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(Circulation Research. 1996;79:1100-1109.)
© 1996 American Heart Association, Inc.

Articles

Regulation of Cardiac Ca²⁺ Release Channel (Ryanodine Receptor) by Ca²⁺, H⁺, Mg²⁺, and Adenine Nucleotides Under Normal and Simulated Ischemic Conditions

Le Xu, Geoffrey Mann, Gerhard Meissner

the Department of Biochemistry and Biophysics and the Department of Physiology, University of North Carolina, Chapel Hill.

Correspondence to Gerhard Meissner, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260.

	Abstract

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In myocardial ischemia, pH_i and [ATP] fall, whereas the free [Ca²⁺] and [Mg²⁺] rise. The effects of these changes on cardiac Ca²⁺ release channel (ryanodine receptor) activity were investigated in [³H]ryanodine binding and single-channel measurements, using isolated membrane and purified channel preparations. In the absence of the two channel ligands Mg²⁺ and ATP, cardiac Ca²⁺ release channels were half-maximally activated at pH 7.4 by 4 µmol/L cytosolic Ca²⁺ and half-maximally inhibited by 9 mmol/L cytosolic Ca²⁺. Regulation of channel activity by Ca²⁺ was modulated by Mg²⁺ and ATP. Single-channel activities were more sensitive to a change of cytosolic pH than SR lumenal pH. Reduction in lumenal and/or cytosolic pH from 7.3 to 6.5 and 6.0 resulted in decreased single-channel activities without a change in single-channel conductance. [³H]Ryanodine binding measurements also indicated that acidosis impairs cardiac Ca²⁺ release channel activity. Mg²⁺ and adenine nucleotide concentrations regulated the extent of inhibition and the Ca²⁺ dependence of binding. In the presence of 5 mmol/L Mg²⁺ and 5 mmol/L ß,-methyleneadenosine 5'-triphosphate (AMPPCP, a nonhydrolyzable ATP analogue), the free [Ca²⁺] for half-maximal [³H]ryanodine binding was increased from 1.9 µmol/L at pH 7.3 to 36 µmol/L at pH 6.5 and to 89 µmol/L at pH 6.2. These results suggest that ionic and metabolic changes that might be expected to affect sarcoplasmic reticulum Ca²⁺ release channel activity in ischemic myocardium include an altered Ca²⁺ sensitivity of the channel, a fall in pH, and a loss of the high-energy adenine nucleotide pool, leading to an increased inhibition by Mg²⁺.

Key Words: excitation-contraction coupling • ischemia

	Introduction

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The rapid intracellular release and sequestration of Ca²⁺ ions by the SR is essential to the process of cardiac muscle contraction and relaxation.^{1 2} The rapid release of Ca²⁺ is mediated by Ca²⁺ release channels, also known as RYRs, because they have the ability to bind the plant alkaloid ryanodine with high affinity and specificity.^{3 4} In cardiac muscle, the influx of Ca²⁺ via a voltage-sensitive dihydropyridine receptor/Ca²⁺ channel (L-type) triggers the massive release of Ca²⁺ by opening the SR Ca²⁺ release channel. This process is known as Ca²⁺-induced Ca²⁺ release. The released Ca²⁺ ions are again sequestered by the SR through the action of a Ca²⁺-transport ATPase, which restores the myoplasmic [Ca²⁺] from 10^-6 to 10^-5 to 10^-7 mol/L.⁵

The cardiac muscle RYR/Ca²⁺ release channel has been purified as a 30S protein complex composed of four identical polypeptides of 5000 amino acids each. The very large subunits presumably allow an extensive pattern of regulation of channel activity, because the channel complex is regulated by, in addition to Ca²⁺, various endogenous and exogenous effector molecules.^{3 4} These include small diffusible endogenous molecules such as Mg²⁺, ATP, and lipid intermediates, proteins such as calmodulin, cAMP-dependent and Ca²⁺/calmodulin-dependent protein kinases, phosphatases, triadin (a 90-kD junctional SR protein), and immunophilins (FK506 binding proteins), as well as exogenous effectors such as caffeine and ryanodine.

Regulation of the cardiac Ca²⁺ release channel by multiple endogenous effectors suggests that changes in the intracellular milieu affect the channel's activity in the heart. Major changes in ionic milieu and metabolites occur especially in myocardial ischemia. These include a fall of pH_i to a value as low as pH 5.8,^{6 7 8} depletion of the high-energy adenine nucleotide pool,^{8 9 10} and increases in cytosolic free [Ca²⁺]^{6 8 10 11 12} and [Mg²⁺].¹³ A change in each of these parameters has been shown to affect cardiac Ca²⁺ release channel activity. SR vesicle–Ca²⁺ efflux, single-channel, and [³H]ryanodine binding measurements have indicated that cardiac Ca²⁺ release channel activity is critically dependent on cytosolic [Ca²⁺], modulated by Mg²⁺ and ATP,^{3 4} and greatly decreased when the pH is lowered from pH 7 to pH 6.^{14 15 16} However, it is unclear how Ca²⁺ release channel activity is affected by a combination of the concurrent ionic and metabolic changes that are thought to occur in ischemic and reperfused hearts.

