Large tetraalkyl ammonium cations produce a reduced conductance state in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel.

The purified Ca(2+)-release/ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) functions as a calcium-activated cation-selective channel under voltage clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effect of large tetraalkyl ammonium (TAA) cations, (CnH2n+1)4N+ (n = 4 and 5) on monovalent cation conduction. These cations modify the conductance of the receptor channel at positive holding potentials from the cytosolic side of the channel. Under these conditions, openings are resolved as a mixture of normal full amplitude events and events of reduced conductance. The amplitude of the reduced conductance state is a fixed proportion of the normal open state. As a proportion of all open events, the occurrence of the tetrabutyl ammonium (TBA+) related subconductance state increases with concentration and increasingly positive holding potential. The TBA+ related subconductance state displays similar conduction properties to the unmodified channel; with a linear current-voltage relationship, a similar affinity for K+ and voltage-dependent block by TEA+. A method was used to quantify the voltage dependence of the occurrence of the TBA+ effect, which yielded an effective gating charge of 1.66. A second method based on kinetic analysis of the voltage dependence of transitions between the full open state and the TBA+ related subconductance state produced a similar value. In addition, this analysis revealed that the bulk of the voltage-dependence resided in the off rate. TBA+ related subconductance events, expressed as a proportion of all open events, saturated with increasing TBA+ concentration. Kinetic analysis revealed that this could be entirely accounted for by changes in the on rate. Tetrapentyl ammonium (TPeA+) causes a qualitatively similar effect with a subconductance state of lower amplitude. The voltage-dependence of the effect was comparable to that displayed by TBA+. These findings are interpreted as a form of partial block in which more than one large TAA cation binds at the extremity of the voltage drop to produce an electrostatic barrier for ion translocation.     

How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel?

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca(2+)-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs+, Li+) have values of fractional conductance in a narrow range (0.60- 0.66). With divalent cations fractional conductance is considerably lower (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum Ca(2+)-release channel produces a conformational alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.     

Electrophysiological effects of ryanodine derivatives on the sheep cardiac sarcoplasmic reticulum calcium-release channel.

We have examined the effects of a number of derivatives of ryanodine on K+ conduction in the Ca2+ release channel purified from sheep cardiac sarcoplasmic reticulum (SR). In a fashion comparable to that of ryanodine, the addition of nanomolar to micromolar quantities to the cytoplasmic face (the exact amount depending on the derivative) causes the channel to enter a state of reduced conductance that has a high open probability. However, the amplitude of that reduced conductance state varies between the different derivatives. In symmetrical 210 mM K+, ryanodine leads to a conductance state with an amplitude of 56.8 +/- 0.5% of control, ryanodol leads to a level of 69.4 +/- 0.6%, ester A ryanodine modifies to one of 61.5 +/- 1.4%, 9,21-dehydroryanodine to one of 58.3 +/- 0.3%, 9 beta,21beta-epoxyryanodine to one of 56.8 +/- 0.8%, 9-hydroxy-21-azidoryanodine to one of 56.3 +/- 0.4%, 10-pyrroleryanodol to one of 52.2 +/- 1.0%, 3-epiryanodine to one of 42.9 +/- 0.7%, CBZ glycyl ryanodine to one of 29.4 +/- 1.0%, 21-p-nitrobenzoyl-amino-9-hydroxyryanodine to one of 26.1 +/- 0.5%, beta-alanyl ryanodine to one of 14.3 +/- 0.5%, and guanidino-propionyl ryanodine to one of 5.8 +/- 0.1% (chord conductance at +60 mV, +/- SEM). For the majority of the derivatives the effect is irreversible within the lifetime of a single-channel experiment (up to 1 h). However, for four of the derivatives, typified by ryanodol, the effect is reversible, with dwell times in the substate lasting tens of seconds to minutes. The effect caused by ryanodol is dependent on transmembrane voltage, with modification more likely to occur and lasting longer at +60 than at -60 mV holding potential. The addition of concentrations of ryanodol insufficient to cause modification does not lead to an increase in single-channel open probability, such as has been reported for ryanodine. At concentrations of > or = 500 mu M, ryanodine after initial rapid modification of the channel leads to irreversible closure, generally within a minute. In contrast, comparable concentrations of beta-alanyl ryanodine do not cause such a phenomenon after modification, even after prolonged periods of recording (>5 min). The implications of these results for the site(s) of interaction with the channel protein and mechanism of the action of ryanodine are discussed. Changes in the structure of ryanodine can lead to specific changes in the electrophysiological consequences of the interaction of the alkaloid with the sheep cardiac SR Ca2+ release channel.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.     

Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)- conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.     

Block of the sheep cardiac sarcoplasmic reticulum Ca2+-release channel by tetra-alkyl ammonium cations

Summary The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (C _n H_2n+1)₄N⁺ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1–3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA ⁺) and 90% for tetraethyl ammonium (TEA⁺) and tetrapropyl ammonium (TPrA⁺) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA⁺ increases the voltage dependence of block to a level similar to that of TEA ⁺ and TPrA⁺. The zero-voltage dissociation constant (K _b(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a K _m of 9.8±1.7 mm (mean±sd) at 40 mV. Larger TAA cations (n = 4–5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.This work was supported by funds from the Medical Research Council and the British Heart Foundation. We would like to thank Richard Montgomery for his considerable help with the chemical synthesis. We are grateful to Drs. John Chambers, Nick Price and staff for showing us the intricacies of NMR spectroscopy.     

Role of the proposed pore-forming segment of the Ca2+ release channel (ryanodine receptor) in ryanodine interaction

In earlier studies we showed that point mutations introduced into the proposed pore-forming segment, GVRAGGGIGD (amino acids 4820-4829), of the mouse cardiac ryanodine receptor reduced or abolished high affinity [3H]ryanodine binding. Here we investigate the effects of these mutations on the affinity and dissociation properties of [3H]ryanodine binding and on ryanodine modification of the ryanodine receptor channel at the single channel and whole cell levels. Scatchard analysis and dissociation studies reveal that mutation G4824A decreases the equilibrium dissociation constant (K(d)) and the dissociation rate constant (k(off)), whereas mutations G4828A and D4829A increase the K(d) and k(off) values. The effect of ryanodine on single G4828A and D4829A mutant channels is reversible on the time scale of single channel experiments, in contrast to the irreversible effect of ryanodine on single wild-type channels. Ryanodine alone is able to induce a large and sustained Ca2+ release in HEK293 cells transfected with the R4822A or G4825A mutant cDNA at the resting cytoplasmic Ca2+ but causes little or no Ca2+ release in cells transfected with the wild-type cDNA. Mutation G4826C diminishes the functional effect of ryanodine on Ca2+ release but spares caffeine-induced Ca2+ release in HEK293 cells. Co-expression of the wild-type and G4826C mutant proteins produces single channels that interact with ryanodine reversibly and display altered conductance and ryanodine response. These results are consistent with the view that the proposed pore-forming segment is a critical determinant of ryanodine interaction. A putative model of ryanodine-ryanodine receptor interaction is proposed.     

Rectification of skeletal muscle ryanodine receptor mediated by FK506 binding protein.

The cytosolic receptor for immunosuppressant drugs, FK506 binding protein (FKBP12), maintains a tight association with ryanodine receptors of sarcoplasmic reticulum (SR) membrane in skeletal muscle. The interaction between FKBP12 and ryanodine receptors resulted in distinct rectification of the Ca release channel. The endogenous FKBP-bound Ca release channel conducted current unidirectionally from SR lumen to myoplasm; in the opposite direction, the channel deactivated with fast kinetics. The binding of FKBP12 is likely to alter subunit interactions within the ryanodine receptor complex, as revealed by changes in conductance states of the channel. Both on- and off-rates of FKBP12 binding to the ryanodine receptor showed clear dependence on the membrane potential, suggesting that the binding sites of FKBP12 reside in or near the conduction pore of the Ca release channel. Rectification of the Ca release channel would prevent counter-current flow during the rapid release of Ca from SR membrane, and thus may serve as a negative feedback mechanism that participates in the process of muscle excitation-contraction coupling.     

