HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 16, 14237-14248, April 18, 2003

This Article Abstract Full Text (PDF) All Versions of this Article: 278/16/14237    most recent M208372200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bidasee, K. R. Articles by Besch, H. R. Search for Related Content PubMed PubMed Citation Articles by Bidasee, K. R. Articles by Besch, H. R., Jr. Social Bookmarking                      What's this?

Diketopyridylryanodine Has Three Concentration-dependent Effects on the Cardiac Calcium-release Channel/Ryanodine Receptor^*

Keshore R. Bidasee^§¶, Le Xu^§, Gerhard Meissner, and Henry R. Besch Jr.^**

From the  Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198, the  Departments of Biochemistry and Biophysics and Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599-7260, and the ^** Departments of Pharmacology and Medicine and Krannert Institute of Cardiology, Indiana Center for Vascular Biology and Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202

Received for publication, August 16, 2002, and in revised form, January 10, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

By interacting with more than one site, ryanoids induce multiple effects on calcium-release channels. To date, the kinetics of interaction of only one of these sites has been characterized. Using C₄,C₁₂-diketopyridylryanodine in both [³H]ryanodine binding and single channel experiments we characterized another site on the cardiac ryanodine receptor (RyR2) with which ryanoids interact. Competitive binding of this ryanoid to RyR2 implied a minimal two-site binding model. At the single channel level, C₄,C₁₂-diketopyridylryanodine induced three distinct effects. At nanomolar concentrations, it increased channel open probability severalfold without inducing a subconductance. This effect was independent of membrane holding potential. As for other ryanoids, low micromolar concentrations of C₄,C₁₂-diketopyridylryanodine readily induced a subconductance state. The major subconductance had a current amplitude of 52% of fully open, it was reversible, and its time to induction and duration were voltage- and concentration-dependent, affording Hill slopes of >2. At higher micromolar concentrations C₄,C₁₂-diketopyridylryanodine induced long lasting, yet reversible shut states. Using a pharmacological strategy we have discerned an additional ryanoid-binding site on RyR2 that triggers an increase in channel activity. This site likely resides outside the strict confines of the transmembrane conducting pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ryanodine receptor calcium release channels (RyRs)¹ are present in almost all mammalian cells. They play an integral role in releasing calcium ions from the internal sarco(endo)plasmic reticulum to mediate distinct cascades of events that culminate in such vital functions as muscle contraction, neurotransmitter release, hormone secretion, and lymphocyte activation (1-7). Three distinct isoforms from mammalian tissues have been cloned from three genes and are designated RyR1 (predominant in skeletal muscle), RyR2 (predominant in cardiac muscle), and RyR3 (first isolated from brain but present in several tissues).

The plant alkaloid ryanodine is the most recognized xenomodulator of RyRs. In calcium efflux assays using junctional sarcoplasmic reticular membrane vesicles from fast skeletal muscle, low micromolar concentrations of ryanodine activate (i.e. open) RyR1, whereas higher concentrations deactivate or close them (8-10). At the single channel level two effects of ryanodine are typically observed, namely induction of a persistent subconductance state and long-lasting channel closure (11-13). The latter is termed the shut state to distinguish it from brief closures that occur spontaneously (14). These two effects are reminiscent of channel activation and deactivation seen at the aggregate channel level in membrane vesicles.

In a recent study using frog twitch fibers we observed that prior to induction of the subconductance state (seen as a steady glow in this preparation) and channel shutting (recorded as inhibition of depolarization-evoked calcium release), ryanodine increased the frequency of spontaneous sparks (15). This increase in sparking frequency, similar to that reported by Gonzales et al. (16), suggests that in addition to the two well documented effects, ryanodine may also induce an earlier effect, namely an increase in channel gating frequency. Two prior studies suggested that nanomolar concentrations of ryanodine may increase the probability of RyR opening (P_o) without inducing the subconductance state (17, 18). Because none of these earlier studies recorded dose dependence, ryanodine per se appears unable to discriminate between functional sites that induce increases in channel gating and those producing the subconductance state. Identifying ryanoids that can discriminate between sites having different functional effects is key to elucidating the phenomena behind ryanoid-induced changes in RyR properties.

In a previous study we found that pyridylryanodine (C₃-O-[pyridylcarbonyl]ryanodol) has activating potency and efficacy similar to those of ryanodine, despite its lower affinity (14). These data suggested that pyridylryanodine or a derivative thereof might be able to discriminate between putative subpopulations of binding sites on RyR. It is also likely that a ryanoid belonging to this group might bind reversibly to RyR2, because like three other reversible ryanoids reported in the literature, it too has a substituent on the A-ring (C₃ carbon) that differs from that of ryanodine (6, 12). We also reasoned that additional reversibility and subsite selectivity might be engineered into pyridylryanodine by relaxing a ring constraint of its skeleton backbone. In the present study we used binding and single channel experiments to show that a modified congener of pyridylryanodine, namely C₄,C₁₂-diketopyridylryanodine (C₃-O-[pyridylcarbonyl]C₄,C₁₂-seco-C₄,C₁₂-dioxoryanodol) induces three distinct and separate concentration-dependent effects on canine RyR2. For comparison, we also detail the binding and single channel properties of its parent ryanoid, pyridylryanodine.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The pyridylryanodine used in this study was isolated from chipped Ryania wood supplied by Integrated Biotechnology Corporation (Carmel, IN) and purified by chromatography to 99% (19). [³H]Ryanodine (specific activity 87 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Precoated silica gel plates (with fluorescence indicator) were obtained from Sigma. Lipids were obtained from Avanti Polar Lipids Inc. (Alabaster, AL). All other reagents, buffers and solvents used were of analytical grade.

Synthesis of C₄,C₁₂-Diketopyridylryanodine-- Synthesis of C₄,C₁₂-diketopyridylryanodine (for structure, see Fig. 1) was carried out as described (20, 21). Briefly, 25 mg (49 µmol) of pyridylryanodine was dissolved in 2 ml of methanol and 113 mg (588 µmol) of periodic acid was added. The solution was stirred for 2 h at room temperature, at the end of which 1 ml of water was added. The reaction mixture was then extracted with 3 × 25 ml of methylene chloride. The pooled methylene chloride fractions were dried over anhydrous sodium sulfate and rotary evaporated to dryness. The residue was redissolved in 2 ml of methylene chloride and chromatographed on a silica gel column (20 g), eluting sequentially with 100 ml each of methylene chloride, methylene chloride/2% methanol, and methylene chloride/4% methanol. The fractions containing the product of interest were pooled and rotary evaporated to dryness before being redissolved in dioxane for freeze drying. The structure of the product was confirmed using ¹H and ¹³C NMR and electrospray mass spectrometry. Purity was ascertained using thin layer chromatography (spraying with 5% ceric ammonium nitrate in 80% phosphoric acid and charring), analytical high performance liquid chromatography, and electrospray mass spectrometry.

