(Ryanodine Receptors) of Cardiac and Skeletal Muscle

Imperatoxin A Induces Subconductance States in Ca2ϩ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Ashutosh Tripathy,* Wolfgang Resch,* Le Xu,* Hector H. Valdivia,‡ and Gerhard Meissner* From the *Department of Biochemistry and Biophysics, and Department of Physiology, University of North Carolina, Chapel Hill, North Carolina 27599; and ‡Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706

abstract Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2ϩ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. -at a holding poten %28ف of the full conductance, whereas it was %43ف The chord conductance at Ϫ40 mV was tial of ϩ40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per ϩ53 mV and the rate constant of dissociation decreased e-fold per ϩ25 mV ap- The IpTxa binding .1.5ف plied holding potential. The effective valence of the reaction leading to the substate was of the voltage drop from the cytosolic side. IpTxa induced substates in %23ف site was calculated to be located at the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2ϩ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site. key words: sarcoplasmic reticulum • imperatoxin activator • substates • scorpion toxin • [3H]ryanodine binding introduction toxins that target various voltage- and ligand-gated channels including the CRCs (Furukawa et al., 1994; Ca2ϩ release channels (CRCs),1 also known as ryano- Morrissette et al., 1995, 1996). Recently, from the dine receptors (RyRs), mediate the release of Ca2ϩ venom of the African scorpion Pandinus imperator, two from an intracellular membrane compartment, the sar- factors, imperatoxin activator (IpTx ) and imperatoxin coplasmic reticulum (SR), into the cytoplasm in stri- a inhibitor (IpTx ), were isolated that selectively activated ated muscle cells. The CRC has been purified as a 30 S i and inhibited, respectively, the CRCs (Valdivia et al., protein complex and shown to be composed of four 1992; El-Hayek et al., 1995). IpTx is a 33 amino acid amino acid residues a 5,000ف large RyR polypeptides of peptide that activated the skeletal CRC in [3H]ryano- and four immunophilins (FK506 binding protein) of dine binding and single channel studies. It did not ex- amino acid residues each. Three tissue-specific 100ف ert a significant effect on cardiac CRC (Valdivia et al., isoforms of RyR have been identified. The conduc- 1992; El-Hayek et al., 1995; Zamudio et al., 1997a). tances and pharmacological properties of the three iso- IpTx has been shown to be a heterodimeric protein forms are, to a large extent, similar (for reviews, see i (subunits of 108 and 27 amino acid residues covalently Meissner, 1994; Sutko and Airey, 1996). linked by a disulfide bond) with lipolytic action, and it Traditionally, venom from many poisonous snakes, inhibited both skeletal and cardiac CRCs by generating lizards, and scorpions has been a rich source of peptide a lipid product (Valdivia et al., 1992; Zamudio et al., 1997b).

Address correspondence to Ashutosh Tripathy, Ph.D., Department of Here, we show that IpTxa induces voltage- and con- Biochemistry and Biophysics, University of North Carolina at Chapel centration-dependent subconductance states in both Hill, Chapel Hill, NC 27599-7260. FAX: 919-966-2852; E-mail: tripathy skeletal and cardiac CRCs when added to the cytosolic @med.unc.edu 1 2ϩ side. Analysis of voltage and concentration dependence Abbreviations used in this paper: CRC, Ca release channel; IpTxa, imperatoxin activator; RyR, ryanodine receptor; SR, sarcoplasmic and stochastic analysis of the events suggests that the in- reticulum. duction of subconductance states corresponds to re-

679 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/98/05/679/12 $2.00 Volume 111 May 1998 679–690 http://www.jgp.org

versible, voltage-dependent binding and unbinding of was in normal gating mode between the closed and full conductance levels) and subconductance states were obtained by man- IpTxa at a single, cytosolically accessible site located in ual positioning of the cursors. the voltage drop across the channel. IpTxa also induced a subconductance state in the ryanodine-modified skeletal muscle CRC and affected [3H]ryanodine binding [3H]Ryanodine Binding to skeletal and cardiac SR vesicles depending on the 3 level of channel activation. These results provide evi- [ H]Ryanodine binding experiments were carried out with rabbit skeletal and canine SR vesicles (protein: 150–250 ␮g/ml) in 0.25 dence for the binding of IpTxa to a channel site differ- M KCl, 20 mM imidazole, pH 7.0, 0.2 mM Pefabloc, 20 ␮M leu- ent from that of ryanodine. peptin, 0.1 mM EGTA, and [CaCl2] necessary to set the free [Ca2ϩ] in the range of 0.1 ␮M to 10 mM. After incubation for 24 h materials and methods at 24ЊC, bound radioactivity was determined by a filter assay as described (Tripathy et al., 1995). Nonspecific binding was deter- Phospholipids were obtained from Avanti Polar Lipids, Inc. (Ala- mined with a 1,000-fold excess of unlabeled ryanodine. baster, AL). All other chemicals were of analytical grade. Preparation of SR Vesicles, and Purification and Other Data Analysis and Conventions 2ϩ Reconstitution of Ca Release Channels Results are given as means Ϯ SD with the number of experiments SR vesicle fractions enriched in [3H]ryanodine binding and Ca2ϩ in parentheses. The SD is included within the figure symbol or release channel activities were prepared in the presence of pro- indicated by error bars if it is larger. Significance of differences tease inhibitors from rabbit skeletal and canine cardiac muscle as was analyzed with Student’s paired t test. Differences were consid- described (Meissner, 1984; Meissner and Henderson, 1987). The ered to be significant when P Ͻ 0.05. CHAPS (3-[(3-cholamido-propyl)dimethylammonio]-1-propane- sulfonate)-solubilized skeletal or cardiac muscle 30 S Ca2ϩ release results channel complex was purified and reconstituted into proteoliposomes by removal of CHAPS by dialysis (Lee et al., 1994). IpTxa Induces Subconductance States in Skeletal and Cardiac

