to 23 pmol/mg protein for skeletal muscle SR vesicles, depending on the preparations.

[3H]ryanodine binding measurements. Unless otherwise indicated, cardiac muscle and skeletal muscle SR vesicles were incubated for 40 and 96 h, respectively, at 12°C in 20 mM imidazole, pH 7.2, 0.2 mM Pefabloc SC, 20 µM leupeptin, 1 nM [3H]ryanodine, and the indicated salts and free Ca2+ concentrations. Nonspecific [3H]ryanodine binding was determined using a 1,000-fold excess of unlabeled ryanodine. Unbound [3H]ryanodine was separated from the protein-bound [3H]ryanodine by filtration of sample aliquots through Whatman GF/B filters presoaked with 2% polyethyleneimine, followed by washing with three 5-ml volumes of ice-cold 0.1 M KCl and 1 mM potassium PIPES (pH 7.0) medium. The radioactivity remaining with the filters was determined by liquid scintillation counting to obtain bound [3H]ryanodine.

Determination of free [Ca2+] and [Sr2+]. Free Ca2+ concentrations of >1 µM were determined with a Ca2+-selective electrode (World Precision Instruments, Sarasota, FL). Free Ca2+ concentrations of <1 µM were obtained by including in the solutions the appropriate amounts of Ca2+ and ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) [or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and nitriloacetic acid] as determined using the stability constants and the mixed solution program published by Schoenmakers et al. (22). Effects of Sr2+ were determined in assay media containing contaminating levels of Ca2+ (2-4 µM) and 1 mM EGTA. Free Sr2+ concentrations were calculated using the EGTA-Sr2+ complexation constants published by Smith and Martell (23) and verified with a Ca2+-selective electrode for Sr2+ concentrations >100 µM.

Data analysis. The Ca2+ dependence of [3H]ryanodine binding was analyzed with the assumption that the RyR2 possesses cooperatively interacting high-affinity Ca2+ activation and low-affinity Ca2+ inactivation binding sites. A simple scheme used to describe the Ca2+ dependence of channel activity was
R <AR><R><C>&mgr;M Ca<SUP>2+</SUP></C></R><R><C>⇌</C></R></AR> A<SUB>Ca</SUB> <AR><R><C>mM Ca<SUP>2+</SUP></C></R><R><C>⇌</C></R></AR> <SUB>Ca</SUB>I<SUB>Ca</SUB> (Scheme 1)
In scheme 1, the RyR ion channel is present in its closed Ca2+-free form (R) at [Ca2+] of <0.1 µM, and its Ca2+-activated (ACa) and Ca2+-inactivated (CaICa) forms are present at micro- and millimolar Ca2+ concentrations, respectively. The tetrameric RyR contains cooperatively interacting Ca2+ activation sites and Ca2+ inactivation sites (see RESULTS); however, only one Ca2+ activation and one Ca2+ inactivation site are shown.

Ryanodine binding (and, by extension, Ca2+-release channel activity) was fitted by the product of an activation and an inactivation variable, each related to [Ca2+] by the Hill formula
B = B<SUB>o</SUB> {[Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>a</SUB></SUP>/([Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>a</SUB></SUP> + <IT>K</IT><SUP><IT>n</IT><SUB>a</SUB></SUP><SUB>a</SUB>)}
· {1 − [Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>i</SUB></SUP>/([Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>i</SUB></SUP> + <IT>K</IT><SUP><IT>n</IT><SUB>i</SUB></SUP><SUB>i</SUB>)} (1)
where B is the [3H]ryanodine binding value at a given [Ca2+], Bo is the binding maximum, Ka and Ki are Hill activation and inactivation constants, respectively, and na and ni are the respective Hill coefficients. In the calculations, Bo was included as one of the variables.