In the present study, using isolated membrane fractions and purified channel preparations, we examined the effects of a changing ionic milieu on cardiac Ca²⁺ release channel activity. The effects of acidosis on channel activity were determined in the presence and absence of the two channel ligands Mg²⁺ and ATP at [Ca²⁺] levels corresponding to those in normal and ischemic hearts. Our results showed that acidosis impairs cardiac muscle Ca²⁺ release channel activity but also suggested that the extent of this impairment is likely determined by the myoplasmic [adenine nucleotide], [Ca²⁺], and [Mg²⁺] in ischemic myocardium.

	Materials and Methods

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Materials
[³H]Ryanodine was purchased from Du Pont–NEN, and unlabeled ryanodine was from Calbiochem. The nonhydrolyzable ATP analogue AMPPCP was obtained from Sigma Chemical Co; leupeptin and Pefabloc (a protease inhibitor), from Boehringer Mannheim; and Ca²⁺ standards, from Molecular Probes. The chemicals to prepare a Mg²⁺-selective membrane were obtained from Fluka. All other chemicals were of analytical grade.

Preparation of SR Vesicles and Purification of Ca²⁺ Release Channels
Canine cardiac muscle SR vesicle fractions enriched in [³H]ryanodine binding and Ca²⁺ release channel activities were prepared in the presence of protease inhibitors (100 nmol/L aprotinin, 1 µmol/L leupeptin, 1 µmol/L pepstatin, 1 mmol/L benzamidine, and 0.2 mmol/L phenylmethylsulfonyl fluoride), as described previously.¹⁴ The CHAPS-solubilized canine heart 30S Ca²⁺ release channel complex was isolated by rate density centrifugation¹⁷ and reconstituted into proteoliposomes by removal of CHAPS by dialysis.¹⁸

Single-Channel Measurements
Single-channel measurements were performed at 23°C to 25°C by fusing proteoliposomes containing the purified cardiac muscle Ca²⁺ release channel with Mueller-Rudin–type bilayers containing phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine at a ratio of 5:3:2 (25 mg of total phospholipid per milliliter n-decane).¹⁸ pH measurements were performed with bilayers containing phosphatidylethanolamine and phosphatidylcholine at a ratio of 1:1. The side of the bilayer to which the proteoliposomes were added was defined as the cis side. A strong dependence of single-channel activities on cis [Ca²⁺] (see "Results") indicated that the large cytosolic ("foot") region of the channel faced the cis chamber in a majority (>98%) of our recordings. The trans side of the bilayer was defined as ground. Single channels were recorded in a symmetrical KCl buffer solution (0.25 mol/L KCl and 20 mmol/L potassium HEPES, pH 7.4) containing the additions indicated in the text. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed using a single threshold level set at 50% of the current amplitude. A missed-events correction was not used. Data acquisition and analysis were performed with a commercially available software package (pClamp 6.0.1, Axon Instruments) using an IBM-compatible 486 computer and 12-bit A/D-D/A converter (Digidata 1200, Axon Instruments). Data files were acquired by continuous Fetchex mode (file length, 2 minutes) or with Clampex pulse protocol (60 episodes of 750 milliseconds with 1-second intervals between episodes). Some data were acquired by using both protocols without a significant difference.

[³H]Ryanodine Binding
Unless otherwise indicated, samples were incubated with 2 nmol/L [³H]ryanodine in media containing 0.1 mol/L KCl, 20 mmol/L imidazole, pH 7.1, 0.2 mmol/L Pefabloc, 20 µmol/L leupeptin, and the indicated concentrations of Ca²⁺, Mg²⁺, and adenine nucleotide. A relatively low incubation temperature of 12°C was used to minimize receptor inactivation during the binding reaction. Nonspecific binding was determined using a 1000-fold excess of unlabeled ryanodine. After 24 to 36 hours, aliquots of the samples were diluted with 20 vol ice-cold water and placed on Whatman GF/B filters preincubated with 2% polyethyleneimine in water. Filters were washed with 3x5 mL medium containing ice-cold 0.1 mol/L KCl and 1 mmol/L potassium PIPES, pH 7.0. The radioactivity remaining with the filters was determined by liquid scintillation counting to obtain bound [³H]ryanodine.

Other Biochemical Assays
Protein concentrations were determined by the Lowry method using BSA as the protein standard. Free [Ca²⁺] levels were obtained by including in the solutions the appropriate amounts of Ca²⁺ and Ca²⁺ chelators. Free [Ca²⁺] levels of >1 µmol/L were determined at 24°C and at the indicated pH values with a Ca²⁺-selective electrode (World Precision Instruments, Inc), using Ca²⁺ standards obtained from Molecular Probes. Free [Ca²⁺] levels of <1 µmol/L were obtained using the stability constants and computer program published by Shoenmakers et al.¹⁹ Free [Mg²⁺] levels were determined with a Mg²⁺ electrode using the Mg²⁺-selective ionophore ETH 7025. Membranes (membrane II) were prepared as described by Spichiger et al.²⁰ Addition of SR vesicles to the binding media did not noticeably change the free divalent cation concentrations. In [³H]ryanodine binding measurements, an incubation temperature of 12°C was used, whereas free [Ca²⁺] and [Mg²⁺] were determined at 24°C. The change in temperature affected the free ion concentrations by changing the pH and the ion complexation constants. These changes were accounted for with the use of a computer program.¹⁹