Purification and Synthesis of IpTxa Muscle CRCs

IpTxa was purified from P. imperator scorpion venom in three The skeletal and cardiac CRCs have been shown to con- chromatographic steps as described (Valdivia et al., 1992; El- duct monovalent ions more efficiently than Ca2ϩ and to Hayek et al., 1995; Zamudio et al., 1997a). Synthetic IpTxa was be essentially impermeant to ClϪ. In 250 mM KCl solu- prepared as described (Zamudio et al., 1997a). tion, the single channel conductances of both isoforms pS (Meissner, 1994; and this study). When 15 770ف Single Channel Measurements and Analysis are nM native IpTxa was added to the cytosolic side (cis bi- Single channel recordings of purified rabbit skeletal muscle or layer chamber) of a single skeletal CRC, the channel canine cardiac muscle CRCs incorporated into planar lipid bilayers were carried out as described (Tripathy et al., 1995; Xu et al., frequently entered a subconductance state (Fig. 1 A, left 1996). Preliminary experiments suggested that the rate of IpTxa- and right). In contrast to the linear current–voltage re- induced subconductance state formation depends on channel lationship of the unmodified CRC, the IpTxa-modified open probability (Po) (see Fig. 4 for a quantitative description). channel showed a slightly rectifying behavior (Fig. 1 B). Therefore, experiments were conducted under “Po-clamp” condi- The chord conductance at Ϫ40 mV holding potential tions with Po values kept in the range of 0.75–1.0. For the skeletal (of the full conductance state %43ف) CRC, in most cases 1–2 mM ATP was added to the cis bilayer was 344 Ϯ 37 pS 2ϩ of the full %28ف) ␮M free Ca to bring the Po in and that at ϩ40 mV was 221 Ϯ 17 pS 5ف chamber that also contained the required range. For the cardiac CRC, where no determina- conductance state) (n ϭ 8). Addition of up to 100 nM tion of the kinetic constants was attempted, one set of experi- IpTxa to the lumenal side (trans bilayer chamber) had (␮M 4ف) ments was conducted using cytosolic contaminant Ca2ϩ no effect (data not shown). This suggested that IpTxa (Po ϭ 0.3–0.5). In a second set, conducted under optimally acti- 2ϩ induced subconductance states by interacting with the vating conditions (10 ␮M cytosolic Ca ), the Po was in the range of 0.8–1. Channel activities were recorded and analyzed using channel at a site accessible from the cytosolic side of commercially available instruments and a software package (Axo- the skeletal CRC. Occasionally, the CRC entered into patch 1D, Digidata 1200, and “pClamp 6.0.3;” Axon Instruments, conductance levels smaller than the main subconduc- Burlingame, CA). Recordings were filtered at 2 or 4 kHz through tance state. However, because of their very infrequent an eight-pole low pass Bessel filter (Frequency Devices Inc., Hav- erhill, MA) and digitized at 10 or 20 kHz. The current values for occurrences, these were not analyzed. the full and subconductance states at different holding potentials Fig. 2 shows similar experiments carried out with the were obtained by constructing all point amplitude histograms cardiac CRC. Cytosolic addition of 15 nM IpTxa in- and subsequent Gaussian fits of data from at least 2 min of chan- duced a substate (Fig. 2 A) with a magnitude and recti- nel recording. The probability of substate occurrence (Psubstate) fying current–voltage relationship (Fig. 2 B) very simi- was calculated as time spent in substate divided by the total re- lar to that of skeletal CRC. As was the case with skeletal cording time. Another parameter CRCsubstate/CRCfull state was calculated as time spent in substate divided by the time in full con- CRC, addition of IpTxa to the lumenal side of cardiac ductance state. The durations of the bursts (when the channel CRC was without effect.

680 Imperatoxin Activator Induces Subconductance States in Ryanodine Receptors Figure 1. IpTxa induces a subconductance state in skeletal CRC. (A) Shown are four recordings from one experiment at the indicated holding potentials. Sin- gle channel currents, shown as downward or upward deflections from closed levels (C), were recorded in symmetrical 0.25 M KCl, 10 mM KHepes, pH 7.3. The cis solution also contained .␮M free Ca2ϩ and 1 mM ATP 5ف The top current traces were obtained under control conditions and the bottom traces after addition of 15 nM native IpTxa to the cis solution. (B) Mean current values of the full conductance (᭺) and IpTxa-induced subconductance (᭹) states vs. holding potential. Data points are mean Ϯ SD of eight experiments. The solid line through the full state current values is the linear regression line. The line through the substate current values was drawn by eye.

