Luminal Ca2+regulated Mg2+ Inhibition of Skeletal RyRs Reconstituted as Isolated Channels or Coupled Clusters
Derek R. Laver1,
Erin R. O'Neill1, and
Graham D. Lamb2
1 School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia
2 Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia
Address correspondence to Derek Laver, School of Biomedical Sciences, University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308, Australia. Fax: 61-2-4921-7406; email: derek.laver{at}newcastle.edu.au
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ABSTRACT
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In resting muscle, cytoplasmic Mg2+ is a potent inhibitor of Ca2+ release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca2+ sites (A-sites) thus preventing Ca2+ activation. We investigate the effects of luminal and cytoplasmic Ca2+ on Mg2+ inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg2+ inhibits RyRs at the A-site in the absence of Ca2+, indicating that Mg2+ is an antagonist and does not simply prevent Ca2+ activation. Cytoplasmic Ca2+ and Cs+ decreased Mg2+ affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca2+ regulation of Ca2+ release whereby increasing luminal [Ca2+] decreases the A-site affinity for cytoplasmic Mg2+ by a noncompetitive, allosteric mechanism that is independent of Ca2+ flow. Ryanodine increases the Ca2+ sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca2+, indicating that ryanodine activates independently of Ca2+. We describe a model for ion binding at the A-sites that predicts that modulation of Mg2+ inhibition by luminal Ca2+ is a significant regulator of Ca2+ release from the SR. We detected coupled gating of RyRs due to luminal Ca2+ permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca2+ and cytoplasmic Mg2+ did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca2+ was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca2+ emanating from their own pore but sensitive to Ca2+ from neighboring channels.
Key Words: ryanodine receptor magnesium calcium skeletal muscle lipid bilayer
Abbreviations used in this paper: BAPTA, 1,2-bis[o-aminophenoxy] ethane-N,N,N',N'- tetraacetic acid; CICR, calcium-induced calcium release; DHPR, dihydropyridine receptor; DIDS, diisothiocyanostilbene-2',2'-di-sulfonic acid; HMM, Hidden Markov Model; SR, sarcoplasmic reticulum.
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INTRODUCTION
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Contraction in skeletal and cardiac muscle occurs when Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca2+ release channels. In striated muscle, excitationcontraction coupling describes the link between depolarization of the transverse tubule and calcium release from the SR. Upon depolarization of the transverse tubule membrane, dihydropyridine receptors (DHPRs, L-type calcium channels) are activated, which trigger the RyRs. In skeletal muscle, the RyRs are stimulated by a direct coupling with the DHPRs (Tanabe et al., 1990
; Melzer et al., 1995
), while in cardiac muscle, it is the influx of extracellular Ca2+ through the DHPR that initiates Ca2+ release.
Calcium release is modulated by a variety of substances, including small diffusible molecules such as ATP, Ca2+, Mg2+, and protons (pH) (Meissner, 1994
). RyRs are activated at µM [Ca2+] and inhibited at mM [Ca2+] in the cytoplasm (Meissner, 1994
). Mg2+ is believed to inhibit RyRs by two mechanisms (the dual-inhibition hypothesis). Mg2+ can inhibit RyRs by competing with Ca2+ for the activation sites (A-sites; Dunnett and Nayler, 1978
; Meissner et al., 1986
). In addition, Mg2+ can close RyRs by binding to low affinity, nonselective divalent cation inhibition sites that also mediate Ca2+ inhibition (I-sites; Meissner et al., 1986
; Soler et al., 1992
; Laver et al., 1997
). However, with the former mechanism, it is not clear if Mg2+ acts as a competitive nonagonist (i.e., prevents Ca2+ from activating the channel) or as an antagonist in its own right. This distinction becomes important when one considers cytoplasmic ATP. Physiological levels of ATP (8 mM) strongly activate skeletal RyRs even in the absence of cytoplasmic Ca2+ (Meissner et al., 1986
; Laver et al., 2001
). Therefore if Mg2+ merely prevents Ca2+ activation, it would not inhibit RyRs that are activated by ATP (Laver et al., 1997
). Here we measure the effects of A-site Mg2+ inhibition on RyRs that are activated by ATP in the absence of Ca2+. The first major finding of this work is that Mg2+ is an RyR antagonist that inhibits in the absence of cytoplasmic and luminal Ca2+.
Ryanodine and the disulfonic stilbene derivatives are widely used pharmacological probes for elucidating the mechanisms of muscle contraction. Diisothiocyanostilbene-2',2'-di-sulfonic acid (DIDS) has two isothiocyanate groups that can form covalent bonds with several amino acid residues. Modification of RyRs by ryanodine and DIDS is used here to help dissect the two mechanisms of Mg2+ inhibition. In the presence of ryanodine and DIDS, the Ca2+ sensitivities of the A- and I-sites are more disparate, producing wider separation of the two Mg2+ inhibition mechanisms and a much clearer manifestation of Mg2+ inhibition at the A-sites (Laver et al., 1997
). DIDS has been shown to activate RyRs by reversible and nonreversible mechanisms (Kawasaki and Kasai, 1989
; Zahradnikova and Zahradnik, 1993
; Sitsapesan, 1999
). Two independent mechanisms for nonreversible activation have been distinguished by their different kinetics (referred to as slow and fast) and their different specific effects (O'Neill et al., 2003
). The fast mechanism increased the degree of Ca2+ activation with no significant change in sensitivity (A-sites) and reduced RyR sensitivity to Ca2+/Mg2+ inhibition (I-sites) by 10-fold. The slow mechanism activated RyRs in the absence of Ca2+ and ATP.
Ryanodine has a profound effect on the regulation of RyRs by intracellular constituents. Ryanodine-modified RyRs are insensitive to cytoplasmic Ca2+ and adenine nucleotides (Rousseau et al., 1987
; Laver et al., 1995
), though they are still inhibited by Mg2+ (Masumiya et al., 2001
) and low pH (Ma and Zhao, 1994
; Laver et al., 2000
), albeit with reduced sensitivity. In spite of the pharmacological importance of ryanodine, its effects on RyR function are not well understood. Earlier studies report that ryanodine-modified RyRs are active (i.e., open probability
1) in the virtual absence of Ca2+ (Rousseau et al., 1987
; Laver et al., 1995
). However, two recent studies on recombinant cardiac RyRs (RyR2) report that ryanodine shifts their Ca2+ activation response to lower concentrations by four orders of magnitude (Du et al., 2001
; Masumiya et al., 2001
). We investigate the effects of cytoplasmic Ca2+ and Cs+ on A-site Mg2+ inhibition in ryanodine-modified RyRs to probe the competitive ion binding kinetics at the A-site. Our second major finding is that ryanodine increases the Ca2+ sensitivity of the A-sites by 10-fold but this is insufficient to explain the level of activation of ryanodine-modified RyRs at nM Ca2+.
The Ca2+ load of the SR is an important stimulator of Ca2+ release (Fabiato and Fabiato, 1977
). It has been shown to regulate Ca2+ release in response to Ca2+ (Ford and Podolsky, 1972
; Endo, 1985
; Meissner et al., 1986
), caffeine (Lamb et al., 2001
), and ATP (Morii and Tonomura, 1983
; Donoso et al., 1995
). Raised luminal Ca2+ increases channel activity in both purified (Tripathy and Meissner, 1996
) and native RyRs (Sitsapesan and Williams, 1995
; Beard et al., 2000
). Activation of RyRs by luminal Ca2+ has been attributed to two quite different mechanisms, and there is as yet no consensus on just how the Ca2+ load in the SR alters RyR activation (Sitsapesan and Williams, 1997
). The "true luminal regulation" hypothesis attributes luminal Ca2+ activation to Ca2+ regulatory sites on the luminal side of the RyR (Sitsapesan and Williams, 1995
). This is supported by the fact that luminal Ca2+ activation is susceptible to tryptic digestion from the luminal side of the membrane (Ching et al., 2000
). The "feedthrough" hypothesis proposes that luminal Ca2+ permeates the pore and binds to the cytoplasmic activation sites (Tripathy and Meissner, 1996
; Xu and Meissner, 1998
). The latter is supported by the close correlation between open probability and Ca2+ flux (lumen to cytoplasm), which is seen under a wide range of experimental manipulations in both cardiac and skeletal RyRs. Here we investigate the effects of luminal Ca2+ on Mg2+ binding to the A-site in order to detect competition between luminal and cytoplasmic ions resulting from Ca2+ feedthrough. Our third and most important finding is a novel mechanism for regulation of Ca2+ release by luminal Ca2+, whereby increasing luminal [Ca2+] decreases the affinity of the A-sites for Mg2+. We show that luminal Ca2+ activates RyRs by both the true luminal regulation and Ca2+ feedthrough mechanisms in a way that depends on whether they are in close proximity to other open RyRs. Finally, we present an ion binding model of Mg2+ inhibition at the A-sites that predicts that modulation of Mg2+ inhibition by luminal Ca2+ is a significant regulator of Ca2+ release from the SR.
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MATERIALS AND METHODS
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Lipid Bilayers, Chemicals, and Solutions
SR vesicles were prepared from the back and leg muscles of New Zealand rabbits killed by captive bolt before muscle removal. The procedure was performed by the holder of a current license granted under ACT State legislation. Native SR vesicles were isolated using techniques based on those of Chu et al. (1988)
, as previously described by Laver et al. (1995)
.
Unless otherwise stated, lipid bilayers were formed from phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (8:2) (Avanti Polar Lipids) dissolved in 20 µl n-decane (50 mg/ml). The bilayers were formed across an aperture of 100200 µm diameter in a Delrin cup. The bilayer separated two solutions: cis and trans (
1 ml). Vesicles were added to the cis solution and vesicle incorporation with the bilayer occurred as described by Miller and Racker (1976)
. Due to the orientation of RyRs in the SR vesicles, RyRs added to the cis chamber incorporated into the bilayer with the cytoplasmic face of the channel orientated to the cis solution.
