Introduction

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The sarcoplasmic reticulum (SR) Ca²⁺ release channels/ryanodine receptors (RyR) play a vital role in the initiation of cellular contraction (for a review, see Meissner, 1994). In cardiac tissues, efflux of calcium from the SR is triggered by an influx of extracellular Ca²⁺ via the L-type Ca²⁺ channels through a process referred to as calcium-induced calcium release (Fabiato, 1983). Incorporation of purified RyR (Lai et al., 1988) into planar lipid bilayers has allowed for the study of the biophysical and pharmacological properties of the RyR. Agents that have been shown to have a direct influence on the RyR activity also affect release of Ca²⁺ from the SR and excitation-contraction coupling (for a review, see Zucchi and Ronca-Testoni, 1997).

The large molecular mass of the RyR (~2000 kDa) has allowed for the visualization of the channel protein (Lai et al., 1988). These studies have revealed that the channel is a 4-fold symmetrical protein, measuring 27 × 27 × 14 nm with a 2-nm diameter pore. Detailed structural information on the channel pore has also been provided using permeant and impermeant organic cations and bis-quaternary ammonium blocking cations (Tinker and Williams, 1993a). These authors suggested the channel pore selectivity filter has a radius of ~3.5Å located 90% into the voltage field from the cytoplasmic face with the entire electrical potential of the channel spanning 10.4Å. Block of RyR by large quaternary ammonium derivatives indicates that the pore may be the site for a number of positively charged agents to affect excitation-contraction coupling. We and other groups have recently demonstrated that local anesthetics can directly block single-channel activity of the RyR (Tinker and Williams, 1993b; Tsushima et al., 1996). Such agents have been shown to inhibit RyR and to possess negative inotropic activity (Bianchi and Bolton, 1967; Chamberlain et al., 1984).

Quinidine, an antiarrhythmic agent, is known to reduce contraction and alter cellular excitability in cardiac tissues (Parmley and Braunwald, 1967). It has been demonstrated that this agent interacts with multiple sarcolemmal voltage-gated ion channels (Lee et al., 1981; Hiraoka et al., 1986; Salata and Wasserstrom, 1987). The interaction of quinidine and other local anesthetic/antiarrhythmic agents with voltage-gated ion channels has been proposed to be the result of binding of these compounds within the inner vestibule of the channel pore (Hille, 1977). Mutagenesis studies have demonstrated critical intracellular residues important for quinidine binding to voltage-gated K⁺ and Na⁺ channels (Snyders et al., 1992; Ragsdale et al., 1995). This would imply that the cytoplasmic milieu is readily accessible to quinidine. The negative inotropic effects of quinidine have been, in part, associated with the inhibition of sarcolemmal L-type Ca²⁺ channels (Hiraoka et al., 1986; Salata and Wasserstrom, 1987); however, it is possible that blockade of RyR may also be involved in the depression of muscle contractility. Earlier studies have demonstrated [³H]quinidine binding to cardiac SR membranes (Besch and Watanabe, 1977) and alterations in cardiac SR Ca²⁺ handling by quinidine (Fuchs et al., 1968; Besch and Watanabe, 1977). Therefore, we examined the effects of quinidine on single-channel activity of RyR. The present study demonstrates that quinidine alters the conductance and activity of these channels in a voltage- and concentration-dependent manner. A more prominent effect is the appearance of a subconductance state. Further analysis revealed that ryanodine modification of the channel has a profound influence on the blocking properties of quinidine.

	Experimental Procedures

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Materials. Quinidine and quinine were obtained from Sigma (St. Louis, MO). Quinidinium (N-methyl quinidine) was a gift from Dr. D. J. Synders (Vanderbilt University, Nashville, TN). Ryanodine was purchased from Calbiochem (San Diego, CA) and phospholipids were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL). All other chemicals were analytical grade and were purchased from Sigma.

Isolation of Junctional SR Membrane Vesicles. Canine cardiac junctional SR membrane vesicles were isolated as described previously (Tsushima et al., 1996). In brief, mongrel dogs were cared for according the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health. Animals were anesthetized with sodium pentobarbital (65 mg/kg; iv), and ventricular tissue was excised and homogenized in 300 mM sucrose, 20 mM PIPES, and 0.5 mM EDTA (pH 7.4), in the presence of the protease inhibitors 100 nM aprotinin, 1 mM benzamidine, 1 mM iodoacetamide, 1 µM leupeptin, 1 µM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. These protease inhibitors were present in all solutions during the isolation of junctional SR membranes and purification of the RyR. The homogenate was centrifuged at 2800g_max for 20 min, and the subsequent supernatant was centrifuged at 120,000g_max to yield a mixed membrane preparation. This membrane preparation was fractionated on a 20 to 40% discontinuous sucrose-density gradient. Junctional SR membranes were recovered at the 30 to 40% interface, diluted with 1.5 volumes of 400 mM KCl, and pelleted at 120,000g_max. The pellet was resuspended in 300 mM sucrose and 5 mM HEPES/Tris solution (pH 7.2). Membranes vesicles were snap frozen and stored in liquid nitrogen.