Data Analysis
Results are given as mean±SE. Significance of differences of data was analyzed with paired Student's t test. Differences were regarded to be statistically significant at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dependence of Single-Channel Activities on Ca²⁺, Mg²⁺, and ATP at pH 7.4
Proteoliposomes containing the purified cardiac Ca²⁺ release channel were fused with planar lipid bilayers and recorded in symmetrical 0.25 mol/L KCl buffer. The use of K⁺ rather than Ca²⁺ as a current carrier resulted in a higher resolution of single-channel events. The use of Ca²⁺ as a current carrier either would have required filtering at a low frequency²¹ or would have resulted in the buildup of high nonphysiological [Ca²⁺] levels near the mouth of the channel on the cytosolic side,²² thus complicating the analysis of the regulation of the cardiac Ca²⁺ release channel by trigger Ca²⁺. The cardiac release channel is impermeant to Cl^-. In the present study, we used preparations that showed infrequent subconductance states for the channel openings (<10%). With K⁺ as the current carrier, single-channel conductance was 770 pS.²³

In Fig 1A, a single cardiac Ca²⁺ release channel was recorded in the presence of three different cis (cytosolic) [Ca²⁺] levels at a holding potential of +35 mV. The free trans (SR lumenal) [Ca²⁺] was 11 µmol/L. Infrequent, brief, and often not fully resolved channel openings were observed with 0.8 µmol/L Ca²⁺ in the cis chamber (Fig 1A, top trace). Elevation of cytosolic [Ca²⁺] from 0.8 to 11 µmol/L (Fig 1A, middle trace) greatly increased P_o. The bottom trace of Fig 1A shows that a further increase from 11 µmol/L Ca²⁺ to 1 mmol/L Ca²⁺ did not substantially affect P_o.

View larger version (31K): [in this window] [in a new window]   Figure 1. Dependence of single-channel activities on cytosolic Ca2+. A, Shown are three current traces of a single purified cardiac Ca2+ release channel at three different cytosolic [Ca2+] levels. Single-channel currents, shown as upward deflections from closed levels (bars on left), were recorded in symmetrical 0.25 mol/L KCl, 20 mmol/L potassium HEPES (pH 7.4) media containing 0.56 mmol/L EGTA, and Ca2+ to yield free cytosolic [Ca2+] of 0.8 µmol/L (top trace), 11 µmol/L (middle trace), and 1 mmol/L (bottom trace). The trans chamber contained 11 µmol/L free Ca2+ (0.5 mmol/L EGTA and 0.5 mmol/L Ca2+), and the holding potential was +35 mV. B, Single-channel Po values were determined as in panel A at +35 mV () and -35 mV (). Values are the mean±SE of 5 to 23 experiments. Solid and dotted lines were obtained according to the following equation: , where Pmax is the maximal Po, and the other terms have their usual meaning. Derived Hill activation (KHa) and inactivation (KHi) constants and coefficients are shown in Table 1.

Fig 1B describes the dependence of single-channel P_o on cytosolic [Ca²⁺] levels ranging from 10^-7 to 10^-2 mol/L at holding potentials of +35 and -35 mV. At both potentials, P_o was close to zero at 10^-7 mol/L Ca²⁺, was maximal in the micromolar [Ca²⁺] range, and decreased again as the cytosolic [Ca²⁺] was raised to 10 mmol/L.

The data of Fig 1B were fitted by a simple scheme assuming that the cardiac Ca²⁺ release channel possesses cooperatively interacting high-affinity activation and low-affinity inactivation Ca²⁺ binding sites.

In the above scheme, the Ca²⁺ release channel (RYR) is present in its closed Ca²⁺-free form (designated _iRYR_a at [Ca²⁺] of <0.1 µmol/L) and its Ca²⁺-activated (_iRYR_Ca) and Ca²⁺-inactivated (_CaRYR_Ca) forms at micromolar and millimolar [Ca²⁺], respectively. The tetrameric Ca²⁺ release channel complex likely contains at least four Ca²⁺ activation and four Ca²⁺ inactivation sites (one each per subunit); however, only one of these is shown in the above scheme.

The Ca²⁺ dependence of P_o at pH 7.4 was calculated according to the equation given in the legend of Fig 1. A good fit was obtained between the measured (solid and open squares in Fig 1B) and calculated (solid and dotted lines in Fig 1B) values. Channels were activated by Ca²⁺ at +35 and -35 mV with K_Ha of 4.5 and 4.0 µmol/L and inhibited with K_Hi of 6.5 and 11.1 mmol/L cytosolic Ca²⁺, respectively (Table 1). n_Ha of 1.3 suggested that Ca²⁺ activated the cardiac channel by a weak cooperative interaction.

View this table: [in this window] [in a new window]   Table 1. Dependence of Single-Channel Activities and [3H]Ryanodine Binding on Ca2+ and Mg2+

The total [ATP] and free [Mg²⁺] in normal intact hearts have been reported to range from 5 to 10 mmol/L^{24 25} and from 0.7 to 1.0 mmol/L,¹³ respectively. Fig 2A shows the dependence of channel activity on total cytosolic [ATP] at a free cytosolic [Ca²⁺] of 15 µmol/L and constant free cytosolic [Mg²⁺] of 1.0 mmol/L. A small increase in P_o was observed when the total [ATP] in the cis chamber was raised from 0 to 1 mmol/L. A further increase in the total [ATP] to 5 mmol/L was without a significant effect on P_o. Fig 2B compares the dependence of channel activity on free cytosolic [Mg²⁺] at a free cytosolic [Ca²⁺] of 15 µmol/L in the presence and absence of a total cytosolic [ATP] of 5 mmol/L. An increase in free [Mg²⁺] was somewhat more effective in inhibiting channel activity in the absence than the presence of ATP. Channel activity was half maximally inhibited at 2.3 mmol/L Mg²⁺ in the absence of ATP and at 5.0 mmol/L free Mg²⁺ in the presence of 5 mmol/L ATP (Table 1). n_Hi values of 1.5 and 2.5, respectively, suggested that Mg²⁺ inhibited channel activity by a cooperative interaction.