The cesium salts were obtained from Sigma-Aldrich and CaCl2 from BDH Chemicals. Unless otherwise stated, solutions were pH buffered with 10 mM N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES, obtained from ICN Biomedicals) and solutions were titrated to pH 7.4 using CsOH (optical grade from ICN Biomedicals). During SR vesicle incorporation the cis (cytoplasmic) solution contained 230 mM CsCH3O3S and 20 mM CsCl (250 mM Cs+ solution) with 1.0 or 0.1 mM CaCl2, while the trans (luminal) solution contained 30 mM CsCH3O3S and 20 mM CsCl (50 mM Cs+ solution) and 01.0 mM CaCl2. The osmotic gradient across the membrane and the Ca2+ in the cis solution also aided vesicle fusion with the bilayer. In experiments using symmetric [Cs+], vesicle fusion was performed using cis and trans baths containing 250 mM Cs+ solution with 500 mM mannitol in the cis bath to produce the necessary osmotic gradient. Cs+ was used as the current carrying ion rather than K+ in order to extinguish interfering signals from the SR K+ channels. During measurements, the composition of the cis solution was altered either by addition of aliquots of stock solutions or by continuous local perfusion of the bilayer via a tube placed in close proximity (O'Neill et al., 2003
).
The required free [Ca2+] was attained by buffering with 4.5 mM BAPTA (1,2-bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid, obtained as a tetra potassium salt from Molecular Probes) and titrated with CaCl2. Free [Ca2+] in excess of 0.1 µM was measured using a Ca2+ electrode (Fluka). At lower concentrations, free [Ca2+] was estimated using published association constants (Marks and Maxfield, 1991
) and the program Bound and Determined (Brooks and Storey, 1992
). Solutions, which mimicked zero Ca2+, were made with 4.5 mM BAPTA and no added Ca2+. In these solutions, the total [Ca2+] arising from impurities was measured to be 15 µM so that with addition of 4.5 mM BAPTA the free [Ca2+] was calculated to be 1 nM.
In solutions containing ATP, which buffers Mg2+, the required free [Mg2+] was determined using the fluorescent magnesium indicator, Mag-fura-2 (tetra potassium salt from Molecular Probes). The ratio of fluorescence intensities at 340 and 380 nm was calibrated in the experimental solutions (50 and 250 mM Cs+ solutions, see above) also containing 5 µM Mag-fura-2, 4.5 mM BAPTA, (free [Ca2+] 1 nM1 µM) and MgCl2 from aliquots of a calibrated stock. This allowed determination of the ATP purity and effective Mg2+ binding constants under experimental conditions, which in turn allowed calculation of free [Mg2+] in solutions containing >1 µM Ca2+, where Mag-fura-2 is not a suitable indicator of [Mg2+].
ATP was obtained in the form of sodium salts from Sigma-Aldrich. Unless otherwise stated, DIDS (sodium salt) was prepared as a stock solution in DMSO (dimethyl sulfoxide) at concentrations between 5 and 50 mM. Care was taken to ensure that DMSO in the bath solutions did not exceed 1% since DMSO concentrations >2% inhibited RyR activity (O'Neill et al., 2003
).
Acquisition and Analysis of Single Channel Recordings
Bilayer potential was controlled and currents recorded using an Axopatch 200B amplifier (Axon Instruments). The cis chamber was electrically grounded to prevent electrical interference from the perfusion tubes, and the potential of the trans chamber was varied. However, all electrical potentials are expressed here using standard physiological convention (i.e., cytoplasmic side relative to the luminal side at virtual ground). Measurements were performed at 23 ± 2°C.
During the experiments, the channel current was recorded after low pass filtering at 5 kHz and sampling at 50 kHz. The data was stored on computer disk using a data interface (Data Translation DT301) under the control of in-house software written in Visual Basic. For measurements of Po, the current signal was digitally filtered at 1 kHz with a Gaussian filter and sampled at 5 kHz. Unitary current and time-averaged currents were measured using Channel2 software (P.W. Gage and M. Smith, Australian National University, Canberra, Australia). To calculate Po from single channel records, a threshold discriminator was set at 50% of channel amplitude to detect channel opening and closing events. For experiments in which bilayers contained several RyRs, the time-averaged current was divided by the unitary current and the number of channels. The number of channels in each experiment could be determined during periods of strong activation (in the absence of Mg2+). Both methods of calculating Po gave similar results.
For measurements of channel open and closed dwell times, the current signal was filtered at 2 kHz and sampled at 10 kHz. Open and closed durations were extracted from single channel recordings using the 50% threshold (see above). Channel gating in multichannel recordings were analyzed using the Hidden Markov Model (HMM; Chung et al., 1990
). The algorithm calculated the idealized, multilevel, current time course (i.e., background noise subtracted) and the transition probability matrix from the raw signal using maximum likelihood criteria. The mean channel opening and closing rates in the presence of no open channels, ko+ and ko, respectively, were calculated from the transition probability matrix, P, by Eq. 1:
 | (1) |
where nT is total number of channels in the bilayer and
t is the sample interval.
Dissecting Mg2+ Inhibition at A-sites and I-sites
RyRs are inhibited when Mg2+ is bound to either A- or I-sites so that the open probability of the channel is the product of the Mg2+ occupancies of each site (Laver et al., 1997
). Consequently, the half-inhibiting [Mg2+], Ki(Mg2+) depends on the Mg2+ affinities of both A- and I-sites (KA and KI, respectively). Provided the KA << KI then KA
Ki(Mg2+), with a relative error of (KA/KI)2 (the second power in this equation stems from the Hill coefficient of
2 for Mg2+ inhibition at the I-sites). Since I-sites are nonselective between divalent ions (Soler et al., 1992
; Laver et al., 1997
; Meissner et al., 1997
), then one can estimate KI from the half-inhibitory [Ca2+] for RyR inhibition, Ki(Ca2+)c. The subscripts c here, and l later, refer to cytoplasmic and luminal solutions, respectively. For example, in the case of ATP-activated RyRs in this study, Ki(Ca2+)c is 2.5 mM (Table I) so that KA
Ki(Mg2+) with <15% error when Ki(Mg2+) < 1 mM. Since the experimental conditions were always chosen to keep the error in KA < 15%, we denote the Mg2+ affinity of the A-sites by either KA or Ki(Mg2+). We found that DIDS and ryanodine are useful pharmacological tools for studying the A-sites because they markedly increased Ki(Ca2+)c and so increased the range of KA that could be determined from Ki(Mg2+).
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TABLE I The Apparent Affinities of the Ca2+ and Mg2+ Binding Sites on the RyRs under Various Experimental Conditions
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Modeling Mg2+ Inhibition at the A-sites
Here we consider competitive and noncompetitive models to explain the modulation of Mg2+ inhibition by the other major ion species present. Within the competitive model, Ki(Mg2+) is related to the Mg2+ binding affinity, Km(Mg2+), and the concentrations, Cj, and binding affinities Km(j) of other ions, j, by Eqs. 2 and 3.
 | (2) |
where
 | (3) |
The parameter nj is equal to the number of ions of species, j, that can bind at that site. The ions considered to compete with Mg2+ in this study are Cs+ and Ca2+ in the cytoplasm and Ca2+ in the lumen. When these ions are explicitly included Eq. 2 becomes
 | (4) |
The term for Cs+ is raised to the second power because it is likely that two monovalent ions can bind to a divalent cation site (Meissner et al., 1997
). Examination of Eqs. 24 reveals that the relative effect of a competing ion on Mg2+ inhibition is significant when its concentration exceeds the value of its affinity and the ratio of its concentration to affinity exceeds that of the other ions present.
In the noncompetitive model, each ion regulates channel activity at independent sites. A specific example of this, used in this work, is where the binding of luminal Ca2+ prevents the binding of cytoplasmic Mg2+ or Ca2+ (denoted by X2+) by an allosteric effect. The apparent affinities for cytoplasmic Ca2+ and Mg2+, Kapp(X2+)c, are related to the affinity Km(X2+)c in the absence of luminal Ca2+ by Eq. 5:
 | (5) |
The Ca2+c dependence of Mg2+ inhibition is analyzed in this study in terms of apparent affinities for Ca2+c and Mg2+, Kapp(Ca2+)c, and Kapp(Mg2+), respectively (see Fig. 4). For this analysis Eqs. 24 are recast into the following form:
 | (6) |
It can be seen from Eq. 6 that the value of Ki(Mg2+) at low [Ca2+]c approximates the value of Km(Mg2+) and the value of Km(Ca2+)c determines the [Ca2+] threshold where Ki(Mg2+) becomes Ca2+ dependent.
Statistics and Curve Fitting
Unless otherwise stated the data are presented as mean ± SEM obtained from N bilayers and n RyRs. Theoretical curves were fitted to the data using the criteria of least squares. The open probability (Po) of RyRs, activated or inhibited by a compound with concentration, C, was fitted by Hill equations, Eqs. 7 and 8, respectively:
 | (7) |
 | (8) |
where Pi and Pmax are RyR open probabilities in the absence of the compound and at maximal activation, respectively. Ka and Ki are half-activation and inhibition concentrations, and na and ni are the associated Hill coefficients. Ion binding models of the A-site were fitted to the logarithm of the data by minimizing the residuals weighted by the experimental error on each datum. Statistical uncertainty in the elements of the transition probability matrix produced by HMM analysis ranged from 5 to 25% relative error. This was estimated by the SEM of transition probability matrices derived from four subsections in four representative experiments.
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RESULTS
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RyR Ca2+ Dependencies
RyRs in the absence of ATP and the modifying agents DIDS and ryanodine (i.e., native RyRs) are inactive in the absence of cytoplasmic [Ca2+] ([Ca2+]c). They are activated by µM Ca2+ and inhibited by mM Ca2+, thus producing the characteristic bell-shaped Ca2+ dependence in open probability (Po) (Fig. 1 A, closed circles).