Purification and Reconstitution of RyR. RyR was purified and reconstituted into unilamellar liposomes as previously described (Tsushima et al., 1996). Junctional SR membranes were solubilized with the zwitterionic detergent Chaps followed by sucrose-density centrifugation. Chaps detergent was removed by dialysis. Purified channels were stabilized in proteoliposomes with phosphatidylcholine and stored in liquid nitrogen.

Planar Lipid Bilayer Measurements. Single-channel activity was recorded from purified RyR incorporated into planar lipid bilayers containing phosphatidylethanolamine and phosphatidylserine (1:1; 40 mg/ml phospholipid in decane). Lipid bilayers were formed across a 200-µm hole in a Delrin partition that separates the cis and trans chambers of the bilayer apparatus. Single-channel activity was recorded under a symmetrical KCl solution (250 mM KCl, 0.15 mM CaCl₂, 0.1 mM EGTA, and 10 mM HEPES/Tris, pH 7.4; free [Ca²⁺] = 50 µM). Channels were incorporated such that the cytoplasmic surface of the channel faced the cis chamber. Potentials given are those experienced at the cis (cytoplasmic) side relative to the trans (luminal) side, such that current flowing at positive holding potentials corresponds to current flow from cis to trans. Single-channel activity was measured using an Axopatch 200 amplifier and was stored directly into a 386 or 486 PC using pClamp software (Axon Instruments, Inc., Foster City, CA). Data were digitized at 10 kHz and filtered at 2 to 5 kHz. Quinidine was present in both the cis and trans chambers to prevent any asymmetrical surface potentials that may arise (Tinker et al., 1992).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Previous work from our laboratory has demonstrated that purified RyR displays a similar sensitivity to the physiologic (Ca²⁺, Mg²⁺, and ATP) and pharmacologic modulators (caffeine, ruthenium red, and ryanodine) as the native channel (Tsushima et al., 1996). The purified channel undergoes the characteristic 40% reduction in single-channel current amplitude with a prolongation in the open probability in the presence of ryanodine. We have demonstrated the pharmacological modulation of RyR channel activity by cocaine and dihydropyridine agonists (Tsushima et al., 1996; Sagawa et al., 2001). In the present study, we have further characterized the properties of RyR by examining the effects of quinidine and its quaternary derivative, quinidinium, on single-channel conductance and gating.

Effects of Quinidine on RyR. The effects of quinidine on the purified RyR are illustrated in Figs. 1 and 2. Quinidine reduced the open probability of the channel in a concentration-dependent manner but only at positive holding potentials (Figs. 1 and 2). The reduction in channel activity was induced only when quinidine was present on the cis (cytoplasmic) side of the channel. When quinidine was present on the trans (luminal) side, no block was observed (data not shown), suggesting the absence of a binding site on the luminal side of the channel. A more pronounced effect was the appearance of a subconductance state (substate). This substate was not the result of a second smaller conducting channel in the bilayer because we never observed this type of channel activity in the absence of quinidine. Previous studies examining the properties of the purified RyR have reported a common occurrence of subconductance activity (Liu et al., 1989). However, we observed little or no incidence of subconductance activity when the channel was solubilized in less Chaps detergent (0.5 versus 1.5%) for a shorter period of time (1 versus 2 h) compared with the studies mentioned above. Any channel displaying such activity under control recording conditions was discarded. Quinidine-induced subconductance activity was enhanced at more positive holding potentials. At negative potentials, we observed neither a change in channel activity nor the appearance of a substate (Figs. 1 and 2). Under symmetrical recording conditions (250 mM KCl), the channel displayed a linear current-voltage relationship with a slope conductance of 721 ± 4 pS (n = 8) (Fig. 3). The quinidine-induced subconductance state had a slope conductance of 204 ± 9 pS (n = 8) (28% of control) at positive holding potentials, whereas the open-state conductance was not affected by quinidine (719 ± 5 pS, n = 8) (Fig. 3). This unidirectional block is similar to our previous observations with cocaine block of this channel (Tsushima et al., 1996).