Methanethiosulfonate ethylammonium block of amine currents through the ryanodine receptor reveals single pore architecture

The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca2+) release channel (ryanodine receptor (RyR)) suggests that the four RyR subunits either combine to form a single pore or that each RyR subunit is an independently conducting pathway. Previously we showed that methanethiosulfonate ethylammonium (MTSEA+) covalently modifies the RyR to reduce current amplitudes in a time-dependent and stepwise manner. To ascertain the number of functionally conducting pores in the RyR, two approaches were combined: modification of the receptor by MTSEA+ and the use of different sized current carriers. Previous reports (Tinker, A., and Williams, A. J. (1993) J. Gen. Physiol. 102, 1107-1129) have shown that the organic cations methylamine, dimethylamine, ethylamine, and trimethylamine are permeant through the RyR but with reduced current amplitude depending upon the diameter of the respective amine. Experiments using the thiol reagent MTSEA+ to modify the channel protein showed that the current amplitudes decrease in steps leading to complete block of the channel when cesium (Cs+) is the current carrier. MTSEA+ modification decreased the number of channel substates as the diameter of the current carrier increased. Comparison of the degree of inhibition of MTSEA+-modified currents allows for differentiation between the two models for channel architecture. These results demonstrate that the conduction pathway for the RyR is comprised of a single central pore.     

Interdependence of ryanodine binding, oligomeric receptor interactions, and Ca2+ release regulation in junctional sarcoplasmic reticulum

We have examined ryanodine binding to its receptor (RR) and compared its effect on Ca2+ release to the Ca2+ release triggered by Ca2+ plus ATP, using vesicular fragments of junctional terminal cisternae (JTC) obtained from skeletal muscle. Ryanodine binding is slow (taking hours or days to complete) and is highly temperature (Q10 = 4) and Ca2+ dependent. At equilibrium, the extent of binding increases as the concentration of ryanodine is raised above 10(-9) M, exhibiting negative cooperativity and reaching the stoichiometry of the 560,000-Da RR chains near 10(-5) M ryanodine. The specificity of the high affinity binding is demonstrated by competitive binding of ryanodine analogs. Kinetic studies using rapid filtration show that, in the absence of ryanodine, rapid (k = 15 s-1) release of Ca2+ follows a triggering exposure of loaded JTC vesicles to perfusion media containing Ca2+ plus ATP. Induction of this release has no lag period and displays minimal temperature dependence. In contrast, prolonged exposure of JTC vesicles to low (10(-7) M) ryanodine concentrations changes the JTC to a state permitting slow (k = 1 s-1) release of Ca2+ even in the absence of the Ca2+ plus ATP trigger. Higher (greater than microM) concentrations of ryanodine do not allow any Ca2+ release and prevent even the release normally triggered by Ca2+ plus ATP. Our data suggest that ryanodine binds to the open state of the tetrameric RR, inducing protein conformational changes and altered oligomeric interactions. Binding of the first molecule of ryanodine to one of the four binding sites on the receptor produces a partially closed and low conductance state of the Ca2+ release channel and reduces the ryanodine binding affinity of the remaining sites. Ryanodine occupancy of all four binding sites on the receptor completes closure of the Ca2+ channel and blocks the triggering action of Ca2+ plus ATP. The tetrameric association of the RR chains is demonstrated by crosslinking with bifunctional reagents, generating crosslinked tetramers that retain ryanodine binding and Ca2+ release functions.     

Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that of ruthenium red. These compounds inhibited the open channel with lower affinities (Kd = 170 microM, 4APd; Kd = 656 microM, 4APt), and had Hill coefficients of close to 1. The results suggest that ruthenium red block of the ryanodine receptor is due to binding to multiple sites located in the conduction pore of the channel.     

Characteristics of cocaine block of purified cardiac sarcoplasmic reticulum calcium release channels.