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FIGURE 1. The dependence of RyR open probability, Po, on cytoplasmic [Ca2+]. (A) RyRs activated by cytoplasmic Ca2+ alone (, N = 30), modified by 10 µM ryanodine ( ) or 4-min exposure to 100 µM DIDS ( , N = 12, n = 64). Luminal [Ca2+] = 1 mM. (B) In the presence of 2 mM ATP with luminal [Ca2+] = 1 mM ( , N = 9, n = 49) or 0.01 mM ( , N = 15, n = 89). The membrane potential was +40 mV. The data points represent means of 220 measurements. The solid curves are Hill fits to the data and the parameter values are shown in Table I. The dashed curve is the Hill fit to the circles in A shown again for comparison.
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Consistent with previous findings (Smith et al., 1986
), cytoplasmic ATP (2 mM) amplified the bell-shaped Ca2+ dependence without greatly altering the half-activating [Ca2+]c, Ka(Ca2+)c (Fig. 1 B and Table I), and activated RyRs in the absence of cytoplasmic Ca2+. We also found that ATP increased the half-inhibitory [Ca2+], Ki(Ca2+)c, by approximately threefold (Table I). Luminal Ca2+ ([Ca2+]l) increased channel activity in 2 mM ATP and 1 nM [Ca2+]c (Pi; Fig. 2, top). The mean Pi increased from 0.25 ± 0.07 in 0.1 mM luminal Ca2+ (number of bilayers, N = 7, and number of channels, n = 21) to 0.50 ± 0.06 in 1 mM (N = 15, n = 37) and 0.63 ± 0.08 in 3 mM (N = 12, n = 43). However, luminal Ca2+ had no effect on the maximum of bell-shaped Ca2+ dependence, Pmax (Table I).

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FIGURE 2. Recordings from one experiment on a single skeletal RyR (RyR1) showing the effects of luminal [Ca2+] and cytoplasmic [Mg2+] on channel gating. Increasing luminal [Ca2+] markedly increased the channel open probability and decreased the channel sensitivity to Mg2+ inhibition. The cytoplasmic solution contained (in mM) 250 CsCH3O3S, 10 TES (pH 7.4), 4.5 BAPTA ( 1 nM free Ca2+), 2 ATP plus the various [Mg2+]. The free Mg2+ is indicated at the left and right of each row. The luminal solution contained (in mM) 30 CsCH3O3S, 20 CsCl, 10 TES, and the indicated [Ca2+]. Under these conditions, Mg2+ is thought to inhibit primarily by binding at the high affinity Ca2+ activation site. Membrane potential was held at +40 mV. The current baselines are shown by dashed lines.
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Ryanodine (10 µM) produced nonreversible activation of RyRs by "locking" them into a conductance substate. Once RyRs were modified by ryanodine in this way, the nonbound ryanodine was washed away from the RyR by local perfusion (see MATERIALS AND METHODS) before measurements. Ryanodine-modified channels were relatively insensitive to [Ca2+]c (Fig. 1, triangles). Ryanodine-modified channels were not inhibited by high [Ca2+]c, neither were they inhibited by low [Ca2+]c in the presence of 1 mM luminal Ca2+. In the absence of both cytoplasmic and luminal Ca2+, the distribution of RyR activity appeared to be bimodal. Most had Po values
1, while a minority had substantially lower Po as follows. In
1 nM [Ca2+]c (impurity Ca2+ plus 4.5 mM BAPTA), Po were 0.71 and 0.66 in 2 of 9 experiments. In
0.1 nM [Ca2+]c (impurity Ca2+ plus 5 mM EGTA), we observed 2 out of 11 RyRs where Po was <0.5. The decrease in activity was associated with an increase in the frequency of short channel closures.
DIDS produces both reversible and nonreversible activation of RyRs (O'Neill et al., 2003
). Here, the effects of permanent RyR modification were measured by applying DIDS (100500 µM) to the cytoplasmic bath for 4 min. DIDS was then removed by local perfusion before measurements commenced. DIDS modification activated RyR in the absence of cytoplasmic Ca2+ (Fig. 1, open circles) and shifted Ca2+ activation and inhibition to higher concentrations by
10-fold and by
3-fold, respectively (Table I).
Regulation of Modified RyRs by Mg2+
We investigated the possibility that Mg2+ inhibits RyRs by occluding Ca2+ from its activation sites on the protein. We did this by measuring the effect of Mg2+ on RyRs activated by ATP, DIDS, or ryanodine in the absence of cytoplasmic Ca2+. In all three cases, Mg2+ could totally and reversibly inhibit RyRs (e.g., see Figs. 2 and 3 for the case of ATP-activated RyRs). An increase in [Ca2+]c produced a corresponding increase in the [Mg2+] required to inhibit the channels (Figs. 3 and 4). Therefore, Ca2+ and Mg2+ compete for a common site (presumably the A-site) where Mg2+ is an antagonist and not merely an inhibitor of Ca2+ activation.

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FIGURE 3. Mg2+ inhibition of RyR Po in the presence of 2 mM ATP and [Ca2+]c = 10 nM ( , N = 7, n = 20), 1 µM (, N = 6, n = 13), and 10 µM ( , N = 7, n = 35). Luminal [Ca2+] = 1 mM. The data points represent means of two to seven measurements. The solid curves are Hill fits to the data. Half-inhibiting Mg2+ concentrations, Ki(Mg2+), are summarized in Fig. 4 and Table III.
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The Mg2+ dependencies of Po for RyRs were fitted with the Hill equation (Eq. 8). Examples of such fits are shown for ATP-modified RyRs in Fig. 3. The half-inhibiting Mg2+ concentration, Ki(Mg2+), in the presence of 1 mM luminal Ca2+ is plotted against [Ca2+]c in Fig. 4. There is a range of [Ca2+]c over which Ki(Mg2+) is relatively insensitive to Ca2+. At higher concentrations, Ki(Mg2+) increases in proportion to [Ca2+]c. The data is fitted with Eq. 6, which describes Mg2+ inhibition in terms of competition between Mg2+ and Ca2+ at a common binding site. The apparent binding affinities for Ca2+ and Mg2+ associated with Mg2+ inhibition are given in Table I. The values for Kapp(Ca2+)c obtained from Mg2+ inhibition and Ka(Ca2+) for Ca2+ activation are the same order of magnitude, suggesting that Mg2+ is inhibiting at the Ca2+ activation site (A-site). The Kapp(Ca2+)c is very much smaller than expected for the I-sites, which have Ca2+/Mg2+ affinities of
1 mM. Interestingly, analysis of the data for the ryanodine-modified channels gives Kapp(Ca2+)c
100 nM (Table I), suggesting that ryanodine modification increases the Ca2+ sensitivity of the A-sites by
10-fold. The increased Ca2+ sensitivity is not sufficient to account for the high Po (Po
1) of ryanodine-modified RyRs at 110 nM cytoplasmic Ca2+. Rather, it is most likely due to a Ca2+-independent activation of RyRs by ryanodine, akin to that produced by DIDS and ATP (Fig. 1).
Effect of Monovalent Ions on Mg2+ Inhibition
In native RyRs, monovalent cations are known to compete with Ca2+ for the activation site to prevent channel activation (Meissner et al., 1997
). Therefore we compared the Mg2+ inhibition of RyRs at high (250 mM) and low (50 mM) Cs+ concentrations in the absence of cytoplasmic Ca2+. Both in the presence and absence of luminal Ca2+, raising [Cs+] by fivefold increased Ki(Mg2+) by 3- and 10-fold for ATP and ryanodine-modified RyRs, respectively (Fig. 5 and Table II, compare entries 1 and 5, 3 and 7; Table III, compare entries 1 and 3, 2 and 4). This is consistent with competition between Mg2+ and Cs+ for common sites. The relatively strong Cs+ dependence of Mg2+ inhibition in the case of ryanodine-modified channels suggests that at least two Cs+ can bind at a Mg2+ inhibition site.

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FIGURE 5. The dependence of Mg2+ inhibition of ryanodine-modified RyRs on cytoplasmic [Cs+] and luminal [Ca2+]. Recordings were made in the absence of cytoplasmic [Ca2+] ( 1 nM). The solid curves are Hill fits to the data. Half-inhibiting Mg2+ concentrations, Ki(Mg2+), and numbers of experiments are summarized in Table II.
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TABLE II The Effect of Cytoplasmic Cs+ and Ca2+ and Luminal Ca2+ on Mg2+ Inhibition of Ryanodine-modified RyRs (+40 mV)
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TABLE III The Effect of Cytoplasmic Cs+ and Ca2+ and Luminal Ca2+ on Mg2+ Inhibition of RyRs in the Presence of 2 mM ATP
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We considered the possibility that monovalent cations can substitute for Ca2+ as an agonist of RyRs that are modified by ATP, DIDS, and ryanodine and that Mg2+ inhibits RyRs by preventing the binding of cytoplasmic Cs+ (Cs+ is the major monovalent cation in our solutions). In ATP-activated RyRs in the absence of Ca2+ and Mg2+, Cs+ (50250 mM) had no effect on channel activation. However, Cs+ did appear to activate ryanodine-modified RyRs because decreasing [Cs+] in the cytoplasm from 250 to 50 mM decreased Po. In ryanodine-modified RyRs, increasing [Ca2+]c from 1 nM to sub-mM levels could not reverse the effect of decreasing cytoplasmic [Cs+] from 250 to 50 mM (see Table V). Thus it appears that Cs + does not activate RyRs by binding at their A-sites. Although Cs+ and Mg2+ do compete for the A-sites, it is unlikely that Mg2+ inhibits RyRs by preventing Cs+ binding to the A-sites.