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Effect of quinidine on purified cardiac RyR. Single-channel activity was recorded under symmetrical 250 mM KCl (free Ca2+ = 50 µM) at holding potential of +60 (A) or 60 mV (B) in the absence or presence of 100, 250, and 500 µM quinidine. The drug was present in the cis and trans chambers. The dashed line represents the closed current level. The marked appearance of the subconductance state is denoted by the arrow. Traces were filtered at 2 kHz.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Voltage dependence of quinidine-induced subconductance activity in purified cardiac RyR. Single-channel activity was recorded as described in Fig. 1. Substate activity elicited by 100 µM quinidine was observed at holding potentials was +40, +60, and +80 mV but not 60 mV. Traces were filtered at 2 kHz.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Current-voltage relationship of the RyR in the absence () and presence (, ) of 100 µM quinidine. The single-channel conductance was 721 ± 4 pS (n = 8) in control. The quinidine-induced substate displayed a conductance of 204 ± 9 pS (n = 8) (). Quinidine did not influence the open conductance state (). The data were fit by linear regression analysis. Each point represents mean ± S.E.M.

Concentration Dependence of Quinidine Block. The quinidine-induced subconductance activity was more apparent with increasing concentrations of quinidine (Fig. 1). The amplitude of the substate did not change with the progressive increase in quinidine levels. Initial inspection of the concentration dependence of quinidine block revealed that drug block did not seem to behave in a simple bimolecular process where occupation of the substate level would be enhanced with more quinidine as described by Scheme 1. 
where k_on and k_off are the on- and off-rate constants for quinidine binding between the open and substate, respectively. At 500 µM quinidine, the open probability of the channel is greatly reduced, whereas channel state alternates only between the subconductance and closed (blocked) state. With increasing concentration of agent, we did not observe an increase incidence of substate events as would be predicted from Scheme 1. Therefore, it seems that quinidine not only elicits a channel substate but also induces channel block as described in Scheme 2. 
where k_on' and k_off' are the on- and off-rate constants of quinidine binding to the blocked state from the substate, respectively. It is difficult to quantify the rate constants of the complex gating scheme above especially for the transitions from the substate to either open or block states using the conventional pClamp software. This prompted us to study the effects of quinidine by examining the probability of each of the different channel states (i.e., open, substate, and block) as a function of quinidine concentration and voltage. Channel-state probabilities were determined from amplitude histograms. Increasing concentrations of quinidine at the cytoplasmic face of the channel resulted in a progressive decline in the open probability (Fig. 4A). Quinidine levels up to 150 µM increased both the substate (0.305 ± 0.005; n = 6) and block probabilities (0.543 ± 0.066). However, higher concentrations resulted in a decrease in substate probability to 0.153 ± 0.015 at 500 µM quinidine, whereas the block-state probability continued to increase (0.705 ± 0.109). This supports the notion that quinidine not only induces the appearance of a subconductance state but, with higher concentrations, an additional quinidine molecule shifts channel transition to the blocked state (Scheme 2).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of channel-state probabilities in the presence of quinidine and quinidinium. The open (), substate (), and block () states are plotted as a function of quinidine (A) or quinidinium (B) concentration. The holding potential was +60 mV. Data represent the mean ± S.E.M. of six (quinidine) or four (quinidinium) experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Dose response of RyR block by quinidine and quinidinium. The probability of the unblocked channels at +60 mV is plotted as a function of quinidine () or quinidinium () concentration. The symbols represent the mean ± S.E.M. of six (quinidine) or four (quinidinium) experiments.

Voltage Dependence of Quinidine Block. We also examined the voltage dependence of quinidine block (Fig. 6). In the presence of 100 µM quinidine, increasing holding potential resulted in the steady decline in the open probability. In contrast, the probability of block was fairly constant from +40 to +55 mV but increased at potentials +60 mV and higher. More interestingly, substate probabilities demonstrated a biphasic response to quinidine showing a steady increase from 0.149 ± 0.031 (n = 8) at +40 mV to 0.293 ± 0.042 at +60 mV, but then declining to 0.185 ± 0.046 at +80 mV. The results suggest that at holding potentials between +40 and +55 mV, the decrease in the open probability is a consequence of quinidine-induced substate activity. With further depolarization, channel activity shifts from substate to block of the channel.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Voltage-dependent block of RyR by quinidine and quinidinium. Channel open-state (), substate (), and block-state () probabilities are plotted as a function of the holding potential in the presence of 100 µM quinidine (A) or 100 µM quinidinium (B). Open channel probability in the absence of drug (control, ) is shown to demonstrate that lack of voltage dependence on channel gating. The symbols represent the mean ± S.E.M. of eight (quinidine) or four (quinidinium) experiments and three to eight for control.