Preparation of Sarcoplasmic Reticular Vesicles and Purification of RyR2-- Animal procedures used in this study were approved by Institutional Animal Care and Use Committees. Crude sarcoplasmic reticular membrane vesicles (a mixture of junctional and longitudinal) were prepared from canine heart as described previously (22), except that aprotonin (0.5 µg/ml), pepstatin A (0.5 µg/ml), and benzamide (80 µM) were added to isolation, homogenization, and storage buffers. These vesicles were used for [³H]ryanodine displacement binding affinity assays to determine the affinity of ryanoids for RyR2. Junctional sarcoplasmic reticular membrane vesicles were prepared in the presence of the protease inhibitors, using discontinuous sucrose gradients as described previously (23). Junctional sarcoplasmic reticular membrane vesicles were solubilized with CHAPS and 30 S RyR2 complexes were isolated by rate density gradient centrifugation and reconstituted into proteoliposomes (24).

Relative Binding Affinities of the Ryanoids-- The affinities of ryanodine and pyridylryanodine for RyR2 were determined from their ability to compete with [³H]ryanodine for binding sites on the receptor (20, 22). The affinity of C₄,C₁₂-diketopyridylryanodine for RyR2 was determined as described previously, except that 1 µM ryanodine was used to determine nonspecific binding to accommodate the lower affinity and limited aqueous solubility of the diketone. The binding displacement data were fit by nonlinear regression to one- and two-site competition models given by equations,

(Eq. 1)

(Eq. 2)

where Y is specific binding, X is the logarithm of the concentration of the unlabeled ligand, and log EC₅₀ is the competitor concentration that displaces half of the specifically bound [³H]ryanodine. Goodness of fit was evaluated by F tests and the simpler model was chosen unless the more complex model provided a better fit at p < 0.05. Equilibrium dissociation constant (K_d) values were ascertained using the Cheng-Prusoff relationship (25) given by,

(Eq. 3)

where L is the concentration of [³H]ryanodine used (6.7 nM) and K_L is the equilibrium dissociation constant of [³H]ryanodine (1.2 nM for RyR2 (22)).

Single Channel Measurements and Analyses-- Phospholipid bilayers were formed from a suspension of phosphatidylethanolamine:phosphatidylserine:phosphatidylcholine in n-decane (in a ratio of 5:3:2 in a total of 35 mg of phospholipid/ml of n-decane) across a 200-µm diameter hole. Proteoliposomes containing the purified RyR2 were fused with the bilayer. The side of the bilayer to which the proteoliposomes were added was designated the cis side. The trans side was defined as ground. Single channels were recorded in symmetric KCl buffer solution (0.25 M KCl, 20 mM K-Hepes, pH 7.4) with 2 µM calcium or as specified. In this study, free calcium concentrations were titered against EGTA. Ryanoids were made up in 100% dimethyl sulfoxide at concentrations up to 500 times higher than that anticipated for use in the bilayer bath. Accordingly, after dilution in recording buffer the dimethyl sulfoxide concentration in the bath was always less than 1%. All experiments were carried out at room temperature (23-25 °C) and for all data shown, the drugs were added only to the cis chamber. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed as described (26, 27). Data acquisition and analyses were performed using commercially available instruments and software packages (Axopatch 1D, Digidata 1200A or 1322A and pClamp 8.2, Axon Instruments, Burlingame, CA).

Normal gating mode is defined to include periods of rapid channel transitions between the closed and full open states, and subconductance mode is defined to include periods in which the channel exhibits a persistent reduced conductance. At high ryanoid concentrations the channel may transition into the shut state and this is distinguished from the brief normal forays to the closed state in the absence of ryanoid. The magnitude of the subconductance state was calculated by dividing the amplitude of the subconductance induced by the ryanoid by the conductance of the channel prior to ryanoid interaction (full conductance). Amplitudes were monitored by manual positioning of cursors at each level corresponding to closed, subconductance, and open conductance states. The probability of the subconductance state (P_s) was calculated from the dwell time in the subconductance divided by the sum of times in normal gating and subconductance modes.

Cumulative dwell time histograms in normal gating and in the subconductance mode were obtained by nonlinear regression fits to single and dual phase exponential functions given by the equations,

(Eq. 4)

(Eq. 5)

where K is the time constant and T is time. _norm and _sub are calculated as 0.69/K. Goodness of fit was evaluated by F tests and the simpler model was chosen unless the more complex model provided a better fit at p < 0.05. From the single exponential fit, K_on and K_off were obtained, according to the following defining equations,

(Eq. 6)

(Eq. 7)

(Eq. 8)

where X is the concentration of ryanoid and V is the transmembrane holding potential. At a given ryanoid concentration in solution and at a fixed holding voltage, the apparent dissociation constant is defined by the following.

(Eq. 9)

Dose-response relationships to determine apparent dissociation constants K_H(V) at voltages other than 0 mV and Hill slopes (n_H) were determined by nonlinear regression fit to the general four parameter logistic equation,

(Eq. 10)

where P_s is the probability of occurrence of the subconductance state (at a given holding potential), P_s(min) and P_s(max) are minimum and maximum probability of substate occurrences, X is the log concentration of the drug, K_H(V) is the log concentration of drug required to elicit P_s(0.5) and n_H is the Hill slope (28). For a reaction that does not exhibit cooperativity, the Hill slope is 1.0. Goodness of fit was evaluated by F tests and the simpler model was chosen unless the more complex model provided a better fit at p < 0.05.

The relationships between K_on and K_off and holding potentials were fitted using the defining following equations.

(Eq. 11)

(Eq. 12)

The relationship between P_s and holding potential was fit to the Boltzmann equation,

(Eq. 13)

where z_total is the voltage dependence of the subconductance state induced by ryanoid interacting with RyR2, F is the Faraday constant, V is the transmembrane voltage, and G_i/RT is constant at a holding potential of 0 mV.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis of C₄,C₁₂-Diketopyridylryanodine-- A strategy similar to that used for the synthesis of C₄,C₁₂-diketoryanodine and C₄,C₁₂-diketo-9,21-didehydroryanodine (20, 21) was used for the synthesis of C₄,C₁₂-diketopyridylryanodine (Fig. 1). Using this method pyridylryanodine underwent rapid and selective modification with periodic acid yielding C₄,C₁₂-diketopyridylryanodine as the primary product. Using gravity silica gel chromatography, the product eluted from the column with methylene chloride/4% methanol and after rotary evaporation and freeze-drying afforded 18 mg (72% yield). The structure of this compound was confirmed using ¹H and ¹³C NMR. The purity of C₄,C₁₂-diketopyridylryanodine was determined to be greater than 99.5% using analytical high performance liquid chromatography (methanol/water, 1:1 as mobile phase, UV detection at 260 nm). Non-UV active contaminants were sought using thin layer chromatography (after spraying the plate with 5% ceric ammonium nitrate in 80% phosphoric acid and charring) and electrospray mass spectrometry. None of these methods revealed contaminants.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Preparation of C4,C12-diketopyridylryanodine from pyridylryanodine. Reaction conditions and reagents are described in the text. Configurations shown represent projections of globally minimized structures (MM2), using the algorithms of the software packages of CS Chem 3D ProTM version 5 (Cambridge Scientific Computing, Inc., Cambridge, MA).