View larger version (13K): [in this window] [in a new window]   Figure 2. Dependence of single-channel activities on [ATP] and free [Mg2+]. A, Single-channel activities were determined at +50 mV as in Fig 1 in the presence of 15 µmol/L free cytosolic Ca2+, the indicated total cytosolic [ATP] levels, and Mg2+ to yield a free cytosolic [Mg2+] of 1.0 mmol/L. Lumenal [Ca2+] was 15 µmol/L. Values are the mean±SE of seven experiments. *P<.05 vs control (-ATP). B, Single channels were recorded as in panel A in the absence (, four experiments) and presence (, five experiments) of a total cytosolic [ATP] of 5 mmol/L and the indicated concentrations of free Mg2+. Solid and dotted lines were obtained according to the following equation: . Derived Hill inhibition constants and coefficients are shown in Table 1. *P<.05 vs control (absence of Mg2+).

Fig 3 shows that the presence of 2 mmol/L cytosolic Mg²⁺-ATP modifies the dependence of the cardiac channel on cytosolic [Ca²⁺]. These experiments were carried out with 5 mmol/L Ca²⁺ in the trans bilayer chamber to better simulate the SR lumenal [Ca²⁺] in the myocardium. In the absence of Mg²⁺-ATP, channel activities showed a similar dependence on cytosolic Ca²⁺ in the presence of 11 µmol/L (Fig 1B) and 5 mmol/L (Fig 3, circles) lumenal Ca²⁺. Channel activities were low at cytosolic [Ca²⁺] of <1 µmol/L, showed a strong Ca²⁺ dependence between 1 and 10 µmol/L, and reached a maximal value at 30 µmol/L cytosolic Ca²⁺. A further increase in cytosolic [Ca²⁺] resulted in reduced channel activities. In the presence of 2 mmol/L cytosolic Mg²⁺-ATP, P_o also reached a maximal value at 30 µmol/L cytosolic Ca²⁺. However, in the presence of 2 mmol/L Mg²⁺-ATP, P_o did not decrease at cytosolic [Ca²⁺] as high as 10 mmol/L (Fig 3, triangles).

View larger version (18K): [in this window] [in a new window]   Figure 3. Dependence of single-channel Po on cytosolic [Ca2+] in the presence of 2 mmol/L Mg2+-ATP. Single-channel activities were determined at +50 mV as indicated in Fig 1 in the presence () and absence () of 2 mmol/L cytosolic Mg2+-ATP and the indicated concentrations of free cytosolic Ca2+. Lumenal [Ca2+] was 5 mmol/L. Values are the mean±SE of six experiments for each condition.

Effect of pH on [³H]Ryanodine Binding
The experiments of Figs 1 through 3 were performed at pH 7.4. The effects of a decrease in pH_i on Ca²⁺ release channel activity were first assessed in [³H]ryanodine binding measurements. The neutral plant alkaloid ryanodine has been shown to bind with high affinity and specificity to the skeletal muscle and cardiac muscle Ca²⁺ release channels. As a general rule, conditions that activated the channel, such as the presence of micromolar Ca²⁺ or millimolar adenine nucleotide, were found to increase the affinity of [³H]ryanodine binding to the channels.^{3 4} We determined the effects of pH on [³H]ryanodine binding before testing the effects of pH on single-channel activities, because it is more difficult to study this parameter at the single-channel level.

The Ca²⁺ activation profiles of [³H]ryanodine binding to cardiac SR membranes were determined in the absence (Fig 4A) and presence (Fig 4B) of 5 mmol/L Mg²⁺ and 5 mmol/L AMPPCP (a nonhydrolyzable ATP analogue) at three different pH values. In the absence of Mg-AMPPCP at pH 7.3, [³H]ryanodine binding levels were close to background at [Ca²⁺] of <1 µmol/L, were strongly dependent on free Ca²⁺ at concentrations between 1 and 10 µmol/L, and reached a maximal level at 50 µmol/L Ca²⁺ (Fig 4A). Free [Ca²⁺] of >100 µmol/L resulted in a reduction of [³H]ryanodine binding. The data (Fig 4A, solid circles) could be well fitted by the equation used to describe the Ca²⁺ dependence of single-channel activities (Fig 1B). Channels were activated with a K_Ha of 2.8 µmol/L and inhibited with a K_Hi of 2.1 mmol/L (Table 1). An n_Ha value of 2.1 suggested that Ca²⁺ activated the cardiac channel by a cooperative interaction. A similar Ca²⁺ dependence of [³H]ryanodine binding (Fig 4A) and single-channel activities (Fig 1B) confirmed that [³H]ryanodine binding was a good probe for assessing the Ca²⁺ sensitivity of the cardiac Ca²⁺ release channel.