With symmetrical 250 mM KCl and lumenal 25 mM Voltage, Concentration, and P Dependence of Ca2ϩ, the current carrier through the CRC is mostly o IpTx -induced Substates Ca2ϩ at a holding potential of 0 mV. Under such re- a cording conditions, IpTxa-induced subconductance The appearance of IpTxa-induced substates was highly states were observed for both the skeletal and cardiac voltage dependent. The probability of substate occur- at a holding potential of Ϫ60 0.05ف muscle CRCs (data not shown). The substate conduc- rence (Psubstate) was ,of the control full conductance val- mV. As the holding potential was made less negative %30ف tances were ues in both cases. Thus, induction of subconductance Psubstate increased and reached a value close to 1 at posi- states in the CRCs by IpTxa does not depend upon the tive holding potentials (40–60 mV) (data not shown). current carrier species. From hereon, we shall describe The concentration dependence of IpTxa effects on experiments carried out on the skeletal CRC. Channel skeletal CRC was investigated at a holding potential of conductances and IpTxa binding constants (see below) Ϫ20 mV by adding increasing concentrations of native of the cardiac CRC are summarized and compared with IpTxa (from 6 to 100 nM) to the cis bilayer chamber. As the skeletal CRC in Table I. shown in Fig. 3, Psubstate reached a value of 0.8 and greater

681 Tripathy et al. Figure 2. IpTxa induces a subconductance state in cardiac CRC. (A) Shown are four recordings from one experiment at indicated holding potentials. Sin- gle channel currents, shown as downward or upward deflections from closed levels (C), were recorded in symmetrical 0.25 M KCl, 20 mM KHepes, pH 7.4. The cis and trans chamber solu- tions contained contaminant- -␮M). The top re 4ف) free Ca2ϩ cordings were obtained under control conditions and the bottom recordings after addition of 15 nM native IpTxa to the cis solution. (B) Mean current values of the full conductance (᭺) and IpTxa-induced subconductance (᭹) states vs. holding potential. Data points are mean Ϯ SD of three experiments. The solid line through the full state current values is the linear regression line. The line through the substate current values was drawn by eye.

as IpTxa concentration was raised to 100 nM. A Line- channel was closed, and the rate of their appearance weaver-Burk plot (Fig. 3, inset) indicated that the data increased linearly with Po. In contrast, IpTxa dissocia- could be described by Michaelis-Menten-type kinetics tion rate from the CRC was independent of Po. with a Psubstate, max close to 1 and a Km of 20 nM. Under 2ف) the same recording conditions, a lower K value m A Kinetic Scheme of IpTx and CRC Interaction, and Testing nM) was observed when experiments were carried out a Its Predictions on skeletal CRC using synthetic IpTxa (data not shown). The rate of IpTxa-induced substate formation was de- The concentration, voltage, and Po dependence of IpTxa pendent on channel open probability. Fig. 4 examines action suggested that IpTxa binds to the open channel this question quantitatively by displaying the rates of at a single site located in the voltage drop across the subconductance state appearance and disappearance channel. Following published procedures (Strecker

(see next section for the determination of IpTxa associ- and Jackson, 1989; Moczydlowski, 1992; Tinker et al., ation and dissociation rates) as a function of Po. The 1992a), we modeled the interaction of IpTxa with CRC subconductance states were rarely observed when the according to Scheme I.

682 Imperatoxin Activator Induces Subconductance States in Ryanodine Receptors fractional electrical distance of the binding site from

the reference state at the cytosolic side, ␦1 is the distance between the transition state and the reference state, and R, T, and F have their usual meanings. Channel activities of skeletal CRC were recorded in the (scheme i) presence of two different concentrations (15 and 100 nM) of native and one concentration (15 nM) of syn-

thetic IpTxa at various holding potentials. Ln (CRCsubstate/ CRC ) values from one experiment in each condi- In Scheme I, V refers to voltage across the mem- full state tion are plotted against holding potential in Fig. 5. As brane, v (V) and k (V) are the observed microscopic on on predicted, the data followed Boltzmann distribution. voltage-dependent association rate and rate constant of The mean z␦ values at 15 and 100 nM native IpTx were IpTx binding to the channel, respectively. The velocity a a 1.44 Ϯ 0.28 (n ϭ 4) and 1.42 Ϯ 0.28 (n ϭ 3), respec- of IpTx dissociation from the subconductance state, a tively. The mean z␦ from two experiments at 15 nM syn- v (V) is a unimolecular microscopic rate constant, k , off off thetic IpTx was 1.66. When all the data were pooled, and is also voltage dependent. Assuming a Boltzmann a the mean z␦ was 1.49 Ϯ 0.25 (n ϭ 9) (Table I). distribution between the full and subconductance states, The voltage dependence of k and k was investi- we can write CRC /CRC ϭ exp [(z␦FV Ϫ G )/ on off substate full state 0 gated for the skeletal CRC at 100 nM native and 15 nM RT], where CRC /CRC refers to time spent substate full state synthetic IpTx at holding potentials varying from Ϫ60 in the subconductance state divided by the time spent a to Ϫ20 mV. Dwell times corresponding to the substate in the full conductance state of the CRC. and the burst mode were calculated from single channel The explicit dependence of k and k on voltage on off records containing 30–150 events for each state. Follow- can be expressed as follows: k (V) ϭ k (0)exp(z␦ FV/ on on 1 ing the predictions of Scheme I, the distribution of dwell RT), and k (V) ϭ k (0)exp(Ϫz␦ FV/RT), where z is off off 2 times in each state followed a single exponential (Fig. 6). the valence of the blocking particle, ␦ (ϭ ␦1 ϩ ␦2) is the