RyR2 Binding Affinities of Ryanodine, Pyridylryanodine, and C₄,C₁₂-Diketopyridylryanodine-- We have yet to synthesize radiolabeled forms of pyridylryanodine or its congener C₄,C₁₂-diketopyridylryanodine. As such, the affinities of these ryanoids for RyR2 were determined from their ability to compete with 6.7 nM [³H]ryanodine for binding to RyR2 using equilibrium displacement binding affinity assays. The affinity data for all three ryanoids used in this study were fit by nonlinear regression to one- and two-site competition models given by Equations 1 and 2 under "Experimental Procedures." The data for ryanodine and pyridylryanodine fit well to the one-site binding model (r² > 0.99 and s_yx < 3.7 for both) yielding IC₅₀ of values of 7.8 ± 0.4 nM (K_d = 1.2 nM ± 0.2 nM) for unlabeled ryanodine and 716.8 ± 13.3 nM (K_d = 108.8 ± 5.4 nM) for unlabeled pyridylryanodine (Fig. 2). For C₄,C₁₂-diketopyridylryanodine, however, the two-site model given in Equation 2 provided a significantly better fit (p = 0.005). The higher affinity site comprises 21% of the total binding sites and has an IC₅₀ of 299.5 ± 49.1 nM (K_d = 45.4 ± 10.4 nM) while the second site constitutes the remainder having an apparent IC₅₀ of 72,170 ± 2169 nM (K_d = 10,962.5 ± 452.8 nM). Extension of the two-site model to a three-site model failed to improve the fit. Also, in preliminary experiments incubation was stopped after 1 and 3 h. Results obtained were similar to those after 2 h, although B_max was optimal after 2 h incubation.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Relative affinities of ryanodine, pyridylryanodine, and C4,C12-diketopyridylryanodine for RyR2. [3H]Ryanodine displacement binding affinity assays were used to determine the affinities of the ryanoids for RyR2. Briefly, SR membranes (0.1 mg of protein/ml) were incubated for 2 h at 37 °C in the presence of 6.7 nM [3H]ryanodine and varying concentrations of the designated unlabeled ryanoids, in 250 mM KCl, 20 mM Tris-HCl, 0.1 mM EGTA, and 0.3 mM CaCl2 (pH 7.4 at 37 °C). At the end of the incubation, the vesicles were filtered and washed. [3H]Ryanodine bound to the receptors was determined by liquid scintillation counting. Data for each compound represent the mean ± S.E. from four experiments using two different SR preparations. Curves were fitted using the binding analysis programs of GraphPad Prism 3.0a (PrismPad Software Inc., San Diego, CA).

Functional Effects of Pyridylryanodine on Single RyR2 Channels-- From previous studies we hypothesized that the biphasic effects of ryanoids on RyRs represent interactions with two functionally distinct binding site classes (10, 29). Pyridylryanodine is unique among ryanoids investigated so far in that with calcium flux assays it functionally separates activation from deactivation while preserving the full extent of both. So separate are the dual concentration-effect curves of pyridylryanodine that their intersection presents a plateau, in marked contrast to the peak typical of other ryanoids (14). The present studies on single RyR2 in bilayers provide an explanation for the plateau: pyridylryanodine increases P_o in two concentration-dependent steps as described below.

At the threshold concentration of 500 nM pyridylryanodine (in a final concentration of 0.02% dimethyl sulfoxide in the cis recording bath) channel P_o was significantly increased within 2 min (Fig. 3) from 0.05 ± 0.01 to 0.14 ± 0.02 at +60 mV, and from 0.06 ± 0.01 to 0.22 ± 0.5 at 60 mV, in four of five experiments. The P_o increase resulted from increases in both the frequency of transitions between closed and full open states (number of events increased from 60 ± 5 to 200 ± 18 per s) and the mean dwell time in the open state (from 0.5 ± 0.1 to 1.5 ± 0.3 ms). P_o increases were seen equally at positive and negative holding potentials. This effect was not observed when dimethyl sulfoxide alone (with 1.0% dimethyl sulfoxide P_o increased by 126 ± 32% as of control, n = 7) or when lower concentrations of pyridylryanodine (50-200 nM in dimethyl sulfoxide) were added to the cis chamber, even after 15 min.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effects of submicromolar concentrations of pyridylryanodine on RyR2. Single channel currents were recorded at +60 mV (left panels, upward deflections) and 60 mV (right panels, downward deflections) in symmetric KCl/K-Hepes buffer, pH 7.4, as described in the text. The upper panels represent control recordings in the presence of 2 µM cytosolic Ca2+ before addition of drug or solvent to the cis chamber (Po = 0.05 at +60 mV and 0.07 at 60 mV). The middle panels represent control recordings in the presence of 2 µM cytosolic Ca2+ after addition of 1.0% dimethyl sulfoxide to the cis chamber (Po = 0.06 at +60 mV and 0.08 at 60 mV). The lower panels show a typical increase in Po induced by 500 nM pyridylryanodine (Po = 0.14 at +60 mV and 0.22 at 60 mV).

Doubling the cis concentration of pyridylryanodine to 1 µM readily induced a persistent subconductance state of RyR2 (Fig. 4). The current amplitude of this subconductance state was 0.32 ± 0.01 (n = 12) of fully open (764 ± 6 versus 246 ± 9 pS). Whereas the subconductance amplitude characterizes different ryanoids, the appearance of a subconductance is common among all ryanoids. Induction of the subconductance state of pyridylryanodine was more likely to occur and persisted for longer times at positive holding potentials. In fact, the time to modification and probability of occurrence of this subconductance state was about 6.5 times more likely to occur at positive holding potentials than at negative potentials.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   The effects of low micromolar concentrations of pyridylryanodine on RyR2. A, single channel currents were recorded at +35 mV (left panels, upward deflections) and 35 mV (right panels, downward deflections) in symmetric KCl/K-Hepes buffer, pH 7.4. The upper panels represent control recording in the presence of 2 µM cytosolic Ca2+ before addition of pyridylryanodine to the cis chamber. Po was 0.11 (left) and 0.06 (right). The lower panels show typical subconductance states induced by 1 µM pyridylryanodine at +35 mV and Po in normal gating mode was 0.19 (left panel) and its reversal at 35 mV (right) and Po in normal gating mode was 0.32. Panel B shows the I/V curves for the experiment. Black circles (behind the open circles) represent channel full open state before pyridylryanodine was added and open circles represent the full open state after addition of 1 µM pyridylryanodine. Open triangles reflect the pyridyl-induced subconductance state. Pyridylryanodine did not significantly change the conductance of full open state and the magnitude of the subconductance state represents 32 ± 1% of full open. Data shown is representative of four experiments.