Quinidinium Block of RyR. Under our experimental conditions, >90% of quinidine is in the protonated state; however, we cannot be certain from the above results whether the charged or uncharged form of the drug is responsible for the blocking behavior. To investigate this further, we examined the effects of the quaternary quinidine derivative, quinidinium (N-methyl quinidine), on RyR activity. The effects of quinidinium on channel activity are illustrated in Fig. 7. When present only on the trans face of the channel, 100 µM quinidinium did not elicit any subconductance activity or block at positive and negative holding potentials. Subsequent addition of quinidinium to the cytoplasmic side elicited substate gating at positive potentials (Fig. 7).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of quinidinium on single-channel RyR activity. Channel activity was recorded at a holding potential of +50 and 50 mV in the presence of 100 µM quinidinium. There is a lack of channel block when quinidinium was present in the trans chamber only. Subsequent addition of quinidinium to the cis chamber resulted in channel block but only at positive holding potentials. Traces were filtered at 2 kHz.

Effects of Quinine on RyR. We examined the stereospecificity of quinidine block using the levoisomer of quinidine, quinine. Quinine (100 µM) reduced the single-channel open probability of the channel with concomitant production of a subconductance state (Fig. 8). As observed with quinidine, block of the channel was not observed at negative holding potentials or when the drug was present on the luminal side of the channel. As a result of the similarity in the phenotypic changes to channel gating at similar drug concentration, we did not study further the kinetics of quinine block. Therefore, these data suggest that there is a lack of stereospecificity of quinidine block of RyR.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Lack of stereospecificity of quinidine block of RyR activity. Single-channel activity in the presence of 100 µM quinine in the cis and trans chambers. The holding potentials were +60 and 60 mV. The dashed lines represent the closed current levels. Quinine elicited a similar substate block of RyR as compared with quinidine.

Ryanodine Modification on Quinidine Block. Ryanodine induces an allosteric modification of RyR leading to changes in single-channel gating and conductance, and increases the sensitivity of the channel to Ca²⁺ (Du et al., 2001; Masumiya et al., 2001) due to marked structural changes in the configuration of the channel protein (Lindsay et al., 1994; Tu et al., 1994). Studies have determined the structural components of the ryanodine molecule responsible for altering channel gating and conductance (Tinker et al., 1996; Welch et al., 1997). In the presence of 1 µM ryanodine, there is a pronounced prolongation in the mean open time and a ~40% reduction in single-channel amplitude, which differs from the quinidine-induced substate (Tsushima et al., 1996). Such changes to the channel by both ryanodine and quinidine may alter the interaction of these agents with the channel when present together. Therefore, we examined the interaction of quinidine on RyR modified with 1 µM ryanodine. In ryanodine-modified channels, quinidine reduced the open channel probability but did not induce subconductance activity (Fig. 9). As with unmodified channels, quinidine had no effect on ryanodine-modified channels at negative holding potentials (data not shown). Kinetic analysis of the on- and off-rates of quinidine binding to the ryanodine-modified channel could be measured because open and closed lifetime histograms were best described by monoexponential functions. The kinetic values for quinidine on- and off-rates, k_on and k_off, respectively, were derived from the time constants of the dwell-time histograms by the following equations.

(1)

(2)

(3)

We interpret k_off to be the reciprocal of the exponential time constant for the blocked dwell time (), k_on to be best described by the reciprocal of the open-state time constant (_O) in the presence of quinidine, and K_d to be the dissociation constant for quinidine binding. The values of k_on and k_off were used to examine the voltage dependence of quinidine binding in ryanodine-modified channels (Fig. 10A). Both rate constants were influenced by the voltage and could be described by a Boltzmann distribution

(4)

(5)

where V is the holding potential, k_on(0) and k_off(0) are the association and dissociation rate constants at 0 mV, respectively, z'_on and z'_off are the equivalent valences, and R, T, and F have their usual thermodynamic meaning. Fit of the voltage dependence of the k_on and k_off data with Boltzmann functions (eqs. 4 and 5) resulted in k_on and k_off values of 0.39 ± 0.04 mM¹ ms¹ and 0.21 ± 0.02 ms¹, respectively, and a K_d at 0 mV of 0.55 mM. The effective valences were 0.82 ± 0.04 (z'_on) and 0.31 ± 0.04 (z'_off) (total effective valence 1.21 ± 0.09) suggesting that even with modification of the channel by ryanodine, more than one quinidine molecule can interact within the channel pore (Hille and Schwarz, 1978). Figure 10B shows that the on-rate of quinidine binding increases linearly as a function of the concentration (slope 1.88 ± 0.12 mM¹ ms¹), whereas the off-rate is unaffected by the levels of quinidine (0.10 ± 0.01 ms¹). Thus, quinidine block of ryanodine-modified channels was both voltage and concentration dependent.