View larger version (18K): [in this window] [in a new window]   Figure 4. Effects of [Ca2+] and pH on [3H]ryanodine binding to cardiac RYRs. Specific [3H]ryanodine binding was determined in the absence (A) and presence (B) of 5 mmol/L Mg2+-AMPPCP at pH 7.3 (), pH 6.5 (), and pH 6.2 () in 0.1 mol/L KCl media containing 20 mmol/L potassium PIPES (, ) or 20 mmol/L potassium MES (), 1 mmol/L EGTA (or 0.5 mmol/L BAPTA and 1 mmol/L nitriloacetic acid), and Ca2+ to yield the indicated concentrations of free Ca2+. Free [Mg2+] levels at <1 µmol/L free Ca2+ were 0.6 mmol/L (at pH 7.3), 1.3 mmol/L (pH 6.5), and 2.1 mmol/L (pH 6.2). Values are the mean±SE of four experiments. The solid line at pH 7.3 in panel A was obtained according to the following equation: . The solid lines in panel B were obtained according to the following equation: B=Bmax/[1+(KHa/[Ca2+])nHa], where Bmax is the maximal [3H]ryanodine binding value, and the other terms have their usual meaning. Derived Hill constants and coefficients are shown in Table 1. In panel A, data at pH 6.5 and 6.2 could not be well fitted by the above equation.

In the absence of Mg²⁺ and AMPPCP, [³H]ryanodine binding was greatly reduced when the pH of the assay media was reduced from pH 7.3 to 6.5 and 6.2 (Fig 4A). At pH 6.5 and 6.2, the Ca²⁺ dependence of [³H]ryanodine binding could not be well described by the equation used to fit the data at pH 7.3.

In the presence of 5 mmol/L Mg-AMPPCP at pH 7.3, [³H]ryanodine binding increased to a maximum value as the free [Ca²⁺] was raised from 1 to 10 µmol/L (Fig 4B). Higher free [Ca²⁺] levels of up to 1 mmol/L did not appreciably inhibit [³H]ryanodine binding. This result coincided with the single-channel measurements, which also showed a lack of inhibition of channel activity by millimolar [Ca²⁺] in the presence of Mg²⁺ and adenine nucleotide (Fig 3). In the presence of 5 mmol/L Mg²⁺-AMPPCP, a decrease in pH had a marked effect on the Ca²⁺ dependence of [³H]ryanodine binding (Fig 4B). At [Ca²⁺] ranging from 1 to 10 µmol/L, [³H]ryanodine binding was highly sensitive to pH, whereas at elevated [Ca²⁺] (0.1 to 1 mmol/L), acidosis was less effective in reducing [³H]ryanodine binding. The data of Fig 4B suggested that acidosis reduced the apparent affinity of Ca²⁺ binding to the high-affinity activation sites. No inactivation was observed at the three pH values. The Hill activation constants and coefficients of [³H]ryanodine binding were 1.9 µmol/L and 4.4 at pH 7.3, 36 µmol/L and 2.1 at pH 6.5, and 89 µmol/L and 1.8 at pH 6.2, respectively (Table 1). Scatchard analysis at the three different pH values in the absence and presence of 5 mmol/L Mg²⁺-AMPPCP showed a change in binding affinity without an appreciable change in B_max value (not shown). This result suggested that the different binding values of panels A and B of Fig 4 reflected changes in binding affinity and not the number of binding sites. Free [Ca²⁺] in excess of 1 mmol/L were not tested because of difficulties of keeping Ca²⁺ in solution in media containing 5 mmol/L AMPPCP.

Effects of [Mg²⁺] and [Adenine Nucleotide] on [³H]Ryanodine Binding
A decrease in pH lowers the binding constant of the Mg²⁺-ATP (AMPPCP) complex. As a consequence, a decrease in pH may directly inhibit the channel as well as reduce channel activity by increasing the concentration of inhibitory Mg²⁺. We assessed the effects of free [Mg²⁺] on channel activity by measuring [³H]ryanodine binding at pH 7.1 and 6.5 in media that lacked or contained a total [AMPPCP] of 5 mmol/L. The two pH values used in the [³H]ryanodine measurements were lower than the pH value used in the single-channel measurements (pH 7.4, Fig 2). We also measured the effects of Mg²⁺ on [³H]ryanodine binding in media that contained a total [ADP] of 5 mmol/L, because extensive hydrolysis of ATP to ADP increases the free [Mg²⁺] in ischemic tissues. Fig 5A shows that at pH 7.1, in media containing 20 µmol/L free Ca²⁺, Mg²⁺ inhibited [³H]ryanodine binding to a greater extent in the absence than in the presence of adenine nucleotides. [³H]Ryanodine binding was half maximally inhibited by 0. 4 mmol/L Mg²⁺ in the absence of adenine nucleotides and by 0.9 and 5.1 mmol/L free Mg²⁺ in the presence of 5 mmol/L ADP and 5 mmol/L AMPPCP, respectively (Table 1).

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of Mg2+ on [3H]ryanodine binding at pH 7.1 and 6.5. Specific [3H]ryanodine binding was determined at pH 7.1 in the presence of 20 µmol/L free Ca2+ (A) and at pH 6.5 in the presence of 110 µmol/L free Ca2+ (B) in 0.1 mol/L KCl, 20 mmol/L potassium PIPES buffer containing a total concentration of 0 (), 5 mmol/L ADP () or 5 mmol/L AMPPCP (), and Mg2+ to yield the indicated free [Mg2+]. Data are the mean±SE of four experiments. Solid lines were obtained according to the following equation: . Derived Hill constants and coefficients are shown in Table 1.