Figure 4. Dependence of von and voff on the skeletal CRC open Figure 2ϩ 3. Concentration dependence of IpTxa-induced substate. probability. The Po was varied by varying free Ca in the cis bilayer Psubstate vs. native [IpTxa] for the skeletal CRC. Channel activities chamber from Ͻ0.1 to 20 ␮M and in some cases adding 1–2 mM were recorded as in Fig. 1 A at a holding potential of Ϫ20 mV. In- ATP. 20 nM synthetic IpTxa was added to the cis bilayer chamber creasing concentrations (6–100 nM) of native IpTxa were added to and channel activities were recorded at a holding potential of Ϫ40 the cis chamber solution. Psubstate was calculated at each [IpTxa]. mV. The data are from three experiments. von (᭺) and voff (᭹) Data points are mean Ϯ SD of five experiments. A Lineweaver- were obtained from exponential fits of dwell-times in the burst and Burk plot of the mean data is shown in the inset, giving a Km of 20 subconductance state (see Fig. 6, legend). The coefficients of de- nM and a Psubstate, max of 0.91. Linear regression line drawn through termination of the linear regression lines through the von and voff the data points had a coefficient of determination of 0.97. data points were 0.98 and 0.17, respectively.

683 Tripathy et al. table i

Channel Conductances and IpTxa Binding Constants of RyR1 and RyR2 RyR1 RyR2

ϪIpTxa ϩIpTxa ϪIpTxa ϩIpTxa

gϪ40 (pS) 795 Ϯ 26(8) 344 Ϯ 37(8) 791 Ϯ 11(3) 298*

gϩ40(pS) 795 Ϯ 26(8) 221 Ϯ 26(8) 791 Ϯ 11(3) 187* ‡ § Km (nM) — 20(5) — 6.7(1) ‡ Kd (nM) — 18.2(4) —ND z␦ 1.49 Ϯ 0.25(9)ʈ¶ 1.32 Ϯ 0.06(3)‡¶ z␦ 1.54 Ϯ 0.21(4)ʈ** ND ʈ z␦1 0.48 Ϯ 0.04(4) ** ND ʈ z␦2 1.06 Ϯ 0.21(4) ** ND The data are mean Ϯ SD of the number of experiments given in parentheses. *Estimated values at Ϯ40 mV from the mean currents vs. voltage curve ‡ § (Fig. 2 B). and Obtained with native and synthetic IpTxa, respectively.

The Km and Kd values of native IpTxa binding to skeletal CRC refer to val-

ues in 250 mM KCI medium and Ϫ20 mV holding potential. The Km of

synthetic IpTxa binding to the cardiac CRC was obtained in 250 mM KCI medium and at Ϫ25 mV holding potential. ʈMean values of experiments ¶ carried out with either native or synthetic IpTxa. Obtained from Boltzmann

fit of CRCsubstate/CRCfull state vs. holding potential curves. **Obtained from dwell-time analysis of bursts and substates. Figure 5. Plots of the natural logarithms of CRCsubstate/CRCfull state vs. holding potential for the skeletal CRC. Channel activities were recorded as in Fig. 1 A at holding potentials of Ϫ60 to ϩ60 mV. tive and two at 15 nM synthetic IpTx ) were 0.48 Ϯ 0.04 IpTxa concentrations in the cis chamber solution were: 15 nM na- a tive (᭹), 100 nM native (᭺), and 15 nM synthetic (᭡). The coeffi- and 1.06 Ϯ 0.21, respectively, with a mean z␦ of 1.54 Ϯ cients of determination and z␦ values, respectively, were as follows: 0.21. This latter value is almost identical to the mean z␦ 15 nM native, 0.97 and 1.65; 100 nM native, 0.98 and 1.25; and 15 value of 1.49 Ϯ 0.25, which was obtained from the plots nM synthetic, 0.99 and 1.47. of the natural logarithms of CRCsubstate/CRCfull state vs. holding potential (Fig. 5 and Table I). The data further show that the unbinding rate is twice as voltage depen-

Plots of the natural logarithms of kon and koff (ob- dent as the binding rate. The mean kon increased e-fold tained from exponential fits of dwell-time data) from per ϩ53.3 (Ϯ5.6 mV, n ϭ 4) of applied holding poten- four experiments against the holding potential fol- tial. The mean koff and mean Kd decreased e-fold per lowed Boltzmann distribution (not shown). The mean ϩ24.7 (Ϯ4.9 mV, n ϭ 4) and ϩ17.4 (Ϯ2.3 mV, n ϭ 4) z␦1 and z␦2 from four experiments (two at 100 nM na- of applied holding potential, respectively.

Figure 6. Single exponential fits of dwell-time data. Data from an experiment with skeletal CRC at a holding potential of Ϫ20 mV and 100 nM cytosolic native IpTxa. (A) Exponential fit of dwell-times in the burst state. Data were sorted into histograms of 100-ms bin width in the cumu- lative binning mode and fitted to the probability distribution function F(t) ϭ A(1 Ϫ exp[Ϫt/␶]) (solid line), where total number of events (A) was 130 and the time-constant ␶ was 965 ms. (B) Exponential fit of dwell-times in the subconductance state. Data were sorted and fitted (solid line) as in A. The time-constant ␶ was 3,376 ms.