In the competition studies, [3H]ryanodine binding was fitted with the equations
B = B<SUB>o</SUB> [Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>a</SUB></SUP>/([Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>a</SUB></SUP> + <IT>K</IT><SUP><IT>n</IT>a</SUP><SUB>Ca,eff</SUB> ) (2a)
<IT>K</IT><SUP><IT>n</IT><SUB>a</SUB></SUP><SUB>Ca,eff</SUB> = <IT>K</IT><SUP><IT>n</IT><SUB>a</SUB></SUP><SUB>Ca</SUB> [(<IT>K</IT><SUP><IT>n</IT><SUB>i</SUB></SUP><SUB>i</SUB> + [I]<SUP><IT>n</IT><SUB>i</SUB></SUP>)/<IT>K</IT><SUP><IT>n</IT><SUB>i</SUB></SUP><SUB>i</SUB>] (2b)
where B is the [3H]ryanodine binding value at a given [Ca2+], Bo is the binding maximum in the absence of the inhibitor (I), KCa is the Ca2+ activation constant, Ki is the inhibition constant of the inhibitor, and na and ni are the respective Hill coefficients.

Results are given as means ± SD, with the number of experiments in parentheses. Unless otherwise indicated, significance of differences of data was analyzed with Student's unpaired t-test. Differences were regarded as statistically significant at P < 0.05.

Materials. [3H]ryanodine was purchased from DuPont NEN. Unlabeled ryanodine was obtained from Calbiochem, and leupeptin and Pefabloc SC (a protease inhibitor) were from Boehringer Mannheim. All other chemicals were of analytical grade.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Determination of the effects of ionic composition on the Ca2+ dependence of [3H]ryanodine binding. In the present study, a majority of the binding experiments was performed at a relatively low incubation temperature of 12°C to minimize loss of RyR activity during the [3H]ryanodine-binding reaction. The Ca2+ dependence of [3H]ryanodine binding to RyR2 was determined in 0.25 M KCl medium at four different time points to obtain the time required for equilibrium binding (Fig. 1). [3H]ryanodine binding had a Ca2+ threshold of ~1 µM, was optimal at 10-100 µM, and showed some inactivation in the 1-10 mM Ca2+ range. In agreement with previous studies (6, 12, 17, 27, 29), these results suggest that the cardiac receptor has both high-affinity Ca2+ activation sites and low-affinity Ca2+ inactivation sites. The [3H]ryanodine binding curves of Fig. 1 show that an incubation time of 40 h was sufficient to obtain close to equilibrium binding conditions.


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Fig. 1.   Time course of [3H]ryanodine binding. Cardiac sarcoplasmic reticulum vesicles were incubated for the indicated times in medium containing 20 mM imidazole, pH 7.2, 0.25 M KCl, protease inhibitors (0.2 mM Pefabloc, 20 µM leupeptin), 1 nM [3H]ryanodine, and the indicated concentrations of free Ca2+. Specific [3H]ryanodine binding was determined as described under EXPERIMENTAL PROCEDURES. Continuous lines were obtained by fitting the data according to Eq. 1 in EXPERIMENTAL PROCEDURES.

Figure 2 compares the Ca2+ dependence of [3H]ryanodine binding to rabbit skeletal muscle and canine cardiac muscle SR vesicles in 0.25 M KCl and 0.25 M choline chloride media. In the KCl medium, two bell-shaped Ca2+ activation/inactivation curves were obtained. Equation 1, which describes the binding of Ca2+ to cooperatively interacting receptor activation and inactivation sites, provided a reasonable fit (lines) to the [3H]ryanodine binding data in the KCl medium. Values for the Hill activation constant for Ca2+ (KCaa) of 1.0 and 2.4 µM were obtained for RyR1 and RyR2, respectively. In agreement with previous reports (6, 17, 19), a substantially higher Ca2+ concentration was required for the inactivation of RyR2 than RyR1 [Hill inhibition constant for Ca2+ (KCai) = 5,860 and 300 µM, respectively]. Table 1 shows for RyR2 the averaged Hill constants and coefficients of several experiments.


