Regulation of Cardiac Ca2+ Release Channel (Ryanodine Receptor) by Ca2+, H+, Mg2+, and Adenine Nucleotides Under Normal and Simulated Ischemic Conditions
In myocardial ischemia, pHi 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 β,γ-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+.
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 pHi 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
[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 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.
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 3×5 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
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.
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 Po. 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 Po.
Fig 1B⇑ describes the dependence of single-channel Po on cytosolic [Ca2+] levels ranging from 10−7 to 10−2 mol/L at holding potentials of +35 and −35 mV. At both potentials, Po 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 iRYRa at [Ca2+] of <0.1 μmol/L) and its Ca2+-activated (iRYRCa) and Ca2+-inactivated (CaRYRCa) 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 Po 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 KHa of 4.5 and 4.0 μmol/L and inhibited with KHi of 6.5 and 11.1 mmol/L cytosolic Ca2+, respectively (Table 1⇓). nHa of 1.3 suggested that Ca2+ activated the cardiac channel by a weak cooperative interaction.
The total [ATP] and free [Mg2+] in normal intact hearts have been reported to range from 5 to 10 mmol/L24 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 Po 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 Po. 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⇑). nHi values of 1.5 and 2.5, respectively, suggested that Mg2+ inhibited channel activity by a cooperative interaction.
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, Po also reached a maximal value at ≈30 μmol/L cytosolic Ca2+. However, in the presence of 2 mmol/L Mg2+-ATP, Po did not decrease at cytosolic [Ca2+] as high as 10 mmol/L (Fig 3⇓, triangles).
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 pHi 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 KHa of 2.8 μmol/L and inhibited with a KHi of 2.1 mmol/L (Table 1⇑). An nHa 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.
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 Bmax 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⇑).
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 Po. Po 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 Po (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. Po 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, Po 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, Po 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).
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, Po 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 Po was observed when the pH was reduced from 7.3 to 6.5. Despite only a small change in Po, 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 Po 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 Po 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+.
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 Po 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 Po, 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).
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 nHa of 1.3 and half maximally inhibited at ≈9 mmol/L cytosolic Ca2+ with an nHi 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 Po.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 pHi in the ischemic myocardium may primarily affect the function of the cardiac Ca2+ release channel by altering its Ca2+ sensitivity.
Selected Abbreviations and Acronyms
|K Ha||=||Hill activation constant|
|K Hi||=||Hill inactivation constant|
|nHa||=||Hill activation coefficient|
|nHi||=||Hill inactivation coefficient|
|Po||=||channel open probability|
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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