684 Imperatoxin Activator Induces Subconductance States in Ryanodine Receptors 3 Next, we tested the predictions pertaining to the IpTxa effects of IpTxa in [ H]ryanodine binding experiments concentration dependence of von and voff (Fig. 7). Skel- using skeletal and cardiac SR vesicles. Fig. 8 shows the etal CRC channel activities were recorded at a holding Ca2ϩ activation profiles of CRCs as measured by [3H]ry- potential of Ϫ20 mV at five different native IpTxa con- anodine binding to skeletal and cardiac SR vesicles in centrations varying from 6 to 100 nM. The regression the absence and presence of 30 nM synthetic IpTxa. As ob- lines drawn through the mean von and voff data points served previously (Valdivia et al., 1992; El-Hayek et al., 3 have coefficients of determination of 0.99 and 0.02, re- 1995; Zamudio et al., 1997a), IpTxa increased [ H]ryano- spectively, thus supporting the prediction that von is lin- dine binding to skeletal SR vesicles. Activation occurred 2ϩ early dependent on IpTxa concentration and voff is in- at all tested Ca concentrations with the possible ex- 3 dependent of it. From the data, a kon (von/[IpTxa]) ception of 10 mM, where [ H]ryanodine binding levels 7 Ϫ1 Ϫ1 Ϫ1 value of 1.7 ϫ 10 M s and a koff value of 0.31 s were close to background. In contrast to its effect on 3 were obtained, which gave a Kd (koff/kon) of 18.2 nM. skeletal SR vesicles, IpTxa decreased [ H]ryanodine This Kd value is very close to the Km value of 20 nM that binding to the cardiac SR vesicles up to twofold at a was obtained from the Michaelis-Menten analysis of Ca2ϩ concentration of 10 ␮M–1 mM. At low (Ͻ1 ␮M) 2ϩ Psubstate vs. native [IpTxa] curve under identical record- and high (Ͼ1 mM) Ca concentration, it was without ing conditions (Ϫ20 mV holding potential and 250 mM a noticeable effect. KCl medium) (Fig. 3 and Table I). [3H]ryanodine binding measurements have been and are extensively used as indicators of the number of 3 IpTxa Modifies [ H]Ryanodine Binding to Skeletal and channels and their activities (Meissner, 1994). We did Cardiac SR Vesicles not observe an increase in Po of the skeletal CRC nor a decrease in P of cardiac CRC in bilayer experiments af- Previous studies had suggested that IpTxa preferentially o binds to and activates the skeletal CRC and that cardiac ter addition of IpTxa (data not shown). Therefore, we were surprised to find that IpTx affected the [3H]ryan- CRC is a poor target of IpTxa (Valdivia et al., 1992; El- a Hayek et al., 1995; Zamudio et al., 1997a). Since we ob- odine binding to skeletal and cardiac CRCs so differ- ently. To gain further insights, we performed Scatchard served that IpTxa induces subconductance states in both the skeletal and cardiac CRCs, we reexamined the analysis of [3H]ryanodine binding to both CRC isoforms in the presence and absence of IpTxa (data not shown). IpTxa (30 nM, synthetic) decreased the Kd of [3H]ryanodine binding to skeletal SR vesicles approximately threefold (Table II). A small increase in maxi-

Figure 7. Dependence of von and voff on [IpTxa]. Increasing con- Figure 3 centrations (6–100 nM) of native IpTxa were added to the cis solu- 8. Effect of IpTxa on [ H]ryanodine binding to SR vesi- tion and channel activities were recorded at Ϫ20 mV. The data are cles. Specific [3H]ryanodine binding to rabbit skeletal (᭹, ᭿) or mean Ϯ SD of three to four experiments except at 100 nM IpTxa, canine cardiac (᭺, ᮀ) SR vesicles in the absence (᭹, ᭺) or pres- which is mean of two experiments. The coefficients of determina- ence (᭿, ᮀ) of 30 nM synthetic IpTxa was determined as described materials and methods tion of the linear regression lines through the mean von (᭺) and in . The data are from one of three simi- voff (᭹) data points were 0.99 and 0.02, respectively. lar experiments with skeletal and cardiac SR vesicles.