Effect of Luminal [Ca2+] on Mg2+ Inhibition
As mentioned above, increasing luminal [Ca2+] from
0 to 1 mM both increased Po in the absence of cytoplasmic Mg2+ and decreased the sensitivity of RyRs to Mg2+ inhibition (e.g., see Fig. 2). The Mg2+ doseresponse curves for ATP-modified RyRs in the absence of cytoplasmic Ca2+ were obtained for three luminal Ca2+ loads (0.1, 1, and 3 mM) (Fig. 6 A). Mean values of Ki(Mg2+) are shown in Fig. 6 B (circles), along with individual values for the 7 out of 12 experiments (crosses) where Ki(Mg2+) was obtained from the same RyRs at two or more [Ca2+]l. These results show that, in spite of some channel-to-channel variations in Ki(Mg2+), the effect of [Ca2+]l was consistently observed. The mean Ki(Mg2+) increased from 20 µM in 0.1 mM luminal Ca2+ to 72 µM at 1 mM Ca2+l and 153 µM in 3 mM Ca2+l (Table III, entries 35). A similar phenomenon was also observed in ryanodine-modified RyRs (Fig. 7; Table II, compare entries 1 and 3, 5 and 7). In ryanodine-modified RyRs, increasing [Ca2+]l from
0 to 1 mM markedly reduced the sensitivity of RyRs to Mg2+ inhibition. It caused a fourfold increase in Ki(Mg2+) in the presence of both 50 and 250 mM Cs+ (Fig. 5).

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FIGURE 6. The effect of luminal [Ca2+] on inhibition of RyRs by cytoplasmic Mg2+. Mg2+ inhibition of RyRs was measured in the presence of 1 nM cytoplasmic [Ca2+], 2mM ATP, and various luminal [Ca2+]. (A) The mean doseresponses to Mg2+ were measured with luminal [Ca2+] of 0.1 mM (, N = 7, n = 21), 1 mM ( , N = 11, n = 26), and 3 mM ( , N = 13, n = 43). The data points represent means of 313 measurements. The solid curves are Hill fits to the data. (B) x, half-inhibiting Mg2+ concentrations, Ki(Mg2+), obtained from seven individual RyRs at two or more [Ca2+]l; , the mean from all experiments. N and n values listed in Table III.
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Ion Competition Models for Mg2+ Inhibition
To further understand the ionic mechanism underlying Ca2+ activation and Mg2+ inhibition of RyRs, we compared our data to the predictions of several models for ion binding at the RyR A-site (Tables II and III). In Table II, we fitted models to the Ki(Mg2+) for ryanodine-modified channels in the presence of various combinations of high and low [Ca2+]c, [Cs+], and [Ca2+]l using a least-squares criterion. In these models, the channel is open in its unbound form or when bound to Ca2+ or Cs+, and the binding of Mg2+ closes the channel. We considered the possibility that cytoplasmic Ca2+, Cs+, Mg2+, and luminal Ca2+ all compete at the A-site (Table II, Model 3). This provided relatively poor fit to the data and was unable to account for Ki(Mg2+) at high [Cs+] and [Ca2+]l (Table II, entry 7) and the effect of [Ca2+]l on Ki(Mg2+) of ATP-activated RyRs (Table III, entries 8 and 9). Several models were investigated that were based on noncompetitive binding between Ca2+l and cytosolic ions and also noncompetitive binding between the cytosolic ions. The only models that gave an improved fit to the data were those in which cytoplasmic Ca2+ and Cs+ compete with Mg2+ at the A-site while luminal Ca2+ inhibits their binding via an allosteric interaction (Models 1 and 2). In Model 1, luminal Ca2+ prevents binding of both cytoplasmic Ca2+ and Mg2+, while in Model 2 it only affects binding of cytoplasmic Mg2+. Both models fit well, but Model 1 fit better with ryanodine-modified RyRs, and Model 2 fit better with ATP-activated RyRs. The predicted ion binding affinities for ryanodine-modified and ATP-activated RyRs were quite different and they are compared Table IV. Note that the affinities in Table IV represent different channel properties to the apparent affinities for the A-sites in the presence of Cs+ in I. Ryanodine increases the A-site affinity for Ca2+ by 40-fold, Cs+ by threefold, and decreases its affinity for Mg2+ by sevenfold and luminal Ca2+ by twofold.
Voltage Dependencies
The voltage dependencies of RyR regulation by Ca2+ and Mg2+ have been used to investigate the mechanism(s) of RyR activation by luminal Ca2+. The rationale is that at 40 mV, the bilayer potential favors the flow of Ca2+ from the luminal to cytosolic sides of the RyR while positive potential inhibits such Ca2+ feedthrough. The voltage dependence of RyR activation by luminal Ca2+ obtained here (in cytoplasmic 2 mM ATP and 1 nM Ca2+) is similar to that seen in previous studies (Sitsapesan and Williams, 1995
; Tripathy and Meissner, 1996
). At +40 mV, the RyR activated with a Ka(Ca2+)l
1 mM, while at 40 mV, Ka(Ca2+)l
0.1 mM.
One might expect that the voltage dependence of Ca2+ flow through the RyR could produce a voltage dependence in their Mg2+ inhibition, because at negative potentials, luminal Ca2+ flowing through the channel should compete with cytoplasmic Mg2+ for the A-sites. In marked contrast with this proposition, we found that Ki(Mg2+) for single RyRs in a bilayer was insensitive to voltage at all luminal [Ca2+] tested. In the case of 1 mM luminal Ca2+ (cytoplasmic 2 mM ATP and 1 nM Ca2+) the difference in Ki(Mg2+) between 40 mV (100 ± 40 µM; N = 5) and +40 mV (130 ± 40 µM; N = 12) was not significant and considerably less than changes in Ki(Mg2+) caused by varying luminal [Ca2+] (this is not the case for coupled groups of RyRs, see below). This is particularly important because it indicates that the effects of luminal Ca2+ on Mg2+ inhibition of single RyRs are not due to Ca2+ feedthrough.
Gating Kinetics of Single RyRs
More detailed information about the mechanisms of channel regulation by cytoplasmic and luminal ligands can be obtained from the rates of channel gating. For example, it has been argued that luminal Ca2+ cannot affect RyR closed dwell times by acting at cytoplasmic sites since they are inaccessible to luminal Ca2+ when the channel is closed (Xu and Meissner, 1998
). We begin with an analysis of mean open and closed dwell times (
o and
c, respectively) of single channels and proceed to analyze the analogous closing and opening rates of RyR from multichannel recordings. The general pattern of RyR activation by Ca2+ (both luminal and cytosolic), Mg2+, and ATP seen here closely follows that previously reported for the adenine nucleotides (see Fig. 7 in Laver et al., 2001
). When Po is <
0.2, channel activation and inhibition was associated primarily with changes in
c, while at higher Po, changes in channel activity were associated primarily with changes in
o (unpublished data). The dependencies of mean open and closed dwell times on luminal [Ca2+] and cytoplasmic [Mg2+] are summarized in Table VI. We find that luminal Ca2+ can modulate channel activity and Mg2+ inhibition via changes in both
o and
c, which implies a mode of action involving luminal sites on the RyR protein.
Gating Kinetics of RyRs in Coupled Groups
On average, 20% of fusion events incorporated groups of four to eight RyRs into the bilayer. Under the right experimental conditions (e.g., 40 mV and the presence of ATP), the opening of one RyR in a group tended to promote the opening of other RyRs. Fig. 8 shows the activity of four, ATP-activated RyRs at positive and negative bilayer potentials. The current trace shows transitions between the current baseline (labeled C) and four equally spaced levels (O1O4) corresponding to one to four open channels. At positive potentials, the weighting of each current level followed a binomial distribution expected from the gating in independent channels in the bilayer (unpublished data). At negative potentials, downward current steps frequently "bypassed" some of the current levels, indicating that several channels were opening in near synchrony. The weighting of current levels in these records markedly deviated from a binomial distribution. HMM analysis of these records (see MATERIALS AND METHODS) shows that the mean RyR opening rate associated with transitions between the current baseline and level O1, k0+, was significantly slower than opening rates associated with transitions between levels O1O4, k1+k4+ (Fig. 9 A,
; P = 0.003, paired t test), while there was no significant difference in the rates associated with transitions between levels O1O4. Channel closing rates, k-, did not depend on the number of open RyRs (Fig. 9 B). Thus, the coupling between RyRs was primarily due to the opening rate, which for each channel depended on whether or not one other channel was open in the bilayer.

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FIGURE 8. Recordings from an experiment with 4 RyRs in the bilayer showing coupled gating at 40 mV. At +40 mV, the channels appeared to gate independently (see text). The baths contained symmetric 250 mM Cs+ solutions with [Ca2+]c = 1 nM and [Ca2+]l = 1 mM. The current baselines are shown by dashed lines and the dashed lines (O1O4) indicate current levels associated with increasing numbers of open channels.
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One measure for the degree of RyR coupling is the difference between k0+ and k1+. The data from individual experiments are summarized in Fig. 10 by comparing k0+ (abscissa) with k1+ (ordinate). Identical RyRs that gate independently would produce data points that lie on the diagonal dashed line, while data from coupled RyRs would lie above the line. Points that lie below the diagonal line can result from channel-to-channel differences in activity, which tends to bias the gating of the low activity RyRs to transitions between the upper current levels. In a large proportion of experiments where [Ca2+]l = 1 mM and bilayer potentials were negative, RyR groups showed coupled gating. The scatter in the data reflects genuine differences between gating kinetics in different experiments rather than statistical uncertainties in the HMM analysis (relative error <25%, see MATERIALS AND METHODS). In the absence of Mg2+ (Fig. 10 A,
) three out of eight experiments exhibited significant coupling effects. This increased to seven out of seven in the same experiments performed in the presence of Mg2+ (Fig. 10 B,
). Fig. 10 shows that RyR coupling is decreased by reducing [Ca2+]l from 1 to 0.1 mM and abolished by positive bilayer potentials that oppose Ca2+ flow from the luminal to cytoplasmic baths (Fig. 10, A and B, empty symbols). Coupling is reversibly abolished by the absence of cytoplasmic ATP when the channels are activated by cytoplasmic Ca2+ (100 µM; Fig. 10 B, crosses).