The effects of Mg²⁺ on [³H]ryanodine binding were also determined at pH 6.5 (Fig 5B). Because of the low [³H]ryanodine binding levels at pH 6.5 in media containing 20 µmol/L free Ca²⁺ (Fig 4B), the effects of free [Mg²⁺] on [³H]ryanodine binding were determined at 110 µmol/L free Ca²⁺. At pH 6.5, [³H]ryanodine binding was half maximally inhibited by 0.2 mmol/L free Mg²⁺ in the absence of adenine nucleotide and by 0.6 and 4.8 mmol/L Mg²⁺ in the presence of 5 mmol/L ADP and 5 mmol/L AMPPCP, respectively (Table 1).

Effect of pH on Single-Channel Activities
The effects of pH on cardiac single-channel activities were studied at three ionic conditions that were shown in Fig 4 to result in a marked to moderate pH dependence of [³H]ryanodine binding. In the left three current traces of Fig 6A, the effects of pH were studied at 100 µmol/L cytosolic Ca²⁺, a condition that resulted in a substantial decrease in [³H]ryanodine binding when the pH was lowered from 7.3 to 6.5 and 6.2 (Fig 4A). Similarly, we observed that a decrease in pH of the two bilayer chambers decreased P_o. P_o fell from .69±.06 at pH 7.3 to .48±.08 at pH 6.5 and .02±.02 at pH 6.0 (n=12). A reduction of pH also had a comparable inhibitory effect on [³H]ryanodine binding (Fig 4B) and P_o (Fig 6A, middle three current traces) when channels were assayed at 10 µmol/L cytosolic Ca²⁺ in the presence of 5 mmol/L cytosolic Mg²⁺-ATP. P_o decreased from .69±.06 at pH 7.3 to .24±.09 at pH 6.5 and .07±.02 at pH 6.0 (n=5). In agreement with [³H]ryanodine measurements, P_o was less affected by a change in pH when the cis chamber contained elevated levels of free Ca²⁺ (0.4 mmol/L) and 5 mmol/L Mg²⁺-ATP (Fig 6A, right three current traces). In this case, P_o decreased from .67±.07 to .42±.07 and .20±.05 as the pH was lowered from 7.3 to 6.5 and 6.0, respectively (n=6).

View larger version (65K): [in this window] [in a new window]   Figure 6. Effect of pH on single-channel activities. A, Shown are three separate recordings at pH 7.3, 6.5, and 6.0 in symmetrical 0.25 mol/L KCl media containing 100 µmol/L free cytosolic and lumenal Ca2+ (on the left, the mean open times were 69.7 and 17.2 milliseconds for the upper and middle traces, respectively), 10 µmol/L free cytosolic Ca2+, 5 mmol/L cytosolic Mg2+-ATP, and 10 µmol/L free lumenal Ca2+ (in the middle, the mean open times were 23.2, 3.0, and 1.5 milliseconds for the upper, middle, and bottom traces, respectively), and 0.40 mmol/L cytosolic Ca2+, 5 mmol/L cytosolic Mg2+-ATP, and 0.4 mmol/L free lumenal Ca2+ (on the right, the mean open times were 4.9, 1.0, and 0.5 milliseconds for the upper, middle, and bottom traces, respectively). Single-channel currents are shown as upward deflections from closed levels (bars on left). Holding potentials were +35 mV (left traces) and +50 mV (middle and right traces). B, Shown are the current-amplitude histograms of the recordings in panel A. Numbers on the ordinate indicate the number of data points per 0.4-pA interval.

Fig 6B shows the current-amplitude histograms of recordings similar to those of Fig 6A. At pH 7.3 and 100 µmol/L cytosolic Ca²⁺ (upper left histogram of Fig 6B), single-channel conductance was 780 pS. In the presence of 10 µmol/L free Ca²⁺ and 5 mmol/L cytosolic Mg²⁺-ATP, conductance decreased to 540 pS (upper middle histogram). Increase of free Ca²⁺ from 10 µmol/L to 0.4 mmol/L further decreased single-channel conductance to 400 pS (upper right histogram). The changes in conductance were due to the blocking action of Ca²⁺ and Mg²⁺.²⁶ The decreases in pH from 7.3 to 6.5 (top and middle rows of histograms) and pH 6.0 (lower middle histogram) did not significantly change single-channel conductances. In the lower right histogram of Fig 6A, only few maximum conductances were observed. A majority of the openings were too brief to be fully resolved, resulting in the absence of a discernible current peak in the lower right histogram of Fig 6B.

The effects of pH on channel activity were analyzed by carrying out a detailed time analysis of single-channel recordings at pH 7.3 and 6.5 similar to those shown in Fig 6. The open-time histograms could be fitted by the sum of two or three exponentials; the closed-time histograms, by three exponential functions (not shown). Table 2 lists the number of events, P_o values, and open- and closed-channel parameters. In the absence of Mg²⁺ and ATP at 100 µmol/L free Ca²⁺, a small (but significant) decrease in the mean P_o was observed when the pH was reduced from 7.3 to 6.5. Despite only a small change in P_o, several major changes in the kinetics of channel opening and closing were observed. Acidosis increased the number of channel events fivefold, decreased the duration of the long open states, and caused a change in the proportion of the open states with a long and intermediate duration. In the presence of 5 mmol/L Mg²⁺-ATP and 10 µmol/L Ca²⁺, disappearance of the longest open state largely accounted for the decrease in P_o at pH 6.5. In the presence of 5 mmol/L Mg²⁺-ATP and 0.4 mmol/L Ca²⁺, lowering the pH to 6.5 also resulted in disappearance of the longest open state. Other significant changes contributing to a decreased P_o included an increase in the proportion of the short open durations and a decrease in the proportion of the short closed durations and increase in the intermediate closed duration (Table 2). At pH 7.3 and 6.5 in the presence of Mg²⁺-ATP, the number of events at 0.4 mmol/L Ca²⁺ were substantially larger than those at 10 µmol/L Ca²⁺.