It should be pointed out that in most experiments (five of seven), the P_o of RyR2 in the normal gating mode immediately prior to and after reversal of the subconductance state was higher than the channel P_o prior to addition of the drug (Fig. 4A, lower panels). As with most other ryanoids, detailed kinetic analyses of the interaction of pyridylryanodine with the site that induces the subconductance state were precluded by its slow dissociation kinetics.

In one of five experiments pyridylryanodine (10 µM) produced an alternate subconductance state, which had a current amplitude of 60% of full open (data not shown). The dwell time in this subconductance state lasted tens of seconds to minutes. This second subconductance state could not have been due to an impurity because three sensitive analytical techniques (see "Experimental Procedures") all indicated the presence of only pyridylryanodine in solution.

At concentrations greater than 10 µM, pyridylryanodine readily induced a reversible shut state of the channel (Fig. 5). Although induction of this shut state occurred both at 60 and +60 mV, the dwell time was shorter at +60 mV (1.1 ± 0.2 compared with 1.9 ± 0.3 s, n = 6). Mechanistic interpretation of the shorter dwell time at positive holding potentials is confounded by the voltage dependence of induction of the subconductance state. At 60 mV, P_s is small so that shutting can occur without prior intervention of the subconductance state. As shown in Fig. 5, at +60 mV the channel reverts from shut back to the subconductance state, whereas at 60 mV it reverts from shut back to the normal gating mode.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   The effects of high micromolar concentrations of pyridylryanodine on RyR2. Single channel currents were recorded at +60 mV (left traces) and 60 mV (right traces) in symmetric KCl/K-Hepes, pH 7.4. These traces show that pyridylryanodine (20 µM) can trigger a reversible closed state of RyR2 at both positive and negative holding potentials. However, at +60 mV RyR2 typically closes from the subconductance state and returns back to the subconductance state, whereas at 60 mV, RyR2 transitions from the normal gating mode (high Po) to the closed state and back to the normal gating mode without entering the subconductance state. It should also be mentioned that the dwell time in the closed state was twice as long at 60 mV than it was at +60 mV. Data shown are representative of six experiments.

Taken together, these results show that pyridylryanodine is able to induce three distinct dose-dependent effects on single RyR2 channels. At nanomolar concentrations it increases P_o without altering unitary conductance, at low micromolar concentrations it induces a major subconductance state, and at higher concentrations it causes a persistent but nevertheless reversible shut state. These three functional effects were not predicted from the binding studies with RyR2 in vesicles, perhaps because of its weak subsite selectivity. Moreover, high affinity of pyridylryanodine precluded detailed kinetic analyses.

Effects of C₄,C₁₂-Diketopyridylryanodine on RyR2 Single Channels at Nanomolar Concentrations-- A pyridylryanodine derivative with greater subsite selectivity and faster kinetics was therefore desirable. We reasoned that both might be achieved by reducing the rigidity of the ryanoid skeletal backbone. Two principal ways to relax the skeletal backbone of pyridylryanodine are through partial oxidation of either (i) the vicinyl diols on C₄ and C₁₂ with concomitant breakage of the carbon-carbon bond that is shared by the A and B rings, or (ii) the hemiacetal moiety of the D-ring, leading to breakage of the C₁,C₁₅ carbon-carbon bond and thereby eliminating the E ring (20, 21). In this study we used the former, affording C₄,C₁₂-diketopyridylryanodine (Fig. 1).

As shown in the example records of Fig. 6A, addition of 50 nM C₄,C₁₂-diketopyridylryanodine to the cis chamber approximately doubled the channel P_o at holding potentials of +35 and 35 mV (184% at +35 mV and by 212% at 35 mV). This increased P_o was independent of the concomitant addition of 0.02% dimethyl sulfoxide. Closer examination of the recordings showed that the increase in P_o resulted from increases in gating frequency (the number of events/time) and mean open time (Table I). Data from nine experiments show that the normalized P_o was significantly increased by 50 nM C₄,C₁₂-diketopyridylryanodine equally at both holding potentials, to mean values of 165 ± 30% at +35 mV and 183 ± 30% at 35 mV (Fig. 6B).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   The effects of submicromolar C4,C12-diketopyridylryanodine concentrations on gating characteristics of RyR2 in the presence of 2 µM cis Ca2+. A, single channel currents were recorded at +35 mV (left panels, upward deflections) and 35 mV (right panels, downward deflections) in symmetric 0.25 M KCl, 20 mM K-Hepes, pH 7.4, buffer containing 2 µM free Ca2+. Top traces show control Po at +35 mV (0.19, left panel) and 35 mV (0.26, right panel). Lower traces show increases in Po after the addition of 50 nM C4,C12-diketopyridylryanodine to the cis chamber. At +35 mV Po increased to 0.35 whereas at 35 mV Po increased to 0.55 (right panel). B, the relationship between normalized Po and C4,C12-diketopyridylryanodine concentration at ±35 mV are shown. The solid (-) line represents the fit of the pooled data at ±35 mV using non-linear regression analysis (four parameter logistic equation). Apparent dissociation constant KH = 68 ± 16 nM (±35 mV) and Hill slope (nH) of 1.7 ± 0.8 were obtained. The dashed and dotted lines show the fit at +35 mV () and 35 mV (). Whereas the data at 35 mV was accommodated by the logit function (Equation 10) affording Po(max) = 386.6 ± 42.2%, KH = 125.7 ± 13.1 nM, and Hill slope = 1.0 ± 0.9, the data at +35 mV did not. Data are the mean ± S.E. of 4 to 11 experiments.

View this table: [in this window] [in a new window]   Table I Channel parameters that are altered by 50 nM C4,C12-diketopyridylryanodine Control: 2 µM Ca2+, Po values were 0.121 ± 0.043 and 0.133 ± 0.044 at 35 mV, and +35 mV, respectively. Topen and Tclosed, mean open time and mean closed time, respectively.