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FIGURE 10. The dependence of opening rate on the presence of open channels in the bilayer obtained from individual experiments. For each experiment, the opening rates, k0+, in the absence of open channels (opening to level O1, see Fig. 8) are shown on the abscissa while opening rates in the presence of one open channel, k1+, (opening to level O2) is on the ordinate. The dashed line indicates independent gating of uniform channels. Datum points above this line indicate coupled channel openings while points below the line can arise independent gating of a heterogeneous group of channels. The legend shows the bilayer potential and luminal [Ca2+] in the absence of cytoplasmic Mg2+ (A) or in the presence of 60230 µM Mg2+ (B). Coupling between channels was promoted by luminal [Ca2+] and cytoplasmic Mg2+ and was abolished by positive bilayer potentials or removing cytoplasmic ATP and using 100 µM Ca2+ as the primary channel activator.
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The degree of coupling is markedly increased by the presence of cytoplasmic Mg2+ (Fig. 9 A and Fig. 10 B), which appears to selectively reduce k0+ (Fig. 11). Fig. 11 shows the Mg2+ dependencies of k0+ () and k1+ (
) in ATP-activated RyRs in the presence of 1 mM [Ca2+]l. At negative potentials (Fig. 11 A), Mg2+ has a much stronger effect on k0+ than k1+, while at positive potentials (Fig. 11 B), the effects of Mg2+ are similar. For comparison, we show the opening rates of single RyRs calculated from their mean closed time (
) which is similar to k0+.
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DISCUSSION
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Effects of Ion Binding at the A-site
The first main finding of this study is that Mg2+ is an RyR antagonist. Until now it was not clear whether Mg2+ was a competitive nonagonist that inhibited RyRs by preventing the agonist (Ca2+) from binding, but itself could not open the channel. We find that Mg2+ closes RyRs that are activated by ATP, DIDS, and ryanodine in the absence of Ca2+ on both sides of the channel. Measurements of Ca2+ release from SR vesicles have previously demonstrated that Mg2+ inhibits RyRs in the absence of externally applied Ca2+ (Meissner et al., 1986
). However, those experiments could not rule out the possibility that Mg2+ inhibited these RyRs by competing with Ca2+ that had been released from the vesicles. We ruled out the possibility that ATP, DIDS, and ryanodine had somehow modified the channel to allow its activation by monovalent ions and that Mg2+ inhibition occurred by occlusion of monovalent ions from the A-sites. Although Cs+ did increase the activity of ryanodine-modified RyRs, it did so by a mechanism different to Ca2+ activation at the A-sites. However, our data indicates that Cs+ did affect Mg2+ inhibition by competing with Mg2+ and Ca2+ at the A-sites (Tables II and III). Meissner et al. (1997)
had come to a similar conclusion regarding competition between Ca2+ and monovalent cations for the A- sites based on [3H]ryanodine assays of Ca2+ activation of RyRs. They found that Cs+ and other alkali cations could inhibit RyRs by preventing Ca2+ from binding to the A-sites and activating the channel. In this regard, the action of Cs+ is like that previously envisaged for Mg2+, i.e., that it is a competitive nonagonist. In summary then, Ca2+ at the A-sites is an agonist, Cs+ is a nonagonist and Mg2+ is an antagonist.
Luminal Ca2+ Alleviates Mg2+ Inhibition by an Allosteric Mechanism
Another major finding of this study is that the sensitivity of RyRs to Mg2+ inhibition is modulated by the level of luminal Ca2+. Increasing [Ca2+]l from 0.1 to 1 mM produced a fourfold decrease in the Mg2+ affinity of the A-sites regardless of whether RyRs were in the presence of high or low cytoplasmic [Cs+] and [Ca2+], or whether they were stimulated by ATP or modified by ryanodine (Tables II and III). A number of our findings indicate that this effect is not due to competition at the A-sites between cytoplasmic Mg2+ and luminal Ca2+ that permeates the channel, but rather due to an allosteric mechanism. First, Ki(Mg2+) for single RyRs was independent of the bilayer potential and hence Ca2+ feedthrough. Second, increased luminal Ca2+ alleviated Mg2+ inhibition via changing both the mean open and closed durations of the channel (Table VI). The effect of luminal Ca2+ on channel closures suggests that it has access to its site of action when the channel is impermeable to Ca2+. Finally, the effect of luminal Ca2+ on Mg2+ inhibition is noncompetitive (Tables II and III). For example, competitive kinetics could not account for the combined effect of luminal Ca2+ and cytoplasmic Cs+ on Ki(Mg2+) (Table II, entries 5 and 7) or the significant effect of luminal Ca2+ on Ki(Mg2+) in the presence 10 µM cytoplasmic Ca2+ (Table III, entries 8 and 9).
Comparison with Cardiac RyRs
Evidence for allosteric modulation of cardiac RyRs by luminal Ca2+ has been reported previously (Gyorke and Gyorke, 1998
). However, the effect of luminal Ca2+ on cardiac RyRs (RyR2 isoform) is quite different from that found here for skeletal RyRs (RyR1). With RyR2, luminal Ca2+ modulates channel function by sensitizing the Ca2+ activation site (A-sites) and by reducing inhibition at the low affinity inhibition sites (I-sites) (Gyorke and Gyorke, 1998
) and a reduction in maximal level of Ca2+ activation (Pmax) (Xu and Meissner, 1998
). In contrast, with ATP-activated RyR1, luminal Ca2+ has no evident effect on the Ca2+ sensitivity of either the A- or I-sites nor does it alter Pmax (Table I). In addition, at low (<1 µM) cytoplasmic Ca2+, luminal Ca2+ causes substantially more activation of RyR1 than of RyR2.
In single RyR1 channels, we did not detect competition between luminal Ca2+ and cytoplasmic Mg2+ for the A-sites, either by measuring the voltage dependence (i.e., Ca2+ flux dependence) of Ki(Mg2+) or its dependence on Cs+ competition. However, evidence of trans-channel competition has been reported in RyR2 by Xu and Meissner (1998)
who noted that cytoplasmic Mg2+ induced a voltage dependence in the effect of luminal Ca2+, which can be interpreted as a voltage-dependent Mg2+ inhibition.
Ca2+ Feedthrough Couples RyRs in Lipid Bilayers
The coupled opening of RyRs that we observe is very similar to the phenomenon reported by Copello et al. (2003)
and Porta et al. (2004)
. They proposed that coupled gating of RyRs occurred in bilayers when Ca2+ flow (luminal to cytoplasm) through one channel raised the local [Ca2+]c sufficiently to activate neighboring RyRs. Several of our findings support their conclusion. First, the coupling of channel openings was promoted under conditions which favored the flow of luminal Ca2+ through the channel (i.e., negative potential and high [Ca2+]l). Second, coupling did not occur in the presence of high (100 µM) cytoplasmic Ca2+ (Fig. 10). At these concentrations, the A-sites are virtually saturated and the feedthrough of luminal Ca2+ would have no significant activating effect. Finally, the opening of one channel in a group of RyRs caused an approximately fivefold reduction in Mg2+ inhibition of the other RyRs (Fig. 11), suggesting that local [Ca2+]c is indeed increased and is competing with Mg2+ for the A-sites. From the effects of cytoplasmic [Ca2+] on Po and Ki(Mg2+) (Figs. 1 and 4), one can estimate [Ca2+]c to be
10 µM at RyRs neighboring an open RyR. The separation of these RyRs in the bilayer may be estimated from theoretical concentration profiles of [Ca2+] in the vicinity of a pore (Stern, 1992
) (Fig. 12). The [Ca2+] near an open channel falls off sharply with distance because Ca2+ emerging from the pore is rapidly chelated by BAPTA (4.5 mM) in the bath. Fig. 12 shows that at 40 mV, 1 mM luminal Ca2+ generates 1 µM [Ca2+] in the cytoplasmic bath at 30 nm from an open channel. Reversing the polarity of the potential or reducing [Ca2+]l to 0.1 mM decreases this distance to
20 nm.
Freeze fracture electron micrographs show that RyRs within the triad junction are organized into square, two-dimensional arrays with the pores of nearest and second nearest neighbors being separated by 31 and 43 nm, respectively (Protasi et al., 1997
). The A-sites could be as close as 20 nm from the pore of another channel. Separations in the range 2030 nm (Fig. 12) would nicely explain the coupling we observe at 40 mV and 1 mM [Ca2+]l and the loss of coupling at 0.1 mM [Ca2+]l and +40 mV. This suggests that during isolation and reconstitution, the RyR arrays in muscle are not completely disrupted and that rafts containing 310 RyRs remain stable in lipid bilayers.
The close packing of RyRs in the membrane makes it seem possible that RyRs could interact physically. In fact, a coupling phenomenon has been identified in CHAPS-purified RyRs (RyR1 and RyR2; Marx et al., 1998
, 2001
) that is likely to stem from a physical interaction between RyRs. Those authors found that protein fractions enriched in RyR multimers produced synchronously gated channels in
10% of instances when reconstituted with lipid bilayers. Functional coupling required FKSO6 binding protein, FKBP 12 and FKBP 12.6, but was independent of luminal Ca2+. Our experimental conditions were not designed to optimize observation of this type of coupling since our membrane preparations were not enriched in RyR multimers. We found such behavior in RyRs to be quite rare, observing it in only two instances out of many hundreds of recordings when using cardiac SR vesicles (Honen, B., personal communication). Therefore, RyR coupling by luminal Ca2+ is the dominant mechanism operating in our experiments.