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Fig. 2.   Ca2+ dependence of [3H]ryanodine binding to skeletal and cardiac muscle ryanodine receptors (RyRs) in KCl and choline chloride (CholCl) media. Specific [3H]ryanodine binding was determined as described in EXPERIMENTAL PROCEDURES in medium containing the indicated salts and concentrations of free Ca2+. Continuous lines for data in KCl (skeletal and cardiac RyRs) and choline chloride media (RyR2) were obtained by fitting data with Eq. 1 in EXPERIMENTAL PROCEDURES. Continuous line for skeletal RyR in choline chloride medium was obtained by assuming that choline ion is a weak Ca2+ agonist of the Ca2+-release channel and fitting data with Eq. 2 of Meissner et al. (18). Data from 1 representative experiment are shown. Averaged Hill constants and coefficients of indicated number of experiments with RyR2 are shown in Table 1.

                              
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Table 1.   Ca2+ dependence of [3H]ryanodine binding in media of different ionic strength and composition

Replacement of K+ with choline ion affected RyR2 differently than RyR1 in that a decrease rather than increase in the maximal level of [3H]ryanodine binding was observed (Fig. 2). Two other differences were that only close to background levels of binding to RyR2 could be detected at <10-7 M Ca2+ and [3H]ryanodine binding showed little inhibition at high Ca2+ concentrations. For RyR1, substantial levels of binding were observed at <10-8 M Ca2+. These could be described assuming that the choline ion is a weak, noncooperative Ca2+ channel agonist of RyR1 (Eq. 2 of Ref. 18) but not RyR2. Table 1 lists the apparent KCaa and na values for RyR2 in 0.25 M choline chloride medium.

Bell-shaped Ca2+ activation/inactivation curves were obtained for RyR2 in medium containing the chloride salts of Li+, Na+, K+, and Cs+ at a concentration of 0.25 M (Fig. 3). The binding levels were highest in KCl medium, intermediate in CsCl and NaCl media, and lowest in LiCl medium. Equation 1 provided a reasonable fit (lines) to the [3H]ryanodine-binding data. Table 1 summarizes the Hill constants and coefficients in the different media. The averaged KCaa in Li+, Na+, K+, and Cs+ media were 32, 7.8, 1.7, and 3.6 µM, respectively. The highest apparent Ca2+ binding affinities to the Ca2+ inactivation sites were obtained in LiCl and KCl media followed by NaCl and CsCl media. An na of close to two indicated that Ca2+ activated [3H]ryanodine binding by cooperative interactions involving at least two Ca2+ binding sites. These results suggest that monovalent cations affect RyR2 activity by interacting with receptor Ca2+ activation and inactivation sites.


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Fig. 3.   Ca2+ dependence of [3H]ryanodine binding to RyR2 in medium containing different inorganic monovalent cations. Specific [3H]ryanodine binding was determined as described in EXPERIMENTAL PROCEDURES in medium containing the indicated salts and concentrations of free Ca2+. Continuous lines were obtained by fitting data with Eq. 1 in EXPERIMENTAL PROCEDURES. One representative experiment is shown. Averaged Hill constants and coefficients of indicated number of experiments are shown in Table 1.

The [3H]ryanodine binding level and Ca2+ dependence of [3H]ryanodine binding were affected by the ionic strength and anionic composition of the assay medium (Fig. 4). An increase in KCl concentration from 0.1 to 1.0 M increased the maximal [3H]ryanodine-binding level about threefold, from 0.3 to 0.9 pmol/mg protein. Replacement of Cl- by MES- in the 0.25 M medium decreased the maximal binding level about twofold. Equation 1 provided a good fit (lines) to the [3H]ryanodine-binding data determined in the five media shown in Fig. 4. Ionic strength and anionic composition primarily affected the binding of Ca2+ to the Ca2+ inactivation sites (Table 1). The KCai increased ~10-fold as the KCl concentration was increased from 0.1 to 0.5 M KCl. A linear correlation coefficient of 0.96 (n = 15) indicated that the increase in KCai was highly significant. Replacement of Cl- by MES- decreased KCai 7.5-fold. Together, the data of Figs. 2-4 suggest that both the actions of monovalent cations and anions must be taken into account to understand the way in which Ca2+ activates and inactivates RyR2.