685 Tripathy et al. table ii at 100 ␮M free Ca2ϩ. The cardiac CRC is nearly fully ac- [3H]Ryanodine Binding to Skeletal and Cardiac SR Vesicles tive at this [Ca2ϩ] (Xu et al., 1996), whereas in the ab- ␮ 2ϩ Bmax (pmol/mg protein) Kd (nM) sence of other activating ligands, 100 M Ca cannot Composition of fully activate the skeletal CRC. To test if the different assay media ϪIpTxa ϩIpTxa ϪIpTxa ϩIpTxa effects of IpTxa on skeletal and cardiac SR vesicles were RyR1 100 ␮M Ca2ϩ 13.5 Ϯ 0.9 17.5 Ϯ 2.1* 16.6 Ϯ 6.0 6.1 Ϯ 2.7* due to the different activation levels of CRCs, we per- ϩ 100 ␮M Ca2 ϩ formed Scatchard analysis of [3H]ryanodine binding 5 mM AMP PCP 17.5 Ϯ 2.1 16.2 Ϯ 0.9 3.5 Ϯ 1.4 4.7 Ϯ 1.5* using maximally activating conditions (100 ␮M Ca2ϩ RyR2 100 ␮M Ca2ϩ 3.5 Ϯ 0.1 3.9 Ϯ 0.6 2.4 Ϯ 0.8 8.4 Ϯ 0.7* and 5 mM AMP-PCP) for both CRC isoforms. Under 100 ␮M Ca2ϩ ϩ 3 5 mM AMP PCP 3.3 Ϯ 0.4 3.6 Ϯ 0.2 1.8 Ϯ 0.2 8.1 Ϯ 1.0* these conditions, IpTxa affected the [ H]ryanodine binding to the skeletal and cardiac SR vesicles in a simi- The experiments were carried out using 30 nM synthetic IpTxa. The data 3 are mean Ϯ SD of three experiments. *Significantly different from con- lar manner. The Kd of [ H]ryanodine binding to the fold and that for-1.3ف trol conditions (ϪIpTxa). skeletal SR vesicles increased fold without a change in-4.5ف the cardiac SR vesicles the Bmax values in both cases (Table II). IpTx Induces Substates in Ryanodine-modified Skeletal mum binding (B ) (from 13.5 to 17.5 pmol/mg) was a max Muscle CRC also observed after IpTxa treatment. In contrast, 30 nM 3 IpTxa increased the Kd of [ H]ryanodine binding to In Fig. 9, we explored the cointeraction of IpTxa and fold without an appreciable effect on ryanodine with the skeletal CRC at the single channel-3.5ف cardiac SR the Bmax value. The above experiments were carried out level. After addition of 100 nM IpTxa to the cis bilayer

Figure 9. Effects of IpTxa and ryanodine on skeletal CRC. (A) IpTxa-induced subconductance states in the absence and presence of ryanodine. Shown are two current traces recorded at Ϫ40 mV. Single channel currents, shown as downward deflections from closed levels, were recorded in media as given in Fig. 1 A, legend, but with 100 nM cytosolic native IpTxa. The top current trace was obtained before and the bottom trace after addition of 5 ␮M ryanodine to the cis solution. The current levels of the different states are indicated. (B) Current–voltage curves of skeletal CRC in presence of both IpTxa and ryanodine. Chan- nels were recorded as in A. The ryanodine- induced substate currents (᭺) and IpTxa-induced substate currents in the presence of ryanodine (᭹) are shown. The data are mean Ϯ SD of three experiments. The dashed and dotted lines are the control and IpTxa-induced substate current– voltage curves, respectively, in the absence of ryanodine, and are same as in Fig. 1 B.

686 Imperatoxin Activator Induces Subconductance States in Ryanodine Receptors From .(1.3ف chamber, the channel current fluctuated between the etal CRC (the value for cardiac CRC was full conductance and IpTxa-induced subconductance the known amino acid sequence of IpTxa (Zamudio et states (Fig. 9 A, top). After further addition of 5 ␮M ry- al., 1997a) and using the program “Isoelectric” of the anodine to the cis chamber, the channel current fluctu- GCG suite (Genetics Computer Group, Inc., Madison, ated between two new levels (Fig. 9 A, bottom). The cur- WI), we calculated a net charge of 6.5 at pH 7.3. Divid- rent–voltage relationships in presence of both IpTxa ing the effective valence by this number gives an electri- -from the cytosolic side for the lo 0.23ف and ryanodine are shown in Fig. 9 B. The higher sub- cal distance of conductance state had linear current–voltage charac- cation of IpTxa binding site. This is based on the as- teristics and was identified as the ryanodine-modified sumption that all the charges of IpTxa enter the CRC subconductance state of the CRC (Lai et al., 1989). The conduction pathway. The unbinding reaction of IpTxa less conducting IpTxa-induced substate of the ryano- from the CRC has a twofold higher voltage depen- dine-modified CRC had a rectifying current–voltage dence than the binding reaction. Higher voltage de- characteristic. For comparison, the control and the IpTxa- pendence for the unbinding rate has also been ob- induced substate current–voltage curves (in the absence served in case of tetrabutyl ammonium (TBAϩ)–induced of ryanodine) from Fig. 1 B are redrawn as a dashed and substate in the cardiac CRC (Tinker et al., 1992a) and dotted line, respectively, in Fig. 9 B. The new substate curare-induced subconductance state in the acetylcho- conductance in the presence of both IpTxa and ryano- line receptor channel (Strecker and Jackson, 1989). dine was significantly smaller than the IpTxa-induced IpTxa is a relatively small peptide of 3,765 D with substate (in the absence of ryanodine) at all applied three pairs of cysteine residues that would stabilize the holding potentials. three-dimensional (3-D) conformation and help it as- sume a compact globular form by forming disulfide bridges (Zamudio et al., 1997a). Preliminary modeling discussion work suggests that IpTxa structure can be roughly ap- nm in diameter. The 2.5ف The present studies explored the effects of IpTxa on proximated as a sphere of purified CRCs in single channel experiments, and on selectivity filter of the skeletal CRC has a diameter of nm, as cholineϩ, Trisϩ, and glucose can permeate 0.7ف SR vesicles in [3H]ryanodine binding experiments. In these studies, we provide the first evidence that IpTxa through the channel (Meissner, 1986; Smith et al., 1988), directly interacts with both skeletal and cardiac CRCs while sucrose cannot (G. Meissner, unpublished obser- to induce voltage- and concentration-dependent sub- vations). Therefore, IpTxa cannot enter the selectivity conductance states in both isoforms by binding at a sin- filter of the CRC. On the other hand, studies with large gle site, located in the voltage drop across the channel. tetraalkyl ammonium ions (Tinker et al., 1992a) and 3 Second, we show that IpTxa affects the [ H]ryanodine charged local anesthetics (Tinker and Williams, 1993) binding characteristics of the two isoforms depending have suggested that the sheep cardiac CRC has a rela- on the level of channel activation. tively large vestibule facing the cytosolic side. Theoreti- cal considerations have suggested that a significant pro- Interaction of IpTxa with CRCs as Revealed by portion of the voltage drop may fall over the wide vesti- Single-Channel Experiments bule of a channel (Jordan, 1986). Our studies showed that the vestibule has to be at least 2–2.5-nm wide to ac- We modeled the interaction of IpTxa with the CRC as of the voltage %23ف outlined in Scheme I. Briefly, we envisaged that when commodate IpTxa, and at least falls over the vestibule. Interestingly, 3-D cryo-electron the channel opened, an IpTxa molecule entered the conductance pathway from the cytosolic side to bind at micrographic reconstruction of the skeletal muscle an internal site. The model presented us with some CRC (Radermacher et al., 1994; Serysheva et al., 1995) nm in the cytoplasmic 5ف testable predictions that were all satisfied. The distribu- shows a central opening of tion of dwell-times in each state (burst and subconduc- domain (facing the transverse tubule), and thus sup- tance) followed a single exponential. The reciprocal ports the presence of a wide vestibule facing the cyto- mean dwell-time in the burst state was linearly depen- plasmic side of the CRC. dent on, and the reciprocal mean dwell-time in the sub- Mechanism of Subconductance State Formation conductance state was independent of, IpTxa concentration. A Kd value of IpTxa binding to skeletal CRC We considered it unlikely that IpTxa would produce could be calculated from the dwell-time analysis, and it subconductance states by a surface charge mechanism was very close to that obtained from the Michaelis- (Green and Andersen, 1991). The estimated decrease Menten-type concentration-dependence curve under in local [Kϩ] using the Guoy-Chapman double-layer identical conditions. theory (McLaughlin, 1977) was only approximately The effective valence of the reaction leading to sub- fourfold (assuming zero net surface charge near the cy- for the skel- tosolic face of the CRC and the IpTxa net positive 1.5ف state formation was determined to be