View this table: [in this window] [in a new window]   Table 2. Effect of pH on Channel Parameters

In control experiments, we tested the reversibility of the effects of pH on channel activity by returning the pH in both chambers to pH 7.3. A 10% to 100% recovery of channel activity was observed when the cytosolic chamber contained Mg²⁺-ATP and the pH was not decreased below 6 (four of five experiments). A decrease in pH to 5.6 in the presence of Mg²⁺-ATP or to 6.0 in the absence of Mg²⁺-ATP resulted in complete channel closure and no recovery of activity on return to pH 7.3 (13 of 13 experiments). These results suggested that conditions leading to complete channel closure at pH 6.0 promoted irreversible loss of cardiac channel activity.

In Fig 6, pH was lowered in both bilayer chambers. Fig 7 compares the P_o values that were obtained when the pH was changed in only one of the two bilayer chambers. Single-channel activities were determined under conditions comparable to those shown in the right panel of Fig 6, ie, at a lumenal and cytosolic [Ca²⁺] of 0.4 mmol/L and at 5 mmol/L cytosolic Mg²⁺-ATP. Decreasing the cytosolic pH from 7.3 to 6.5 and 6.0 resulted in an approximately twofold and fourfold decrease of P_o, respectively (n=5). A corresponding decrease of lumenal pH was less effective in inhibiting channel activity (n=7). Neither a change in cytosolic nor lumenal pH affected single-channel conductance (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 7. Effect of cytosolic and lumenal pH on single-channel activities. Single-channel Po values were determined in symmetrical 0.25 mol/L KCl media containing 0.4 mmol/L free cytosolic Ca2+, 5 mmol/L cytosolic Mg2+-ATP, and 0.4 mmol/L free lumenal Ca2+. The pH was decreased from pH 7.3 to 6.0 in the trans (lumenal) (, n=7) or cis (cytosolic) (, n=5) chamber.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effects of Ca²⁺, Mg²⁺, H⁺, and adenine nucleotides on cardiac Ca²⁺ release channel activity were investigated in single-channel and [³H]ryanodine binding measurements. In these studies, we used purified canine cardiac Ca²⁺ release channel preparations and SR membrane fractions enriched in [³H]ryanodine binding activities.

Regulation by Ca²⁺, Mg²⁺, and Adenine Nucleotides at Physiological pH
It is widely accepted that Ca²⁺ entering the cell during an action potential serves as the trigger for SR Ca²⁺ release in cardiac muscle excitation-contraction coupling.^{1 2} In the absence of Mg²⁺-ATP, single cardiac Ca²⁺ release channels were half maximally activated at 4 µmol/L cytosolic Ca²⁺ with an n_Ha of 1.3 and half maximally inhibited at 9 mmol/L cytosolic Ca²⁺ with an n_Hi of 0.9 (Fig 1B). [³H]Ryanodine binding measurements indicated a similar dependence of channel activity on [Ca²⁺] (Fig 4A). Our results confirmed previous SR vesicle Ca²⁺ flux,^{14 28} [³H]ryanodine binding,^{28 29} and single-channel³⁰ measurements; these measurements also showed an activation and inactivation of cardiac Ca²⁺ release channel activity by Ca²⁺, but they are at variance with a study that failed to show an inhibition of single-channel activity.²⁸

The effects of Mg²⁺ and ATP on single-channel (Figs 2 and 3) and [³H]ryanodine binding (Figs 4 and 5) activities were analyzed under conditions that were thought to approximate those in both relaxed and contracting muscles. These included cytosolic [Ca²⁺] ranging from submicromolar to millimolar and, in single-channel measurements, a lumenal [Ca²⁺] of 5 mmol/L. The addition of 2 mmol/L cytosolic Mg²⁺-ATP (Fig 3) or 5 mmol/L Mg²⁺-AMPPCP (Fig 4) rendered the cardiac Ca²⁺ release channel less sensitive to inhibition at [Ca²⁺]s of >100 µmol/L. The addition of 2 mmol/L Mg²⁺-ATP and 5 mmol/L Mg²⁺-AMPPCP to the assay media yielded free [Mg²⁺] (0.5 to 1.0 mmol/L) close to those measured in normal intact hearts.¹³ At a free [Ca²⁺] of 10 to 20 µmol/L, Mg²⁺ was more effective in inhibiting channel activity in the absence than in the presence of adenine nucleotide. Taken together, our results suggested a major role for Ca²⁺ and a more modest role for Mg²⁺ and adenine nucleotides in regulating cardiac SR Ca²⁺ release channel activity.