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Concentration dependence of quinidine block on single-channel activity of ryanodine-modified RyR. Channels were modified with 1 µM ryanodine. Single-channel records in the absence and presence of 50 and 250 µM quinidine. The holding potential was +60 mV. The closed state (C) levels are indicated by the dashed lines. Traces were filtered at 2 kHz.

View larger version (15K): [in this window] [in a new window]   Fig. 10.   A, the voltage dependence of the association (kon ) and dissociation (koff ) rate constants for 100 µM quinidine block of ryanodine-modified channels. RyR were modified with 1 µM ryanodine. The data were fit with Boltzmann functions as described under Results. B, concentration dependence of the blocking (1/O, ) and unblocking (1/B, ) rates for quinidine block of ryanodine-modified channels. Each point represents the mean ± S.E.M. of four experiments.

	Discussion

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Quinidine block of RyR elicits a distinct subconductance state that seems to be precluded in channels previously modified by ryanodine. Further application of quinidine results in complete channel block due to an additional quinidine molecule interacting with the channel. Narrowing of the channel vestibule on the cytoplasmic face (Lindsay et al., 1994) or alterations in the cationic binding site in the pore due to conformational changes in RyR by ryanodine (Tanna et al., 2001) may explain the lack of substate activity in the presence of quinidine. This blockade differs from that we have observed previously with cocaine where a single blocking molecule binds within the pore to completely occlude ion flow (Tsushima et al., 1996).

Interpretations of the Quinidine-Induced Substate. Many ion channels have inherent substate activity (for review, see Fox, 1987). Recording of purified RyR has demonstrated a frequent appearance of subconductance states, resulting from the presence of multiple conductance pathways (Liu et al., 1989). It is also possible that solubilization of the channel removes a regulatory protein involved in coordinating the gating of the homotetrameric protein. The FK506-binding protein, a cis-trans peptidy-prolyl isomerase has been recently shown to be associated with the RyR (Jayaraman et al., 1992) and, furthermore, to regulate the gating of expressed RyR (Brillantes et al., 1994). Interactions of this protein by FK506 or mice deficient in the FK506-binding protein result in the induction of subconductance activity of RyR (Ahern et al., 1994; Brillantes et al., 1994; Shou et al., 1998). With our purification conditions, we have a low frequency of substate activity, suggesting that the harsher solubilization technique used by the previous studies may account for the higher probability of substate activity (Lai et al., 1988). Our findings demonstrating quinidine-induced subconductance activity cannot be explained by an alteration in channel gating, wherein each individual subunit operates independently of one another or as a result of the loss of the FK506-binding protein. In such an instance, up to three subconductance states should be detected, each being a fraction of the open state. Only one quinidine-induced substate was observed having a conductance of 28% of the open state.

Implications of Changes in Quinidine Block by Ryanodine Modification. Analysis of the ryanodine-modified RyR revealed that the channel undergoes a number of structural transformations (Lindsay et al., 1994; Tu et al., 1994). Alterations included a widening of the selectivity filter located deeper within the channel, a decreased density of the negative charges lining the pore and a narrowing of the outer vestibular region on the cytoplasmic face of the channel. Profound alterations such as these make it reasonable to speculate that channel block could be affected to some degree. Quinidine block was markedly altered after modification of the channel by ryanodine. Ryanodine modification of RyR resulted in the absence of a subconductance state in the presence of quinidine, while still allowing for up to two quinidine molecules to interact with the channel as observed in ryanodine-unmodified channels. The reduction in drug sensitivity after ryanodine modification (550 versus 75 µM) is similar to our previous observations in which the affinity for cocaine binding was reduced in ryanodine-modified channels (Tsushima et al., 1996). These effects could be the result of ryanodine-induced structural changes in RyR eliciting a change in the affinity of quinidine binding or could be due to the presence of only the lower affinity site remaining while the high-affinity site is occupied by ryanodine or not accessible. Similar findings with tetraalkylammonium and QX314 block of the ryanodine-modified RyR were observed, which were suggested to be the result of structural reorganizations in the conduction pathway with a relocation of the cationic drug site (Tinker and Williams, 1993c; Tanna et al., 2001).