Increasing the concentration of C₄,C₁₂-diketopyridylryanodine incrementally above 50 nM up to 1000 nM increased mean channel P_o, apparently in a concentration-dependent manner. However, because of experimental variation, no statistical differences could be shown between mean values at +35 and 35 mV at each concentration. Thus, for initial dose-response analyses these values were pooled. Concentration-effect relationships were assessed by nonlinear regression fit to the general four parameter logistic equation with minimal P_o = 100%. Goodness of fit was compared between the version in which n_H was constrained to 1 and one in which regression was used to fit n_H. For the pooled data at ±35 mV (Fig. 6B, solid line, r² = 0.73, s_xy = 39.2), P_o(max) was 328.3 ± 25.5%, the apparent dissociation constant K_H was 68 ± 16.0 nM and n_H was 1.7 ± 0.8.

It was of interest to evaluate the possibility of concentration dependence of the normalized P_o data independently at the two holding potentials. The interrupted lines of Fig. 6B show this analysis. Above 50 nM C₄,C₁₂-diketopyridylryanodine, dependence of normalized P_o on drug concentration was apparent but only at the negative holding potential. The data at 35 mV are well accommodated by the logit function (Equation 10) with values of P_o(max) = 386.6 ± 42.2%, K_H = 125.7 ± 13.1 nM, and Hill slope = 1.0 ± 0.9. This site likely corresponds to the higher affinity site detected in displacement binding assays. These data represent the first clear example of a ryanoid that increases channel P_o in a concentration-dependent manner without a transitioning into a subconductance state.

Effects of C₄,C₁₂-Diketopyridylryanodine at Micromolar Concentrations on RyR2 Single Channels-- C₄,C₁₂-diketopyridylryanodine at 1 µM was the threshold concentration for induction of the subconductance state, given prolonged incubation times in the cis chamber. With advancing concentrations of C₄,C₁₂-diketopyridylryanodine (2-20 µM) RyR2 quickly entered the reduced conductance state with a current amplitude of 52 ± 1% of full open (n = 9, Fig. 7). This subconductance state was frequent at positive but only rarely seen at negative voltages. On examination of the recordings at 5 µM, the increased channel gating previously seen at nanomolar concentrations was apparent prior to induction and following reversal of the subconductance state at +35 mV and also at 35 mV where the subconductance did not occur (Fig. 7, lower panels). P_o in the normal gating mode increased from 0.11 to 0.44 at +35 mV and from 0.12 to 0.54 at 35 mV. The subconductance amplitude was not different at the different drug concentrations nor was the unitary conductance (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   The effects of a low micromolar concentration of C4,C12-diketopyridylryanodine on RyR2 in the presence of 2 µM cis Ca2+. Single channel currents were recorded at +35 mV (left panels, upward deflections) and 35 mV (right panels, downward deflections) in symmetric 0.25 M KCl, 20 mM K-Hepes, pH 7.4, buffer containing 2 µM free Ca2+. The upper panels show the control recording before addition of 5 µM C4,C12-diketopyridylryanodine to the cis chamber. Po was 0.11 at + 35 mV (upper left panel) and 0.12 at 35 mV (upper right panel). The lower left panel (+35 mV) shows that after 5 µM C4,C12-diketopyridylryanodine was added to the cis chamber, RyR2 entered into a reversible subconductance state (52 ± 1% of full open). This subconductance state was not observed at 35 mV (lower right panel). Po in normal gating mode was 0.44 at +35 mV (lower left panel) and 0.54 at 35 mV (lower right panel).

The ready reversibility of the C₄,C₁₂-diketopyridylryanodine subconductance state at positive holding potentials afforded the opportunity to evaluate reversibility of this ryanoid under experimental conditions designed to vitiate subtle P_o effects. To achieve a high initial P_o, we used a bathing calcium concentration of 25 µM, producing a control P_o of 0.90 ± 0.03 (n = 8, Fig. 8). Over a period of 9 s of recording, six transitions from the normal gating mode to the subconductance state occurred (Fig. 8B).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Multiple subconductance states and reversibility C4,C12-diketopyridylryanodine on RyR2. Single channel currents were recorded at +35 mV in symmetric 0.25 M KCl, 20 mM K-Hepes, pH 7.4, buffer containing 25 µM free Ca2+. A, the upper two traces show a continuous control recording with Po = 0.95. B, these continuous traces show that after 5 µM C4,C12-diketopyridylryanodine was added to the cis chamber, RyR2 entered and reversed from the subconductance state of 52 ± 1% of full open. C, this panel shows an infrequently occurring subconductance state of 75% of full open conductance.

In addition to the major subconductance of 52%, C₄,C₁₂-diketopyridylryanodine occasionally induced a second subconductance of 75% of fully open (Fig. 8C). This subconductance is the largest yet reported for a ryanoid. On one occasion, a third subconductance of ~25% was observed. To evaluate whether these two additional subconductance states might have resulted from contamination we used analytical high performance liquid chromatography, thin layer chromatography, and electrospray mass spectrometry. By all three of these techniques we could find only C₄,C₁₂-diketopyridylryanodine in solution.

Kinetics of Interaction of C₄,C₁₂-Diketopyridylryanodine with the Binding Site That Triggers the Major Subconductance State-- The reversibility of C₄,C₁₂-diketopyridylryanodine permitted evaluation of its kinetics of interaction with the binding site that induces the major channel subconductance. In the first series of these experiments, the dwell times for a channel in the normal gating and major subconductance states were monitored over 6 min at +35 mV in the presence of 10 µM C₄,C₁₂-diketopyridylryanodine and 25 µM calcium. The distribution of dwell times in the normal and subconductance modes fit best to single exponentials (r²  0.98, s_yx  2.88) and afforded a mean dwell time in the unmodified state (_norm) of 2450 ms, and in the modified state (_sub) of 335 ms (Fig. 9). In the presence of 10 µM C₄,C₁₂-diketopyridylryanodine K = 0.0408 s¹ µM¹ and K_off = 2.98 s¹ were obtained using Equations 6-8. At 35 mV the apparent dissociation constant K_H for C₄,C₁₂-diketopyridylryanodine is 73.0 µM (Equation 9). This value is slightly larger but in reasonable agreement with the aggregate K_d of 10.9 ± 0.5 µM at 0 mV for the lower affinity fraction of sites obtained using displacement binding assays at a calcium concentration optimized for binding.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Dwell time histograms in normal and subconductance modes after addition of C4,C12-diketopyridylryanodine. Cumulative dwell time histograms in normal gating mode (left panel) and in subconductance mode (right panel) were obtained from a 6-min recording recorded at 35 mV in symmetric 0.25 M KCl buffer containing 25 µM cytosolic Ca2+. Both dwell time histograms were fitted to single exponential function with time constants of norm 2450 ms (left panel) and sub 335 ms (right panel), respectively.