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Fig. 4.   Effects of ionic strength and anionic composition on Ca2+ dependence of [3H]ryanodine binding. Specific [3H]ryanodine binding was determined as described in EXPERIMENTAL PROCEDURES in medium containing the indicated concentrations of KCl, K+-MES, and free Ca2+. Continuous lines were obtained by fitting experimental data according to Eq. 1 in EXPERIMENTAL PROCEDURES. One representative experiment is shown. Averaged Hill constants and coefficients of indicated number of experiments are shown in Table 1.

Scatchard analysis indicated the presence of a single high-affinity [3H]ryanodine binding site. Changes in binding affinity without a major change in maximal binding value (3.0 ± 0.5 pmol/mg protein) were observed in 0.25 M KCl, 0.25 M choline chloride, and 0.25 M K+-MES media (all at 100 µM free Ca2+; not shown). This result suggests that the different binding levels of Figs. 2-4 reflect changes in binding affinities rather than the number of high-affinity binding sites.

Dependence of [3H]ryanodine binding on Sr2+ and Ba2+. Figure 5 compares the effects of Ca2+, Sr2+, and Ba2+ on [3H]ryanodine binding in 0.25 M KCl medium. Contaminating levels of Ca2+ (2-4 µM) were kept below 0.1 µM free Ca2+ by including 1 mM EGTA in the assay media. [3H]ryanodine binding had a higher threshold for Sr2+ than for Ca2+ (~10 µM vs. ~1 µM), was optimal at ~1 mM Sr2+, and showed some inactivation at 5 mM Sr2+. Ba2+ did not noticeably activate RyR2. The KCaa for Sr2+ (283 ± 21 µM, n = 3) and Ca2+ (1.7 ± 0.7 µM, Table 1) differed by more than a factor of 100. The Hill inactivation constant and Hill coefficients for Sr2+ did not significantly differ from those shown for Ca2+ in Table 1.


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Fig. 5.   Dependence of [3H]ryanodine binding on divalent cations. Specific [3H]ryanodine binding was determined as described in EXPERIMENTAL PROCEDURES in 0.25 M KCl medium containing the indicated concentrations of free divalent cations (X2+). Continuous lines were obtained by fitting data with Eq. 1 in EXPERIMENTAL PROCEDURES. One representative experiment is shown. Averaged Hill constants and coefficients for Sr2+ were as follows: activation constant = 283 ± 21, activation coefficient = 2.4 ± 0.6, inhibition constant = 7,021 ± 1,258, and inhibition coefficient = 1.5 ± 0.4 (n = 3).

Effects of AMP and caffeine on Ca2+ dependence of [3H]ryanodine binding. The effects of two channel activators, AMP and caffeine (3, 15), on the Ca2+ dependence of [3H]ryanodine binding were determined in 0.25 M KCl medium. In these studies, AMP rather than ATP or a nonhydrolyzable ATP analog was used because AMP, in contrast to adenosine triphosphates, binds Ca2+ with negligible affinity. AMP (5 mM) did not significantly affect the Ca2+ binding affinity of the Ca2+ activation sites (Table 1). Ca2+ binding affinity to the Ca2+ inactivation sites of the receptor decreased approximately twofold. Caffeine (20 mM) increased the apparent Ca2+ affinity of the Ca2+ activation sites of the receptor by more than 15-fold. KCai increased approximately twofold (Table 1). Similar changes in the Hill constants and coefficients were obtained when the effects of 5 mM AMP and 20 mM caffeine were examined in 0.25 M LiCl, 0.25 M NaCl, or 0.25 M CsCl media (not shown).