687 Tripathy et al. nm di- drotoxin-I (DTX-I)-induced substate formation in a-2.5ف charge of 6.5 spread out over a sphere of ameter). This decrease (from 250 to 62.5 mM) was not maxi Ca2ϩ-activated Kϩ channel, a conformational sufficient to cause the subconductance state as the Kd change mechanism was also favored as the toxin converted the linear control current–voltage relationship 20ف of Kϩ for the cardiac CRC has been shown to be mM (Tinker et al., 1992b). However, if the positive to a rectifying one, though in this case von and voff were charges of IpTxa are clustered in a much smaller area linearly dependent on and independent of the toxin (leading to a higher charge density), the local [Kϩ] concentration (Lucchesi and Moczydlowski, 1990). In could be much lower, which may explain the formation the present case, IpTxa binding converted an ohmic of subconductance state. The above possibility is also CRC to a rectifying one. Furthermore, IpTxa-modified not very likely as the ratios of subconductance to full CRCs (both skeletal and cardiac) had higher Kds of conductance values were similar, and there was no indi- [3H]ryanodine binding under fully activating condi- cation of any relief of block, when the [KCl] in the bi- tions (conditions similar to bilayer recording condi- layer chambers was increased symmetrically from 250 tions) (see below for further discussion on this aspect). to 500 mM (data not shown). However, we observed an These observations favor an IpTxa-induced conforma- increase in the Kd of IpTxa binding to CRC when [KCl] tional change in both the skeletal and cardiac CRCs as was raised from 250 to 500 mM (our unpublished stud- the mechanism causing the subconductance state. Al- ies). Similar effects of increasing [KCl] on Kd have ternatively, resultant conformational changes by the been observed by other investigators studying other membrane electric field may cause IpTxa to differen- toxins (Anderson et al., 1988; Lucchesi and Moczyd- tially occlude the CRC at positive and negative holding lowski, 1990). potentials to produce substates of different conduc- In our experiments with the purified CRCs in bilayer tances. Regardless of whether partial occlusion of the experiments, substates of different amplitudes are some- pore or a conformational change is the mechanism times observed even in control recordings. These sub- causing subconductance state formation, our studies states show linear current–voltage relationships. There- strongly suggest the existence of a cytosolically accessi- fore, it is conceivable that IpTxa preferentially binds to ble IpTxa binding site in the conductance pathway of and stabilizes a normally occurring CRC substate. How- both the skeletal and cardiac CRC. ever, we think this to be an unlikely mechanism as the