Effects of Acidosis on Cardiac Ca²⁺ Release Channel Activity
The present study showed that acidosis affects [³H]ryanodine binding differently in the presence and absence of 5 mmol/L Mg²⁺-AMPPCP. In the absence of Mg²⁺ and adenine nucleotide, acidosis appeared to lower the affinity of [³H]ryanodine binding. Major changes in the apparent Ca²⁺ affinity of the Ca²⁺ regulatory site(s) were not evident. In contrast, in the presence of 5 mmol/L Mg²⁺-AMPPCP, major changes in the apparent affinity of the Ca²⁺ activation site(s) were observed when the pH was lowered. Exactly how this change was achieved is unclear, but possibilities include a stronger binding of free Mg²⁺ to the Ca²⁺ activation sites (Table 1) or a nucleotide-induced change in the pH sensitivity of the Ca²⁺ regulatory sites.

An advantage of the planar lipid bilayer method is the direct access to the two sides of the SR membrane. A decrease in cis (SR cytosolic) pH from 7.4 to 6.5 resulted in nearly full closing of the cardiac Ca²⁺ release channels without a change in single-channel conductance, whereas a decreasing lumenal pH resulted in a reduction of the current amplitudes without a significant change of P_o.¹⁵ In the present study, channel activity was decreased to a greater extent by an acidification of the SR cytosolic than SR lumenal side. A decrease in neither cytosolic nor lumenal pH had a significant effect on the single-channel conductance of the cardiac channel.

Because of the high H⁺ permeability of the cardiac SR membrane,³¹ it is unlikely that [H⁺] can be controlled independently on the two sides of the SR membrane. Therefore, the pH was also simultaneously reduced in both bilayer chambers. We focused on three ionic conditions that, in agreement with the [³H]ryanodine binding measurements, resulted in a moderate to severe reduction of channel activity as the pH was reduced from 7.3 to 6.5 and 6.0. Analysis of single-channel parameters showed, with 100 µmol/L free Ca²⁺ as the sole activating ligand, major changes in channel gating. Reduction of pH from 7.3 to 6.5 resulted in a fivefold increase in the number of single-channel events and shift from long to intermediate open durations. On the other hand, in the presence of 5 mmol/L cytosolic Mg²⁺-ATP and at free [Ca²⁺] of 10 µmol/L and 400 µmol/L, the same reduction in pH elicited no significant effect on the number of channel events. In this case, a major effect of acidosis was to cause the disappearance of the long open-channel events. These results suggested that the cytosolic [Ca²⁺] or the level of ATP may determine the extent of inhibition and the way in which acidosis affects Ca²⁺ release channel activity in ischemic hearts.

SR Ca²⁺ Release Channel Function in Ischemic and Postischemic Myocardium
Elimination of the flow of oxygenated blood to the myocardium leads to a rapid decline in contractile function during the first 1 to 2 minutes of ischemia.²⁴ Our data suggest that the modest changes in [H⁺], [Ca²⁺], [Mg²⁺], or [ATP]^{6 7 8 9 10 11 12 13 24 32} that occur during early ischemia are insufficient to cause a substantial impairment of Ca²⁺ release channel activity. However, SR Ca²⁺ release might be inhibited by the multiple changes in ionic milieu that occur during sustained ischemia.^{6 7 8 9 10 11 12 13 24 32} According to our data, a major effect of a decreasing pH might be to lower the Ca²⁺ sensitivity of the channel so that higher [Ca²⁺] would be required to activate the channel (Fig 4). An attenuation of Ca²⁺-induced Ca²⁺ release might be beneficial, because it would reduce ATP-dependent Ca²⁺ accumulation by the SR, thus helping the heart to recover more readily from brief ischemic episodes by slowing the fall in high-energy phosphates.

The relative insensitivity of the cardiac Ca²⁺ release channel to the level of cellular ATP (Fig 2A) may be important when the myocardium recovers from an ischemic episode. Brief periods of ischemia followed by reperfusion of the ischemic myocardium lead to myocardial dysfunction (myocardial stunning).³³ Although the cellular mechanisms of myocardial stunning are not well understood, one major factor may be a depressed level of cellular ATP in the reperfused myocardium.^{8 34} In digitonin-lysed cardiomyocytes, a decline in cytosolic [ATP] has been suggested to interfere primarily with the SR Ca²⁺ release channel by causing an increase in cytosolic [Mg²⁺].²⁵ Our single-channel and [³H]ryanodine binding measurements provide direct evidence for this idea and furthermore suggest that a decrease in pH during ischemia may enhance the inhibitory action of Mg²⁺.

In conclusion, we have described the regulation of the cardiac Ca²⁺ release channel at Ca²⁺, H⁺, Mg²⁺, and adenine nucleotide concentrations thought to be present in normal, ischemic, and postischemic myocardium. The results of the present study suggest that imbalances in the intracellular milieu occurring during brief ischemic episodes do not result in a major impairment of SR Ca²⁺ release channel activity. However, it seems equally likely that changes in channel function take place during sustained ischemia. Our data pointed to one of these changes, namely, that a falling pH_i in the ischemic myocardium may primarily affect the function of the cardiac Ca²⁺ release channel by altering its Ca²⁺ sensitivity.

	Selected Abbreviations and Acronyms

 

AMPPCP = ß,-methyleneadenosine 5'-triphosphate KHa = Hill activation constant KHi = Hill inactivation constant nHa = Hill activation coefficient nHi = Hill inactivation coefficient Po = channel open probability RYR = ryanodine receptor SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-27430. We thank Dr Erno Lindner (Duke University) for his advice and assistance in preparing the Mg²⁺-selective electrode.

Received March 14, 1996; accepted September 17, 1996.

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