Coupled and Single RyRs are Regulated Differently by Luminal Ca2+
Our data shows that Mg2+ inhibition is highly dependent on the situation of the RyR. Mg2+ inhibition of single RyRs, measured by open probability and opening rate, was not affected by bilayer potential. However, in clusters, RyRs responded quite differently to Mg2+. Although the opening of the first RyR in a group was similar to the single channel situation (Fig. 11, compare and
), Mg2+ had a markedly reduced effect on subsequent openings, which we explained in terms of the raised local [Ca2+]c reducing Ki(Mg2+) of the neighboring closed RyRs (see above). However, the fact that Ca2+ feedthrough does not also decrease Ki(Mg2+) for the first channel opening means that Mg2+ inhibition for any particular channel is somehow unaffected by the raised [Ca2+] originating from its own pore. This is surprising since the local [Ca2+] near the open channel will be much higher than near its closed neighbors.
The reason for this curious phenomenon probably lies in the different ways that the A-sites of single RyRs and RyR rafts access luminal Ca2+. With a single RyR, the A-sites are only accessible to luminal Ca2+ while the channel is open but with an RyR in a raft, even when the channel is closed, the A-sites can still have access to luminal Ca2+ via adjacent open channels. With single RyRs, it is then possible that luminal Ca2+ feedthrough will not affect closed dwell times since the A-sites are inaccessible to luminal Ca2+ when the channel is closed. However, luminal Ca2+ would be able to modulate both open and closed durations of an RyR in a raft. Therefore there is scope for luminal Ca2+ to have a bigger effect on RyRs in rafts than individually. This difference should become quite significant when RyR Po < 0.2 where changes in Po are mediated almost entirely through changes in closed dwell time. The differing accessibility of A-sites to luminal Ca2+ in single and coupled RyRs could also influence the binding of luminal Ca2+ and cytoplasmic Mg2+ at the A-sites. The unbinding rate of Mg2+ might not be fast enough to allow luminal Ca2+ and cytoplasmic Mg2+ to reach equilibrium at the A-sites during the open time of a single RyR (Zahradnikova et al., 2003
). This could be the reason why luminal Ca2+ and cytoplasmic Mg2+ do not exhibit competitive binding kinetics with single RyRs. The situation with coupled RyRs would be quite different. When an RyR is exposed to luminal Ca2+ via neighboring channels, there is more time for Ca2+ and Mg2+ to attain equilibrium at the A-site before channel opening.
In any case, A-sitemediated Mg2+ inhibition of an RyR is effectively "immune" to the Ca2+ emanating from its own pore. This can be seen, albeit to a lesser degree, for luminal Ca2+ activation in the absence of Mg2+. The first channel opening in a cluster frequently has a slower rate than the second opening (compare k0+ and k1+ in Fig. 10 A). Therefore, RyRs can be more strongly activated by the Ca2+ from neighboring channels than by the Ca2+ from their own pore.
Mechanism of Luminal Ca2+ Activation
In this study, we found evidence supporting both the feedthrough and the true luminal regulation hypotheses for luminal Ca2+ regulation of RyRs. We observed coupled gating of RyRs that is due to feedthrough of luminal Ca2+ to the cytoplasmic Ca2+ sites on the RyR. We also find that luminal Ca2+ alters Mg2+ inhibition by a noncompetitive mechanism, indicating the presence of luminal Ca2+ sites that regulate RyR activity. Both mechanisms are probably important for the stimulation of Ca2+ release in muscle by increased store load (see below). Though there is strong evidence that the Ca2+ feedthrough activates RyR rafts, the question of how luminal Ca2+ activates single RyRs is not totally resolved since it remains possible that RyRs are "immune" to their own Ca2+ flux.
The fact that RyR activation by luminal Ca2+ requires the presence of specific secondary activators of the channel such as ATP, suramin (Sitsapesan and Williams, 1994
), or caffeine (Xu and Meissner, 1998
) has been taken as evidence against the Ca2+ feedthrough mechanism (Sitsapesan and Williams, 1997
). However, our finding that ATP is required for Ca2+ coupling of RyRs leads to an alternative interpretation of this phenomenon. In the absence of secondary activators, any opening of RyRs would be associated with Ca2+ binding at the A-sites. Thus when luminal Ca2+ gains access to the A-sites through the open channel, Ca2+ will not augment channel opening because the A-sites are already occupied. However, when ATP is present, the RyRs can open even when the A-sites are unoccupied, leaving scope for luminal Ca2+ to enhance RyR activation.
The nature of the luminal Ca2+sensing sites is still not determined. It is known that the luminal proteins calsequestrin, triadin, and junction are associated with RyRs and modulate their activity (Beard et al., 2004
). These proteins can confer on RyRs a means of sensing luminal [Ca2+] by either Ca2+-dependent dissociation from the RyR complex or by regulating RyRs in a Ca2+-dependent manner, as is the case for calmodulin. It is unlikely that calsequestrin dissociation underlies the effects of luminal Ca2+ in Mg2+ inhibition because luminal Ca2+ was kept in the range that stabilizes calsequestrin binding to the RyR complex.
Ryanodine Modification of RyRs
The effects of ryanodine modification of RyRs were broadly similar to those seen in previous studies. Ryanodine markedly stabilized channel openings to a lower than normal conductance, channel activity was relatively insensitive to cytoplasmic Ca2+ (Fig. 1). There are two interpretations for the loss of Ca2+ sensitivity. One is that the Ca2+ activation dependence is shifted to such low [Ca2+] that RyRs are fully activated over the experimentally attainable [Ca2+] range. The alternative is that ryanodine-modified RyRs do not require cytoplasmic Ca2+ to open. The decisive experiment is to find Ca2+ levels sufficiently low to deactivate ryanodine-modified RyRs. Unfortunately, on this key experiment, reports are divided. Experiments on purified, recombinant cardiac RyRs show that ryanodine-modified RyRs deactivate (Po
0.2) in the presence of 1 mM EGTA and no added Ca2+ (Du et al., 2001
; Masumiya et al., 2001
) while ryanodine-modified sheep cardiac RyRs remained fully active in the presence of Ca2+ buffers and no added Ca2+ (Rousseau et al., 1987
; Laver et al., 1995
) (the actual [Ca2+]free in these studies is uncertain because impurity Ca2+ levels were not specified).
In the present study, we did not see reproducible deactivation of ryanodine-modified RyRs at low [Ca2+]. In only a few instances did these RyRs show a clear decrease in activity at 0.11 nM Ca2+. The reason why reduced activity at low [Ca2+] was frequently seen in studies on recombinant RyR2 is not clear. It could be due to differences between native RyRs and those purified from nonmuscle cells or it could be due to the different bathing solutions used for single channel recording (KCl vs. CsCH3O3S). However, in spite of these differences, sensitivity of RyRs to Mg2+ inhibition seen here is consistent with the study of Masumiya et al. (2001)
, which obtained a Ki(Mg2+)
2 mM in the presence of 100 nM cytoplasmic Ca2+, zero luminal [Ca2+], and symmetric 250 mM KCl (our model predicts Ki(Mg2+) = 2.0 mM). Du et al. (2001)
also found that Mg2+ inhibition was independent of membrane potential over the range ± 30 mV.
The ion selectivity of the A-site is strongly and reproducibly altered by ryanodine. In the presence of 250 mM Cs+, the apparent affinity of the A-sites for Ca2+ is 100 nM (see Kapp(Ca2+)c in Table I), which means that RyRs should always deactivate at cytoplasmic [Ca2+] < 100 nM if Ca2+ at the A-sites is required for channel activation. This was not the case in most RyRs studied here. The decrease in activity seen by others at nM Ca2+, and occasionally seen here, probably does not involve the same mechanism as Ca2+ activation in native RyRs.
Control of Calcium Release by Luminal Calcium and Cytoplasmic Mg2+
The results of this study give new insight into how SR luminal Ca2+ and cytoplasmic Mg2+ influence Ca2+ release from the SR under physiological conditions in muscle fibers. Mg2+ exerts inhibitory effects on RyR1 by acting at the I-site as well as the A-site (see INTRODUCTION). We have extended the dual-inhibition model to include the effects of luminal Ca2+ on the ion binding properties of the A-sites. The inhibitory effect of Mg2+ at the I-sites, which is evidently not affected by luminal [Ca2+] (note similar Ca2+ affinity of the I-sites at 0.01 and 1 mM luminal Ca2+ in Table I), tempers the effects of luminal Ca2+ on the overall Mg2+ inhibition. This is because the overall Ki(Mg2+) for both Mg2+ inhibition mechanisms is always less than the Ki(Mg2+) for either A- and I-sites alone (Laver et al., 1997
). For the I-sites, Ki(Mg2+) is
250 µM at physiological ionic strength (
100 mM) in the presence of ATP (Table I). At the normal resting cytoplasmic [Ca2+] (
100 nM), the Ki(Mg2+) of the A-sites and overall Mg2+ inhibition are similar, ranging from
20 µM at very low luminal Ca2+ (0.1 mM) to
100 µM at quite high luminal Ca2+ (3 mM) (i.e., in this situation, the overall inhibition is largely set by the A-site properties). Thus, even though the ATP present in the cytoplasm has a strong stimulatory action on RyR1 (Fig. 1), the presence of physiological free Mg2+ (1 mM) will keep the channel Po very low even if the SR is very loaded with Ca2+ (Mg2+ is present at four times the Ki(Mg2+) of I-site and 10 to 50 times the Ki(Mg2+) of the A-site). Moreover, as the SR becomes progressively more depleted of Ca2+, increased Mg2+ inhibition would cause a large reduction (>25-fold) in channel activity and Ca2+ release. This is much larger than the two to fivefold decrease in activity that one would expect in the absence of Mg2+ (Tables III and VI). Therefore, modulation of Mg2+ inhibition by luminal Ca2+ is a significant regulator of Ca2+ release from the SR.