Interaction of mono- and divalent cations with high-affinity Ca2+ activation sites of RyR2. We determined whether monovalent cations inhibit RyR2 activity by competing with Ca2+ for the Ca2+ activation sites, as observed for RyR1 (18). In these studies, we took advantage of the observation that, at low Ca2+ concentrations, the [3H]ryanodine-binding level was the highest in choline chloride medium (Fig. 2) by assessing the inhibitory effects of K+ in 0.5 M choline chloride medium containing varying low concentrations of free Ca2+ (Fig. 6). To interpret the [3H]ryanodine-binding data, three simple alternative types of inhibition were considered, namely, that K+ was a competitive, noncompetitive, or uncompetitive inhibitor. Choline ion was assumed to be without effect on RyR2 activity. As observed for RyR1 (18), the RyR2 binding data could not be fitted assuming non- or uncompetitive inhibition (not shown) but could be well fitted when it was assumed that K+ inhibited [3H]ryanodine binding by a competitive mechanism (Eqs. 2a and 2b, EXPERIMENTAL PROCEDURES) (Fig. 6). Table 2 summarizes the derived Hill Ca2+ activation and inactivation constants and coefficients for K+, as well as for Li+, Mg2+, and Ba2+. Similar Ca2+ activation constants were obtained for the four cations. Mg2+ was the most effective in inhibiting [3H]ryanodine binding (Ki = 0.007 mM). Ba2+ was ~100-fold less effective than Mg2+ in competing with Ca2+ for the Ca2+ activation sites. Of the two monovalent cations tested, Li+ was more effective than K+ in inhibiting [3H]ryanodine binding (Ki = 21 and 31 mM, respectively). The latter data are consistent with the observation that Ca2+ concentrations required to half-maximally activate [3H]ryanodine binding were higher for LiCl than for KCl medium (Fig. 3 and Table 1).


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Fig. 6.   Inhibition of [3H]ryanodine binding by K+. Specific [3H]ryanodine binding was determined in 0.5 M choline chloride medium containing the indicated concentrations of K+ and free Ca2+. Total Cl- concentration was kept constant by adjusting choline ion concentration so that choline ion concentration + K+ concentration = 0.5 M. In A and B, continuous lines were obtained with Eqs. 2a and 2b in EXPERIMENTAL PROCEDURES, using a single set of parameters for all data. In B, data were plotted using derived Hill inactivation coefficient of 1.6. One representative experiment is shown. Averaged Hill constants and coefficients of 3 separate experiments are shown in Table 2.

                              
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Table 2.   Inhibition of [3H]ryanodine binding by mono- and divalent cations

Effects of magnesium beta ,gamma -methyleneadenosine 5'-triphosphate and glutathione on Ca2+ dependence of [3H]ryanodine binding. Attempts were made to determine the Ca2+ dependence of [3H]ryanodine binding under more physiologically relevant conditions by performing experiments at room temperature in 0.125 M KCl medium in the presence of magnesium beta ,gamma -methyleneadenosine 5'-triphosphate (AMP-PCP) and/or glutathione. In rat heart muscle, the myofibrillar concentrations of K+, Na+, and Cl- were estimated to be 74-128, 4-11, and 11-25 mM, respectively (25). The total ATP and free Mg2+ concentrations in intact hearts ranged from 5 to 10 mM (8, 10) and from 0.7 to 1.0 mM (20), respectively. Figure 7 compares the Ca2+ dependence of [3H]ryanodine binding at room temperature (~24°C) in 0.125 M KCl medium in the absence and presence of 5 mM AMP-PCP and 5 mM Mg2+ (~0.7 mM free Mg2+ at ~1 µM Ca2+). Addition of 5 mM MgAMP-PCP shifted the Ca2+ activation curve to the right and rendered the channel less sensitive to inactivation at Ca2+ concentrations of up to 1 mM. Free Ca2+ concentrations in excess of 1 mM were not tested because of difficulties of keeping Ca2+ in solution in 5 mM AMP-PCP medium. RyRs contain sulfhydryl groups that are important to their function (30). These may be in a reduced state in vivo because cells maintain a reducing environment by containing thiol-reducing compounds, the most abundant being glutathione (4). Figure 7 shows that the addition of a relatively high concentration of glutathione (5 mM) had a minimal effect on the levels and Ca2+ dependence of [3H]ryanodine binding in the 0.125 M KCl medium containing and lacking 5 mM MgAMP-PCP.