We have examined the effects of cocaine on the SR Ca2+ release channel purified from canine cardiac muscle. Cocaine induced a flicker block of the channel from the cytoplasmic side, which resulted in an apparent reduction in the single-channel current amplitude without a marked reduction in the single-channel open probability. This block was evident only at positive holding potentials. Analysis of the block revealed that cocaine binds to a single site with an effective valence of 0.93 and an apparent dissociation constant at 0 mV (Kd(0)) of 38 mM. The kinetics of cocaine block were analyzed by amplitude distribution analysis and showed that the voltage and concentration dependence lay exclusively in the blocking reaction, whereas the unblocking reaction was independent of both voltage and concentration. Modification of the channel by ryanodine dramatically attenuated the voltage and concentration dependence of the on rates of cocaine block while diminishing the off rates to a lesser extent. In addition, ryanodine modification changed the effective valence of cocaine block to 0.52 and the Kd(0) to 110 mM, suggesting that modification of the channel results in an alteration in the binding site and its affinity for cocaine. These results suggest that cocaine block of the SR Ca2+ release channel is due to the binding at a single site within the channel pore and that modification of the channel by ryanodine leads to profound changes in the kinetics of cocaine block.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex

Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle.     

Evidence for a Ca2+ channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum

The solubilized [3H]ryanodine receptor from cardiac sarcoplasmic reticulum was centrifuged through linear sucrose gradients. A single peak of radioactivity with apparent sedimentation coefficient of approximately 30S specifically comigrated with a high molecular weight protein of apparent relative molecular mass approximately 400,000. Incorporation of the ryanodine receptor into lipid bilayers induced single Ca2+ channel currents with conductance and kinetic behavior almost identical to that of native cardiac Ca2+ release channels. These results suggest that the cardiac ryanodine receptor comprises the Ca2+ release channel involved in excitation-contraction coupling in cardiac muscle.     

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).     

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.     

Probing the structure of the conduction pathway of the sheep cardiac sarcoplasmic reticulum calcium-release channel with permeant and impermeant organic cations

The sarcoplasmic reticulum Ca(2+)-release channel plays a central role in cardiac muscle function by providing a ligand-regulated pathway for the release of sequestered Ca2+ to initiate contraction following cell excitation. The efficiency of the channel as a Ca(2+)-release pathway will be influenced by both gating and conductance properties of the system. In the past we have investigated conduction and discrimination of inorganic mono- and divalent cations with the aim of describing the mechanisms governing ion handling in the channel (Tinker, A., A.R. G. Lindsay, and A.J. Williams. 1992. Journal of General Physiology. 100:495-517.). In the present study, we have used permeant and impermeant organic cations to provide additional information on structural features of the conduction pathway. The use of permeant organic cations in biological channels to explore structural motifs underlying selectivity has been an important tool for the electrophysiologist. We have examined the conduction properties of a series of monovalent organic cations of varying size in the purified sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel. Relative permeability, determined from the reversal potential measured under bi- ionic conditions with 210-mM test cation at the cytoplasmic face of the channel and 210 mM K+ at the luminal, was related inversely to the minimum circular cation radius. The reversal potential was concentration-independent. The excluded area hypothesis, with and without a term for solute-wall friction, described the data well and gave a lower estimate for minimum pore radius of 3.3-3.5 A. Blocking studies with the impermeant charged derivative of triethylamine reveal that this narrowing occurs over the first 10-20% of the voltage drop when crossing from the lumen of the SR to the cytoplasm. Single-channel conductances were measured in symmetrical 210 mM salt. Factors other than relative permeability determine conductance as ions with similar relative permeability can have widely varying single-channel conductance. Permeant ions, such as the charged derivatives of trimethylamine and diethylmethylamine, can also inhibit K+ current. The reduction in relative conductance with increasing concentrations of these two ions at a holding potential of 60 mV was described by a rectangular hyperbola and revealed higher affinity binding for diethylmethylamine as compared to trimethylamine. It was possible to describe the complex permeation properties of these two ions using a single-ion four barrier, three binding site Eyring rate theory model. In conclusion, these studies reveal that the cardiac Ca(2+)-release channel has a selectivity filter of approximately 3.5-A radius located at the luminal face of the protein.(ABSTRACT TRUNCATED AT 400 WORDS)