In the second series of experiments, the effect of C₄,C₁₂-diketopyridylryanodine concentration on P_s was evaluated at three increasingly positive holding potentials. Increasing the drug concentration at a given holding potential decreased the mean dwell time in the normal gating mode and increased the mean dwell time in the subconductance state (Table II). Thus as the drug concentration increased, the rate at which ryanoid molecules become bound increases and this in turn decreases the time to induction, as well as increasing the duration of the subconductance state. Increasing the holding potential from +20 to +50 mV at a given concentration of C₄,C₁₂-diketopyridylryanodine did not affect the number of transitions between the subconductance and normal gating mode (Table III). These results are consistent with previous reports that the time to onset of the subconductance state decreases as the holding potential increases (12, 26, 30). They are also consistent with the general notion that positive holding potentials facilitate ryanoid interactions with RyR2.

In a third set of experiments association and dissociation rates were investigated at 35 mV over the concentration range of 5-20 µM. Consistent with the data of Table II above, the association rate constant of C₄,C₁₂-diketopyridylryanodine with RyR2 increased as a function of concentration at +35 mV (r² = 0.99), affording an association rate (slope of the curve) of 0.044 ± 0.004 s¹ µM¹ (Fig. 10, ). On the other hand, dissociation from RyR2 was not statistically related to the concentration of C₄,C₁₂-diketopyridylryanodine as its slope was not significantly different from zero (p = 0.07), because of the large standard errors. The rate of dissociation of C₄,C₁₂-diketopyridylryanodine concentration (K_off) was determined to be 2.8 s¹. Using the values obtained, K_H approximates 63.6 µM (35 mV) as calculated from Equations 11 and 12. This value is in close agreement with the K_H obtained using probability density function curves.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   The dependence of association (open circles) and dissociation (filled circles) rates on concentration of C4,C12-diketopyridylryanodine. Kon and Koff were obtained using Equations 6 and 8. K was derived using Equation 7. Whereas Kon was dependent on concentration of C4,C12-diketopyridylryanodine, Koff was not. Each point represents the mean ± S.E. from three to five experiments.

The dose-response relationships between concentration of C₄,C₁₂-diketopyridylryanodine and P_s at three holding potentials are summarized in Fig. 11. The data fit well to the four parameter logistic function (Equation 13) giving r² > 0.90 and s_yx  0.092 at all three holding potentials. P_s values at +20 mV were doubled at +35 mV and nearly doubled again at +50 mV. The apparent dissociation constant K_H (i.e. voltage-dependent K_d) values were 29.0 ± 1.2 µM at 20 mV, 15.8 ± 1.2 µM at 35 mV, and 8.9 ± 1.0 µM at 50 mV and corresponding Hill slopes of 2.1 ± 0.1, 2.1 ± 0.1, and 2.3 ± 0.2. These data strongly suggests that induction of the subconductance state results after binding of more than one molecule of C₄,C₁₂-diketopyridylryanodine to RyR2. These results are internally consistent, in that the first molecule bound increases channel P_o, whereas a subsequent molecule induces the subconductance state.

View larger version (25K): [in this window] [in a new window]   Fig. 11.   The relationship between Ps and concentration of C4,C12-diketopyridylryanodine at increasing positive holding potentials. Ps was calculated from recordings similar to those shown in Fig. 8A (25 µM Ca2+). Po in the absence of the drug was 0.90 ± 0.03 (n = 8). The lines represent the logit fits to the data using Equation 13. KH values of 29.0, 15.8, and 8.9 µM and nH values of >2 were obtained for 20, 35, and 50 mV, respectively. Values shown represent the mean ± S.E. of three to eight experiments.

Because the channel dwell time in the subconductance state is voltage-dependent, we further evaluated the relationship between P_s and transmembrane holding potential at 10 µM C₄,C₁₂-diketopyridylryanodine. Fig. 12A represents the nonlinear regression best fit of ln(P_s) as a function of transmembrane holding potential (Equation 13). A slope of 0.055 (r² = 0.99) was obtained and from this value, an effective gating charge (z_total) of 1.4 was derived. The z_total was also calculated from the rates of association and dissociation of C₄,C₁₂-diketopyridylryanodine at the three holding potentials. As shown in Fig. 12B, K_on increases as the holding potential is made more positive; at the same time as K_off decreases. The lines shown were obtained by nonlinear regression fit to the first-order polynomial equation. Values for z_on and z_off obtained from the slopes of these lines were 0.60 and 1.1, respectively. This afforded a total valence, z_total of 1.7. 

View larger version (14K): [in this window] [in a new window]   Fig. 12.   Relationship between Ps and holding potential at 10 µM C4,C12-diketopyridylryanodine. A, Ps was obtained from single channel currents similar to those in Fig. 8B. Each point represents the mean ± S.E. of three to five experiments. Points were fitted using the Boltzmann equation (Equation 13 in text) and afforded a ztotal of 1.4. B, the relationship between ln(Kon) an ln(Koff) at various positive holding potentials. Each point represents the mean ± S.E. obtained from three to five experiments (on open circles, S.E. values are too small to be seen). zon of 0.6 and zoff of 1.1 were obtained using Equations 11 and 12, respectively. ztotal = zon + zoff = 1.7. ztotal obtained from A and B are very similar.

Effect of a High Micromolar Concentration of C₄,C₁₂-Diketopyridylryanodine on RyR2 Single Channel Activity-- At a concentration of 250 µM C₄,C₁₂-diketopyridylryanodine induced a long lasting closed state that was nonetheless reversible (Fig. 13). Such shutting was observed in three separate experiments. In each experiment, the dwell time in the shut state lasted for tens of seconds. It should be pointed out that the shutting closures induced by C₄,C₁₂-diketopyridylryanodine at positive holding potentials were much longer lasting (about 8 times) than that caused by parent pyridylryanodine.

View larger version (18K): [in this window] [in a new window]   Fig. 13.   The effects of high micromolar concentrations of C4,C12-diketopyridylryanodine on RyR2. These panels show continuous recordings obtained after 250 µM C4,C12-diketopyridylryanodine was added to the cis chamber. Three separate recording were carried out using 25 µM Ca2+ in the cis chamber. Note the long lasting, but reversible shut state.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The principal finding of the present study is that C₄,C₁₂-diketopyridylryanodine and its parent pyridylryanodine induce three distinct, concentration-dependent effects on RyR2 single channels. These ryanoids differ from ryanodine in that their C₃ hydroxyl carries a pyridyl substituent instead of a pyrrole. At nanomolar concentrations the diketo derivative and pyridylryanodine first increase channel P_o (by ~3-fold) without inducing a persistent subconductance state. The initial P_o increase consists of an increase in both gating frequency and mean open dwell time. At higher concentrations, they induce the hallmark ryanoid subconductance state and at even higher concentrations they produce a persistent shut state that nevertheless is reversible. These effects are summarized in Scheme 1, where RyR2_n represents the normal gating of the channel, RyR2 reflects the voltage-independent increase in channel gating frequency, RyR2 represents induction of the subconductance state, and RyR2 represents the long lasting but reversible transition to the shut state. These three effects were more easily identified with C₄,C₁₂-diketopyridylryanodine than they were with pyridylryanodine. The first effect was hardly discernable when control P_o had been elevated to ~0.9 by adjusting the calcium concentration to 25 µM.