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Fig. 7.   Effects of magnesium beta ,gamma -methyleneadenosine 5'-triphosphate (MgAMP-PCP) and glutathione on Ca2+ dependence of [3H]ryanodine binding. Specific [3H]ryanodine binding was determined at room temperature (~24°C) in 0.125 M KCl medium containing the indicated concentrations of MgAMP-PCP, glutathione (GSH), and free Ca2+. Continuous lines were obtained by fitting data with Eq. 1 in EXPERIMENTAL PROCEDURES. One representative experiment is shown. Averaged Hill constants and coefficients of indicated number of experiments are shown in Table 1.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, the effects of mono- and divalent ions on the cardiac muscle RyR (Ca2+ release) channel were investigated by determining the Ca2+ dependence of [3H]ryanodine binding. These studies led to two new observations that may have important implications for the mechanism of SR Ca2+ release in cardiac muscle. First, the results of this study show that the affinity of Ca2+ binding to the Ca2+ activation sites and inactivation sites of RyR2 is dependent on ionic composition. Second, competition studies indicate that physiologically relevant mono- and divalent cations such as K+ and Mg2+ inhibit [3H]ryanodine binding by competitive binding to Ca2+ activation sites.

The Ca2+ dependence of Ca2+-release channel activity was examined by measuring [3H]ryanodine binding to SR vesicles. A low [3H]ryanodine concentration was used to limit binding to a single high-affinity receptor site. Under these conditions, [3H]ryanodine binding is a sensitive method in assessing RyR activity (3, 15). Although single-channel measurements provide more direct information on Ca2+-release channel activity than [3H]ryanodine binding measurements and channels gate on a much faster time scale than they bind [3H]ryanodine (µs to ms vs. min to h), an advantage of [3H]ryanodine binding is that it allows the study of a large number of ionic conditions.

One goal of this study was to find out how mono- and divalent ions affect the cardiac RyR. A second goal was to compare their effects on the cardiac and skeletal RyRs. Such a comparison is of interest because the two receptor isoforms are differently regulated by Ca2+ and other effectors. Because our studies with the skeletal muscle RyR were mostly performed with 0.25 M salt solutions (18), we chose 0.25 M solutions for most of our studies with the cardiac RyR as well. The binding parameters of RyR1 and RyR2 are summarized in Table 3 and are compared below.

                              
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Table 3.   Regulation of skeletal muscle and cardiac muscle RyRs by Ca2+ and monovalent ions

The effects of monovalent cations on the Ca2+ dependence of [3H]ryanodine binding were determined by using the chloride salts of Li+, Na+, K+, and Cs+. The same order of apparent Ca2+ binding affinities was obtained for the two receptor isoforms, however, with RyR1 displaying higher apparent Ca2+ binding affinities than RyR2 (Table 3). Values of na of 1.9 and greater indicate that Ca2+ activated RyR2 by a cooperative interaction involving at least two Ca2+ activation sites. In choline chloride medium, the Ca2+ dependence of [3H]ryanodine binding differed in that, at free Ca2+ <10-8 M, significant levels of [3H]ryanodine binding were observed for RyR1 but not RyR2, which suggests an apparent agonist action of choline ion for RyR1 (18) but not RyR2. Another difference was that at micromolar Ca2+ concentrations, the [3H]ryanodine binding level of RyR2 was higher in KCl than choline chloride medium, whereas the opposite was observed for RyR1 (Fig. 2). This result suggests that monovalent cations may influence the activity of RyR2 in additional ways, not indicated by the scheme in EXPERIMENTAL PROCEDURES.

Differences were observed in apparent binding affinities of Ca2+ to the inactivation sites of the cardiac and skeletal muscle RyR and the order of the affinities in the various monovalent cation media (Table 3). For RyR2, the Ca2+ affinities were similar in LiCl, NaCl, and KCl media. In CsCl and choline chloride media, decreased affinities were observed. Values for ni of Ca2+ binding to the cardiac Ca2+ inactivation sites ranged from 0.7 to 1.4, suggesting that Ca2+ bound with no or low cooperativity to the Ca2+ inactivation sites. Our results are consistent with previous SR vesicle Ca2+ flux (2, 17), single-channel (12, 27), and [3H]ryanodine binding (2, 6, 27) measurements; these studies also showed an activation and inactivation of RyR2 by Ca2+ in K+ or Cs+ media, but they are at variance with a study that failed to show an inhibition of single-channel activities by 10 mM Ca2+ (2).