It is also apparent that heavy loading of the SR with Ca2+ will make RyRs more prone to activation by cytoplasmic Ca2+ and/or caffeine. If the cytoplasmic [Ca2+] rises above the resting level, the Ki(Mg2+) at the A-site will increase substantially. For the case of high SR luminal [Ca2+] (3 mM), Ki(Mg2+) for the A-sites will increase from
100 µM at 100 nM cytoplasmic Ca2+ to 180 µM at 1 µM Ca2+, 340 µM at 3 µM Ca2+, and 900 µM at 10 µM Ca2+. As a result, the overall Ki(Mg2+) will increase from
80 µM at 100 nM cytoplasmic Ca2+ to 110 µM at 1 µM Ca2+c, 150 µM at 3 µM Ca2+c, and 200 µM at 10 µM Ca2+c. Consequently, if the cytoplasmic [Ca2+] is raised above the resting level, or if caffeine is applied (caffeine increases the sensitivity of the A-site for Ca2+ relative to Mg2+; Balog et al. 2001
), the total inhibitory effect of Mg2+ will decrease and the channel will activate to some extent. Nevertheless, peak activation will still be ultimately limited by the prevailing Mg2+ inhibition at the I-site.
Thus, the above interplay of I-site and A-site properties would seem adequate to account for the findings that (a) appreciable calcium-induced calcium release (CICR) cannot be induced at physiological [Mg2+] if the SR is loaded at <
25% of its maximum capacity ([Ca2+]l
1 mM, Endo, 1985
; Saiki and Ikemoto, 1999
), (b) caffeine-induced release and CICR and are greatly potentiated by heavily loading the SR with Ca2+ and attenuated by raising the free [Mg2+] above 1 mM (Endo, 1985
; Nelson and Nelson, 1990
; Lamb et al., 2001
), (c) the peak rate of caffeine-induced Ca2+ release in muscle fibers is very much lower than that produced by action potential stimulation (Lamb et al., 2001
; Posterino and Lamb, 2003
), and (d) the SR can be depleted of some but not all of its Ca2+ if the cytoplasmic [Mg2+] is lowered to 50 µM, and fully depleted by lowering [Mg2+] or by applying caffeine in the presence of low [Mg2+] (Lamb and Stephenson, 1994
; Fryer and Stephenson, 1996
).
Voltage Sensor Control of Calcium Release in Skeletal Muscle
The above considerations show how the levels of luminal Ca2+ and cytoplasmic Mg2+ can affect the sensitivity of skeletal muscle fibers to CICR and caffeine-induced Ca2+ release. However, these are not the normal mechanisms triggering Ca2+ release in vertebrate skeletal muscle. Instead, Ca2+ release is controlled largely by the DHPRs in the T-tubules (see INTRODUCTION). At rest, CICR is greatly down-regulated by strong Mg2+ inhibition exerted at physiological [Mg2+] (Meissner et al., 1986
; Laver et al., 1997
). DHPRs must be able to overcome this inhibition because RyRs are near maximally activated (i.e., Po
1) during action potential stimulation (Posterino and Lamb, 2003
, and references therein). The DHPRs do not appear to bypass Mg2+ inhibition of RyRs, because raising the [Mg2+] to >3 mM considerably inhibits the ability of the DHPRs to trigger Ca2+ release (Lamb and Stephenson, 1991
, 1994
; Westerblad and Allen, 1992
; Anderson and Meissner, 1995
; Blazev and Lamb, 1999
). Furthermore, the DHPRs are not able to activate the RyRs unless the RyRs are also stimulated by ATP in the cytosol (see above references). The preceding two observations show that DHPR activation is insufficient to activate RyRs independently of cytoplasmic factors acting on the RyR. This raises the possibility that DHPRs activate RyRs by raising the overall Ki(Mg2+), thereby reducing their inhibition by Mg2+ and allowing cytosolic ATP to activate the channel, with the released Ca2+ then able to reinforce this activation (Lamb and Stephenson, 1991
, 1994
).
Not all published data can be easily explained solely by the removal of RyR Mg2+ inhibition. O'Brien et al. (2002)
investigated the ability of the DHPRs to activate mutated RyR1s (E4032A) in which Ca2+ and ATP activation was virtually abolished. They found that DHPR activation of these RyRs could still produce
20% of the Ca2+ release of wild-type RyRs, which suggests that Ca2+ and ATP activation was not absolutely necessary for DHPR controlled Ca2+ release. However, Ca2+ and ATP activation of the E4032A mutant RyR was determined in bilayer experiments where luminal Ca2+ was absent. Therefore, it is possible that in the presence of physiological luminal [Ca2+] (1 mM), the mutant RyRs show substantially more activation by ATP as is known to be the case for the wild-type RyRs.
Exactly how DHPRs modulate the dual mechanism of Mg2+ inhibition is not clear. At the very least, the DHPRs must overcome the Mg2+ inhibition exerted at the I-site (Laver et al., 1997
). Importantly, the results here also show that reducing the I-site inhibition would not be enough by itself to open the RyRs because, unless the SR was very loaded with Ca2+, RyR1 would still be inhibited by Mg2+ on the A-sites. However, if the DHPRs could reduce the inhibitory effect of Mg2+ at the A-sites as well as at the I-sites, the RyR would be potently activated irrespective of the level of SR Ca2+ loading and the resting cytoplasmic [Ca2+]. This immediately raises the intriguing prospect that the DHPRs are able to commandeer the mechanism by which SR luminal Ca2+ reduces Mg2+ inhibition at the A-sites. In fact this seems quite plausible, as this mechanism must involve an allosteric interaction mediated from the luminal side to the cytoplasmic side of the RyR, and it is apparent that DHPR activation must also involve long-range allosteric effects within the RyR. Moreover, this "commandeering" would readily explain why DHPRs can potently activate RyRs irrespective of the level of Ca2+ loading in the SR and thereby, unlike applied caffeine or cytoplasmic Ca2+, completely empty the SR of Ca2+ in the presence of physiological [Mg2+] (Kurebayashi and Ogawa, 2001
; Posterino and Lamb, 2003
).
In conclusion, cytoplasmic Mg2+ is an important regulator of RyRs in muscle, which is very sensitive to both the cytoplasmic and luminal milieu. The effect of luminal Ca2+ on the Mg2+ affinity of the A-sites may well be the trigger for Ca2+ release from internal stores while feedthrough of luminal Ca2+ to the cytoplasmic A-sites would further promote Ca2+ release. Finally, measurements of single RyRs in artificial bilayers have had a major impact on our understanding of the mechanisms of Ca2+ release. In addition, this study demonstrates that RyR arrays like those found in muscle can be regulated by mechanisms that are different to those identified in single channels.
 |
ACKNOWLEDGMENTS
|
---|
Thanks to Paul Johnson and Melanie Laver for assisting with the experiments and to Angela Dulhunty, Suzy Pace, and Joan Stivala (Australian National University, Canberra, ACT, Australia) for supplying SR vesicles.
This work was supported by the National Health & Medical Research Council of Australia (234420) and by infrastructure grant from New South Wales Health through Hunter Medical Research Institute.
Olaf S. Andersen served as editor.
Submitted: 7 May 2004
Accepted: 21 October 2004
 |
REFERENCES
|
---|
Anderson, K., and G. Meissner. 1995. T-tubule depolarization-induced SR Ca2+ release is controlled by dihydropyridine receptor- and Ca2+-dependent mechanisms in cell homogenates from rabbit skeletal muscle. J. Gen. Physiol. 105:363383.[Abstract]
Balog, E.M., B.R. Fruen, N.H. Shomer, and C.F. Louis. 2001. Divergent effects of the malignant hyperthermia-susceptible arg(615)cys mutation on the Ca2+ and Mg2+ dependence of the RyR1. Biophys. J. 81:20502058.[Abstract/Free Full Text]
Beard, N.A., A.F. Dulhunty, and D.R. Laver. 2000. The effect of increasing luminal calcium on skeletal muscle. Proceedings of the Australian Physiological and Pharmacological Society. 31:22P.
Beard, N.A., D.R. Laver, and A.F. Dulhunty. 2004. Calsequestrin and the calcium release channel of skeletal and cardiac muscle. Prog. Biophys. Mol. Biol. 85:3369.[CrossRef][Medline]
Blazev, R., and G.D. Lamb. 1999. Low [ATP] and elevated [Mg2+] reduce depolarization-induced Ca2+ release in rat skinned skeletal muscle fibres. J. Physiol. 520:203215.[Abstract/Free Full Text]
Brooks, S.P., and K.B. Storey. 1992. Bound and determined: a computer program for making buffers of defined ion concentrations. Anal. Biochem. 201:119126.[Medline]
Ching, L.L., A.J. Williams, and R. Sitsapesan. 2000. Evidence for Ca2+ activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex. Circ. Res. 87:201206.[Abstract/Free Full Text]
Chu, A., M.C. Dixon, A. Saito, S. Seiler, and S. Fleischer. 1988. Isolation of sarcoplasmic reticulum fractions referable to longitudinal tubules and junctional terminal cisternae from rabbit skeletal muscle. Methods Enzymol. 157:3650.[Medline]
Chung, S.H., J.B. Moore, L.G. Xia, L.S. Premkumar, and P.W. Gage. 1990. Characterization of single channel currents using digital signal processing techniques based on Hidden Markov Models. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 329:265285.[Medline]
Copello, J.A., M. Porta, P. Diaz-Silvester, A. Nani, A.L. Escobar, S. Fleischer, and M. Fill. 2003. Coordinated gating of multiple ryanodine receptor channels (RyRs). Biophys. J. 84:17a.