View larger version (17K): [in this window] [in a new window]   Scheme 1.

Another major finding of the present study is that unlike its parent, the chemically modified ryanoid C₄,C₁₂-diketopyridylryanodine (having a backbone-relaxed structure) is able to discriminate two populations of ryanoid binding sites on RyR2. Analysis of the displacement binding data revealed that of the total binding sites, 21% exhibit nanomolar affinity for C₄,C₁₂-diketopyridylryanodine whereas the remaining 79% exhibit micromolar affinity. Because RyR2 is the only isoform detected in canine left ventricle, either (a) the single isoform comprises two populations of binding sites or (b) among the several energetically favored conformations of C₄,C₁₂-diketopyridylryanodine, there are conformers that can discriminate between multiple members of a single class of binding sites on RyR2. Data from several laboratories are supportive of the notion that RyR2 has more than one ryanoid binding site. Because each effect recorded in the present study is distinct and concentration-dependent (and one is also voltage-dependent), it is likely that each effect is induced by a ryanoid molecule binding to a separate site on a single RyR2 molecule. Three concentration-dependent effects then should result from serial binding of three ryanoid molecules. The competition displacement studies (Fig. 2) discern only two. Because effects on single channels in bilayers are more discriminant than aggregate effects on multiple channels in vesicles, it is not unlikely that within the two populations of binding sites, subpopulations may exist that remain poorly discernable with binding affinity assays.

From here on, the discussion will focus primarily on the effects induced by C₄,C₁₂-diketopyridylryanodine on RyR2. References to the effects of pyridylryanodine will occasionally be made for comparison. Within the nanomolar concentration range, C₄,C₁₂-diketopyridylryanodine induces a voltage-independent increase in P_o (of 300%) without a step-change to a channel subconductance. This increase in P_o first became evident with channels that were submaximally activated at the start of the experiment but persisted even when control P_o had been elevated. The dose-response data at 35 mV taken alone gave a Hill slope (n_H) of 1.0 suggesting that this effect requires binding of only a single molecule of C₄,C₁₂-diketopyridylryanodine to RyR2. However, given the data variance the alternate fit to the pooled (±35 mV) data was equally valid statistically. The fit for the pooled data yielded a n_H of 1.7. The latter interpretation suggests the possibility that more than one ryanoid molecule may participate in the increased P_o effect. Pyridylryanodine also increased the frequency of channel gating, but this required a concentration 10 times higher than that for C₄,C₁₂-diketopyridylryanodine. Consistent with recent reports (31-33) it seems reasonable to view the ryanoid-induced increase in P_o as resulting from destabilization of the closed state of the channel.

At low to intermediate micromolar concentrations C₄,C₁₂-diketopyridylryanodine induces a voltage-dependent major subconductance state of 52% of fully open. Pyridylryanodine induced a smaller subconductance state. These data are consistent with previous studies showing that the magnitude of the subconductance states varies as a function of ryanoid structure and that the frequency of occurrence of the substate increases with increasing positive holding potentials (6, 12, 26). When probability of occurrence of the major substate (P_s) was plotted against concentration of C₄,C₁₂-diketopyridylryanodine at several membrane holding potentials, Hill slopes of ~2 or greater were obtained. These data suggest that substate induction occurs as a consequence of the binding of more than one ryanoid molecule to RyR2. These data differ from those for ryanodol (30) and 21-amino-9-hydroxyryanodine (26), as the two latter compounds display apparent bimolecular kinetics, consistent with there being only a single ryanoid molecule needed for induction of the subconductance state.

Both C₃-modified ryanoids used in this study induced more than one subconductance state (32 and 60% for pyridylryanodine and 25, 52, and 75% for C₄,C₁₂-diketopyridylryanodine). As contamination was analytically ruled out, considerations of other means by which a single ryanoid can induce more than one subconductance state are in order. Two explanations are likely. First, there may be alternate ryanoid-induced conformations of RyR2, depending on how many ryanoid molecules have become bound per molecule of RyR2. Second, multiple conformers of the ligand may each induce distinct conformations of the receptor.

Functional effects suggest at least three ryanoid binding sites per RyR2 homotetramer. If the conductance amplitude of the subconductance state ratchets down as each ryanoid molecule becomes bound to RyR2, then one would expect that P_s should serially diminish as each ryanoid molecule thereafter gets bound. With C₄,C₁₂-diketopyridylryanodine, the observed subconductance state of 75% could reflect binding of one ryanoid molecule (in addition to the one bound to induce the increase in channel P_o), the subconductance of 52% could reflect binding of a second additional molecule and the subconductance of 25% could reflect binding of a third additional ryanoid molecule. This leaves unanswered the question of why the 52% subconductance state should be the most frequent. It can be speculated that it is the most stable. In parallel reasoning, the subconductance state of 60% could reflect binding of one molecule of pyridylryanodine and the subconductance of 32% could reflect binding of two such molecules. The latter subconductance state might be more stable as it is more frequently observed. The stepwise decrease in subconductance amplitude with an increasing number of ryanoid molecules bound is supportive of this hypothesis and is also consistent with the model described by Pessah and co-workers (34).

On the other hand, multiple subconductances might reflect alternate steric configurations of the ligand. Schliefer (35) suggested that pyridylryanodine can exist in two alternate low energy conformers, based on the arrangement of the pyridine nitrogen relative to that of the carbonyl oxygen on C₂₂ as indicated in Fig. 14A. These two conformers might induce two distinct substates. The major substate would presumably be the one that is induced by the predominant conformer. However, it is indeterminate whether this may be the E-/syn-conformer (oxygen and the nitrogen on the same sides) or Z-/anti-conformer (oxygen and the nitrogen on opposite sides).

View larger version (30K): [in this window] [in a new window]   Fig. 14.   Energy minimized conformations of pyridylryanodine and C4,C12-diketopyridylryanodine. The illustrations depict the lowest steric energy forms for pyridylryanodine and diketopyridylryanodine. These views are elaborated from the studies of Jefferies et al. (21) and Schleifer (35). The conformations were achieved by taking two-dimensional representations of the ryanoids generated using ChemDraw Ultra software (ChemOffice, version 5) and minimizing steric interactions using MM2 minimization to define the given configurations of the molecules. After minimization, the molecules were rotated to a standard presentation of the C3 substituent. MOPAC data files were then generated and physical parameters were obtained for pyridylryanodine (A) and its modified congener C4,C12-diketopyridylryanodine (B(i)). Green lines are drawn to reflect an arbitrary dihedral angle between the skeletal backbone and the C3 substituent. The structures on the right differ from those on the left only with respect to the orientation of the nitrogen of the pyridyl ring relative to the C22 carbonyl. Panel B (ii) shows conformational inversion of the C3 pyridine ring (from the -side to the -side) and of the isopropyl group on C2 (from the -side to -side) following breakage of the C4-C12 carbon bond.