Major differences were observed in the action of divalent cations. Ca2+ and Sr2+ activated RyR2, whereas Mg2+ and Ba2+ inhibited [3H]ryanodine binding by competing with Ca2+ for the Ca2+ activation sites. Divalent cations similarly affected [3H]ryanodine binding to RyR1 (D. A. Pasek and G. Meissner, unpublished observations) (Table 3). The results of the present study also indicate competitive binding of monovalent cations to the Ca2+ activation sites of RyR2 to affect the receptor's Ca2+ sensitivity. The inhibition constants of K+ and Mg2+ were 42 and 0.013 mM for RyR1 and 31 and 0.007 mM for RyR2, respectively (Table 3). The intracellular K+ and free Mg2+ concentrations in cardiomyocytes have been estimated to be 74-128 mM (25) and 0.4-0.8 mM (20), respectively. Therefore, in cardiomyocytes a small fraction of the Ca2+-release channel's Ca2+ activation sites may be occupied by K+ instead of Mg2+ at rest. Occupation of some sites by K+ may be of physiological significance because Ca2+ may bind faster to channel sites occupied by K+ than sites occupied by more tightly bound Mg2+.

The activity of RyR1 is greatly affected by the anionic composition of assay medium (6, 7, 13, 18). In 0.25 M K+-MES, RyR1 bound only close to baseline levels of [3H]ryanodine (18). Substitution of Cl- by MES- also reduced the [3H]ryanodine binding levels of RyR2 but to a lesser extent. Substitution of Cl- by MES- appeared to primarily affect [3H]ryanodine binding to RyR2 by causing a decrease in KCai. Increase of KCl concentration from 0.1 to 0.5 M had an opposite effect by increasing KCai of RyR2. It therefore appears that RyR2 is also sensitive to regulation by anions.

The Ca2+ dependence of [3H]ryanodine binding was analyzed under conditions that were thought to approximate those in the myocardium. These included the use of a 0.125 M KCl medium containing 5 mM MgAMP-PCP and/or 5 mM glutathione (Fig. 7). The addition of 5 mM MgAMP-PCP modified the Ca2+ dependence of RyR2 by rendering the channel less sensitive to activation at Ca2+ concentrations of <10 µM and less sensitive to Ca2+ inactivation at Ca2+ concentrations of >100 µM. Glutathione has recently been shown to reduce the affinity and maximal binding values but not Ca2+ dependence of [3H]ryanodine binding to skeletal muscle RyR (28). At variance with this result, the addition of 5 mM glutathione had a minimal effect on both the level and Ca2+ dependence of [3H]ryanodine binding to cardiac RyR.

In conclusion, the results of this study show that monovalent ions affect in a similar, but not identical, manner the in vitro regulation of the cardiac and skeletal muscle Ca2+-release channels by Ca2+. In the absence of other activating ligands, Ca2+ activates the cardiac channel to a greater extent than the skeletal channel (17). Two possible explanations for this difference that we considered in our experiments were an increased Ca2+ affinity of the Ca2+ activation sites and decreased Ca2+ affinity of the Ca2+ inactivation sites. A consequence of a widened "Ca2+ window" is that higher levels of receptor activation can be achieved. Our results indicate that Ca2+ bind to the Ca2+ inactivation sites of the cardiac receptors with a lower apparent affinity than those in the skeletal muscle receptor, whereas Ca2+ bind with a somewhat higher apparent affinity to the Ca2+ activation sites of the skeletal than cardiac muscle RyR. Accordingly, differences in Ca2+ binding affinity to the Ca2+ inactivation sites may be primarily responsible for the different activity levels of the two receptors.

    ACKNOWLEDGEMENTS

Support by National Institutes of Health Grants HL-27430 and AR-18687 is gratefully acknowledged.

    FOOTNOTES

Address for reprint requests: G. Meissner, Dept. of Biochemistry and Biophysics, Univ. of North Carolina, Chapel Hill, NC 27599-7260.

Received 14 May 1997; accepted in final form 12 September 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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