INTRODUCTION

Top Abstract Introduction Procedures Results 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 (C, SCN, I, ) 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

Top Abstract Introduction Procedures Results Discussion References

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(-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

(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 (A_Ca) and Ca²⁺-inactivated (_CaI_Ca) 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.

(1)

(2a)

(2b)

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

Top 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.

View larger version (19K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

7

8

View larger version (24K): [in this window] [in a new window]   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.

View larger version (25K): [in this window] [in a new window]   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.

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 K^Ca_a 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.

View larger version (14K): [in this window] [in a new window]   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. K^Ca_i 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 (K_i = 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 (K_i = 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).

View larger version (20K): [in this window] [in a new window]   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.

Effects of magnesium ,-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 ,-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.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effects of magnesium ,-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

Top 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.

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 n_a 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⁺ <10⁸ 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 n_i 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 K^Ca_i. Increase of KCl concentration from 0.1 to 0.5 M had an opposite effect by increasing K^Ca_i 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.