Donoso, P., H. Prieto, and C. Hidalgo. 1995. Luminal calcium regulates calcium release in triads isolated from frog and rabbit skeletal muscle. Biophys. J. 68:507515.[Abstract]
Du, G.G., X. Guo, V.K. Khanna, and D.H. MacLennan. 2001. Ryanodine sensitizes the cardiac Ca2+ release channel (ryanodine receptor isoform 2) to Ca2+ activation and dissociates as the channel is closed by Ca2+ depletion. Proc. Natl. Acad. Sci. USA. 98:1362513630.[Abstract/Free Full Text]
Dunnett, J., and W.G. Nayler. 1978. Calcium efflux from cardiac sarcoplasmic reticulum: effects of calcium and magnesium. J. Mol. Cell. Cardiol. 10:487498.[CrossRef][Medline]
Endo, M. 1985. Calcium release from sarcoplasmic reticulum. Current Topics and Membrames and Transport. 25:181230.
Fabiato, A., and F. Fabiato. 1977. Calcium release from the sarcoplasmic reticulum. Circ. Res. 40:119129.[Medline]
Ford, L.E., and R.J. Podolsky. 1972. Calcium uptake and force development by skinned muscle fibres in EGTA buffered solutions. J. Physiol. 223:119.[Medline]
Fryer, M.W., and D.G. Stephenson. 1996. Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle. J. Physiol. 493:357370.[Abstract]
Gyorke, I., and S. Gyorke. 1998. Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys. J. 75:28012810.[Abstract/Free Full Text]
Kawasaki, T., and M. Kasai. 1989. Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum. J. Biochem. (Tokyo). 106:401405.[Abstract]
Kurebayashi, N., and Y. Ogawa. 2001. Depletion of Ca2+ in the sarcoplasmic reticulum stimulates Ca2+ entry into mouse skeletal muscle fibres. J. Physiol. 533:185199.[Abstract/Free Full Text]
Lamb, G.D., and D.G. Stephenson. 1991. Effect of Mg2+ on the control of Ca2+ release in skeletal muscle fibres of the toad. J. Physiol. 434:507528.[Abstract]
Lamb, G.D., and D.G. Stephenson. 1994. Effects of intracellular pH and [Mg2+] on excitation-contraction coupling in skeletal muscle fibres of the rat. J. Physiol. 478:331339.[Abstract]
Lamb, G.D., M.A. Cellini, and D.G. Stephenson. 2001. Different Ca2+ releasing action of caffeine and depolarisation in skeletal muscle fibres of the rat. J. Physiol. 531:715728.[Abstract/Free Full Text]
Laver, D.R., L.D. Roden, G.P. Ahern, K.R. Eager, P.R. Junankar, and A.F. Dulhunty. 1995. Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. J. Membr. Biol. 147:722.[Medline]
Laver, D.R., T.M. Baynes, and A.F. Dulhunty. 1997. Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. J. Membr. Biol. 156:213229.[CrossRef][Medline]
Laver, D.R., K.R. Eager, L. Taoube, and G.D. Lamb. 2000. Effects of cytoplasmic and luminal pH on Ca2+ release channels from rabbit skeletal muscle. Biophys. J. 78:18351851.[Abstract/Free Full Text]
Laver, D.R., G.K. Lenz, and G.D. Lamb. 2001. Regulation of the calcium release channel from rabbit skeletal muscle by the nucleotides ATP, AMP, IMP and adenosine. J. Physiol. 537:763778.[Abstract/Free Full Text]
Ma, J., and J. Zhao. 1994. Highly cooperative and hysteretic response of the skeletal muscle ryanodine receptor to changes in proton concentrations. Biophys. J. 67:626633.[Abstract]
Marks, P.W., and F.R. Maxfield. 1991. Preparation of solutions with free calcium concentration in the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Anal. Biochem. 193:6171.[Medline]
Marx, S.O., K. Ondrias, and A.R. Marks. 1998. Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors). Science. 281:818821.[Abstract/Free Full Text]
Marx, S.O., J. Gaburjakova, M. Gaburjakova, C. Henrikson, K. Ondrias, and A.R. Marks. 2001. Coupled gating between cardiac calcium release channels (ryanodine receptors). Circ. Res. 88:11511158.[Abstract/Free Full Text]
Masumiya, H., P. Li, L. Zhang, and S.R. Chen. 2001. Ryanodine sensitizes the Ca2+ release channel (ryanodine receptor) to Ca2+ activation. J. Biol. Chem. 276:3972739735.[Abstract/Free Full Text]
Meissner, G. 1994. Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Annu. Rev. Physiol. 56:485508.[CrossRef][Medline]
Meissner, G., E. Darling, and J. Eveleth. 1986. Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides. Biochemistry. 25:236244.[Medline]
Meissner, G., E. Rios, A. Tripathy, and D.A. Pasek. 1997. Regulation of skeletal muscle Ca2+ release channel (ryanodine receptor) by Ca2+ and monovalent cations and anions. J. Biol. Chem. 272:16281638.[Abstract/Free Full Text]
Melzer, W., A. Herrmann-Frank, and H.C. Luttgau. 1995. The role of Ca2+ ions in excitation-contraction coupling of skeletal muscle fibres. Biochim. Biophys. Acta. 1241:59116.[Medline]
Miller, C., and E. Racker. 1976. Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers. J. Membr. Biol. 9:283300.
Morii, H., and Y. Tonomura. 1983. The gating behavior of a channel for Ca2+-induced Ca2+ release in fragmented sarcoplasmic reticulum. J. Biochem. (Tokyo). 93:12711285.[Abstract]
Nelson, T.E., and K.E. Nelson. 1990. Intra- and extraluminal sarcoplasmic reticulum membrane regulatory sites for Ca2+-induced Ca2+ release. FEBS Lett. 263:292294.[CrossRef][Medline]
O'Brien, J.J., W. Feng, P.D. Allen, S.R. Chen, I.N. Pessah, and K.G. Beam. 2002. Ca2+ activation of RyR1 is not necessary for the initiation of skeletal-type excitation-contraction coupling. Biophys. J. 82:24282435.[Abstract/Free Full Text]
O'Neill, E.R., M.M. Sakowska, and D.R. Laver. 2003. Regulation of thecalcium release channel from skeletal muscle by suramin and the disulfonated stilbene derivatives DIDS, DBDS, and DNDS. Biophys. J. 84:16741689.[Abstract/Free Full Text]
Porta, M., P.N. Diaz-Sylvester, A. Nani, M. Fill, S. Fleischer, and J.A. Copello. 2004. Modulation of coordinated gating of ryanodine receptor (RyR) channels in planar lipid bilayers. Biophys. J. 86:241a.
Posterino, G.S., and G.D. Lamb. 2003. Effect of sarcoplasmic reticulum Ca2+ content on action potential-induced Ca2+ release in rat skeletal muscle fibres. J. Physiol. 551:219237.[Abstract/Free Full Text]
Protasi, F., C. Franzini-Armstrong, and B.E. Flucher. 1997. Coordinated incorporation of skeletal muscle dihydropyridine receptors and ryanodine receptors in peripheral couplings of BC3H1 cells. J. Cell Biol. 137:859870.[Abstract/Free Full Text]
Rousseau, E., J.S. Smith, and G. Meissner. 1987. Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am. J. Physiol. 253:C364C368.[Medline]
Saiki, Y., and N. Ikemoto. 1999. Coordination between Ca2+ release and subsequent re-uptake in the sarcoplasmic reticulum. Biochemistry. 38:31123119.[CrossRef][Medline]
Sitsapesan, R. 1999. Similarities in the effects of DIDS, DBDS and suramin on cardiac ryanodine receptor function. J. Membr. Biol. 168:159168.[CrossRef][Medline]
Sitsapesan, R., and A.J. Williams. 1994. Regulation of the gating of the sheep cardiac sarcoplasmic reticulum Ca2+-release channel by luminal Ca2+. J. Membr. Biol. 137:215226.[Medline]
Sitsapesan, R., and A.J. Williams. 1995. The gating of the sheep skeletal sarcoplasmic reticulum Ca2+-release channel is regulated by luminal Ca2+. J. Membr. Biol. 146:133144.[Medline]
Sitsapesan, R., and A.J. Williams. 1997. Regulation of current flow through ryanodine receptors by luminal Ca2+. J. Membr. Biol. 159:179185.[CrossRef][Medline]
Smith, J.S., R. Coronado, and G. Meissner. 1986. Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation by Ca2+ and ATP and modulation by Mg2+. J. Gen. Physiol. 88:573588.[Abstract]
Soler, F., F. Fernandez Belda, and J.C. Gomez Fernandez. 1992. The Ca2+ release channel in junctional sarcoplasmic reticulum: gating and blockade by cations. Int. J. Biochem. 24:903909.[CrossRef][Medline]
Stern, M.D. 1992. Buffering of calcium in the vicinity of a channel pore. Cell Calcium. 13:183192.[Medline]
Tanabe, T., K.G. Beam, B.A. Adams, T. Niidome, and S. Numa. 1990. Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling. Nature. 346:567569.[CrossRef][Medline]
Tinker, A., A.R. Lindsay, and A.J. Williams. 1992. A model for ionic conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum. J. Gen. Physiol. 100:495517.[Abstract]
Tripathy, A., and G. Meissner. 1996. Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel. Biophys. J. 70:26002615.[Abstract]
Westerblad, H., and D.G. Allen. 1992. Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle. J. Physiol. 453:413434.[Abstract]
Xu, L., and G. Meissner. 1998. Regulation of cardiac muscle Ca2+ release channel by sarcoplasmic reticulum lumenal Ca2+. Biophys. J. 75:23022312.[Abstract/Free Full Text]
Zahradnikova, A., and I. Zahradnik. 1993. Modification of cardiac Ca2+ release channel gating by DIDS. Pflugers Arch. 425:555557.[Medline]
Zahradnikova, A., M. Dura, I. Gyorke, A.L. Escobar, I. Zahradnik, and S. Gyorke. 2003. Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions. Am. J. Physiol. Cell Physiol. 285:C1059C1070.[Abstract/Free Full Text]