With respect to C₄,C₁₂-diketopyridylryanodine, relaxing the ring constraint on the skeletal backbone induces two major structural changes to the molecule. First, the conformation of the C-ring changes from chair to boat/half-boat and this alters the orientation of the methyl functionality on the C₉ carbon. Second, merging of A and B rings into a single 8-membered ring can also result in conformational inversion of the pyridine ring on the C₃ carbon (from  to  side) and the isopropyl group on the C₂ carbon (from  to  side) (21). For each of these two major conformations, the E-/syn and Z-/anti forms can exist. Thus, a total of four conformers are likely (Fig. 14, B(i) and B(ii)). If each conformation induces a single substate, then C₄,C₁₂-diketopyridylryanodine should be capable of inducing four subconductance states. In the simplest case, the frequency of occurrence of each substate should be proportional to the ratio of the conformers in solution. Thus far, we have detected only three of the four subconductances suggested by this physiochemical rationalization.

When exposed to low micromolar concentrations of C₄,C₁₂-diketopyridylryanodine at +35 mV, RyR2 spends a minor percentage of its time in the major subconductance state and the majority of its time in the normal gating mode. Plots of P_s as a function of holding potentials in the presence of 10 µM C₄,C₁₂-diketopyridylryanodine fit well to the logit function and afford an effective gating charge (z_total) of 1.4. A similar z_total (1.7) was obtained from plots of ln K_off/ln K_on as a function of holding potentials. Because C₄,C₁₂-diketopyridylryanodine carries a formal charge of +1, and if the voltage dependence of the interaction is derived solely from transmembrane movement of the charge, then at least two molecules will be needed to accommodate the effect of the voltage drop. Whereas this hypothesis is at least tangentially supported by Hill slopes of >2, it seems unlikely on physical grounds.

Both pyridylryanodine and C₄,C₁₂-diketopyridylryanodine induced long lasting shut states of RyR2 that were nonetheless reversible. As previously shown for other ryanoids (6, 12, 26, 31), channels in the normal gating mode can transition to the subconductance state and from the latter back either to the full open or to the closed state. In addition, we found that at negative holding potentials pyridylryanodine caused RyR2 to directly transit from normal gating to the closed state and back to normal gating without the intermediate intervention of a subconductance. Thus, it is clear that induction of a subconductance state is not a necessary prerequisite for ryanoid-induced shutting of RyR2.

Another interesting question raised by the present results is whether reducing the rigidity of the skeletal backbone alone is sufficient to induce subsite selectivity and reversibility among ryanoids. We tested this by preparing C₄,C₁₂-diketo derivatives of ryanodine and didehydroryanodine. In competition binding affinity assays neither compound was able to discern two classes of ryanoid binding sites on RyR2 (data not shown). In single channel measurements, C₄,C₁₂-diketodidehydroryanodine induced a persistent subconductance state of 39% of full open, which was not reversible on the time scale of our bilayer experiments (30-60 min) (data not shown). These results suggest that subsite selectivity and reversibility require not only a reduced rigidity of the ryanoid skeletal backbone, but also alterations in the C₃ substituent of the A-ring of ryanodine.

Photoaffinity labeling, protein degradation, and mutation studies have shown that at least one site of ryanoid interaction is located in the C-terminal, membrane spanning region of RyR (36-40). Zhao et al. (40) showed that point mutations around a probable pore-forming region, GVRAGGGIGD (amino acids 4820-4829), significantly impair high affinity ryanodine binding. These data, as well as others by the same group (41) led to the conclusion that a high affinity ryanodine site resides within the pore-forming region of RyR2. Fessenden et al. (34) found that the E4032A point mutation on RyR1 significantly impaired high affinity ryanodine binding but the channel remained sensitive to 500 µM ryanodine. This observation led these workers to conclude that ryanodine may occlude the pore on RyR1 by an allosteric mechanism. Data from the present study support the existence of more than one ryanoid binding site on RyR. One of these sites (or class) resides outside the strict confines of the transmembrane voltage gradient whereas a second binding site may be within the pore.

In summary, the present results show that C₄,C₁₂-diketopyridylryanodine and its parent pyridylryanodine induce three distinct concentration-dependent effects on RyR2, the first of which is to increase P_o without a step change to a subconductance. We have also shown that C₄,C₁₂-diketopyridylryanodine is able to discriminate between two classes of binding sites on RyR2. This suggests the intriguing possibility that further discrete modifications on ryanoid molecules will produce even more discriminate, subsite selective ryanoids.

	ACKNOWLEDGEMENTS

We thank Ashutosh Tripathy and Mirko Stange for helpful suggestions and Daniel Pasek for preparation of some of the proteoliposomes.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants HL66898 (to K. R. B.) and HL27430 (to G. M.), and the Showalter Trust (to H. R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors are considered first authors.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, NE 68198. Tel.: 402-559-9018; Fax: 402-559-7495; E-mail: kbidasee@unmc.edu.

Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M208372200

	ABBREVIATIONS

The abbreviations used are: RyRs, ryanodine receptor calcium-release channels; pyridylryanodine, C₃-O-[pyridylcarbonyl]ryanodol; C₄, C₁₂-diketopyridylryanodine, C₃-O-[pyridylcarbonyl]C₄,C₁₂-seco-C₄,C₁₂-dioxoryanodol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. Cherednichenko, C. W. Ward, W. Feng, E. Cabrales, L. Michaelson, M. Samso, J. R. Lopez, P. D. Allen, and I. N. Pessah Enhanced Excitation-Coupled Calcium Entry in Myotubes Expressing Malignant Hyperthermia Mutation R163C Is Attenuated by Dantrolene Mol. Pharmacol., April 1, 2008; 73(4): 1203 - 1212. [Abstract] [Full Text] [PDF]

				E. A. Sobie, L.-S. Song, and W. J. Lederer Local recovery of Ca2+ release in rat ventricular myocytes J. Physiol., June 1, 2005; 565(2): 441 - 447. [Abstract] [Full Text] [PDF]

				C. Paolini, J. D. Fessenden, I. N. Pessah, and C. Franzini-Armstrong Evidence for conformational coupling between two calcium channels PNAS, August 24, 2004; 101(34): 12748 - 12752. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 278/16/14237    most recent M208372200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bidasee, K. R. Articles by Besch, H. R. Search for Related Content PubMed PubMed Citation Articles by Bidasee, K. R. Articles by Besch, H. R., Jr. Social Bookmarking                      What's this?