IpTxa-induced substates had different conductances at Difference in Potency Between Native and Synthetic IpTxa positive and negative holding potentials (Figs. 1 and 2, Most of our experiments were conducted using native, and Table I). purified IpTx . In some experiments carried out using How then does IpTx reduce channel conductance? a a synthetic IpTx , we observed that the synthetic toxin Is it by partial occlusion of the channel pore or by a a fold more effective in inducing substates in-10ف was conformational change? Tetrabutyl and tetrapentyl am- skeletal and cardiac CRCs than its native counterpart. monium–induced substates in the cardiac CRC have The reasons for this difference are not clear. One possi- been explained in terms of partial occlusion of the con- bility is that during purification the native toxin lost duction pathway (Tinker et al., 1992a). Convincing ar- part of its biological activity. guments have been put forward in favor of partial block of acetylcholine receptor channel by curare (Strecker Cointeraction of IpTx and Ryanodine with CRCs and Jackson, 1989) and complete block of a maxi Ca2ϩ- a activated Kϩ channel by charybdotoxin (Anderson et [3H]ryanodine binding correlates reasonably well with al., 1988; MacKinnon and Miller, 1988). In the former CRC activation levels. Because of its simplicity and con- case, displacing curare from the agonist binding sites of venience, it is extensively used to explore the effects of acetylcholine receptor channel had no effect on the ki- many drugs/toxins on CRC. But as this study shows, di- netics of subconductance state occurrence. In the latter rect measurements of channel activity can provide in- case, enhancement of external toxin dissociation by insights that might not be detected by [3H]ryanodine ϩ ternal K and competition for the toxin-binding site by binding measurements. When the effects of IpTxa were external tetraethylammonium ions gave strong support assessed at 100 ␮M free Ca2ϩ, an increase in [3H]ryano- to the occlusion model. On the other hand, Zn2ϩ-induced dine binding level for the skeletal but a decrease for substates in canine cardiac sarcolemma sodium chan- the cardiac SR vesicles was observed (Fig. 8), though nel (Schild et al., 1991) and proton and deuterium ion- the net effect is the formation of subconductance states induced substates in a voltage-dependent Ca2ϩ channel in both isoforms in bilayer experiments. Furthermore, (Prod’hom et al., 1987; Pietrobon et al., 1989) have using purified skeletal CRC, we demonstrated that IpTxa been explained in terms of conformational changes; as can still bind and induce substates of slightly smaller in the above two cases, voff was dependent on the conductances in the ryanodine-modified channel (Fig. blocker concentration. Furthermore, in case of den- 9, A and B). Similar paradoxical results were observed

688 Imperatoxin Activator Induces Subconductance States in Ryanodine Receptors in studies exploring the effects of polyamines on the tive at 100 ␮M Ca2ϩ, the substates provided no further CRCs. Polyamines increased [3H]ryanodine binding to improvement. On the contrary a decrease was ob- skeletal SR vesicles approximately fivefold (Zarka and served, probably because the IpTxa-modified channel Shoshan-Barmatz, 1992). However, in single-channel may have reduced affinity for ryanodine. In favor of studies, it caused a blockade of the cardiac CRC, and this idea, we observed that under maximally activating 2ϩ no increase in Po was observed (Uehara et al., 1996). conditions for both CRC isoforms (100 ␮M Ca and 5 3 3 Though [ H]ryanodine binding and single channel ex- mM AMP-PCP), IpTxa decreased the Kds of [ H]ryano- periments were done on different CRC isoforms, the dine binding to both skeletal and cardiac SR vesicles 3 above experiments and the present study call for cau- (Table II). Furthermore, IpTxa increased [ H]ryano- tion in interpreting [3H]ryanodine binding results in dine binding to cardiac SR vesicles under minimally ac- ␮M Ca2ϩ) just as it increased 1ف) the presence of other drugs and toxins. tivating conditions 3 The apparently paradoxical effects of IpTxa on skele- [ H]ryanodine binding to skeletal SR vesicles under tal and cardiac SR vesicles in [3H]ryanodine binding suboptimally activating conditions (100 ␮M Ca2ϩ) (our measurements led us to investigate its effects in detail. unpublished studies). Thus, our data show that the IpTxa- 2ϩ At 100 ␮M Ca , IpTxa caused a decrease in Kd (with a modified CRC (both skeletal and cardiac) has a Kd of 3 small increase in Bmax) for the skeletal SR vesicles, but [ H]ryanodine binding intermediate between those of an increase in Kd with little or no change in Bmax for the the minimally and maximally activated channel. cardiac SR vesicles (Table II). The affinity of [3H]ryan- In conclusion, the present work demonstrates a di- odine binding to SR vesicles correlates with the CRC ac- rect, high affinity interaction of IpTxa with both the tivation level, suggesting that ryanodine binds to an skeletal and cardiac CRCs. The use of IpTxa should aid open channel (Meissner, 1994). The IpTxa-induced in the structural elucidation of the ion-conductance long-lived substates in the skeletal CRC would thus pro- pathway of the skeletal and cardiac muscle CRCs. Fur- vide a favorable environment over the rapidly gating thermore, we show that experiments exploring effects control low channel activity (at 100 ␮M Ca2ϩ) for ryan- of drugs/toxins on CRCs by [3H]ryanodine binding odine entry into the CRC interior and subsequent measurements must be interpreted with caution and be binding. For the cardiac CRC, which is maximally ac- correlated with measurements of channel activity.

The authors express their appreciation to Dan Pasek for carrying out the [3H]ryanodine binding experiments and Ling Gao for help with the GCG program.

This work was supported by National Institutes of Health (NIH) grant HL-55438 and Grant-in-Aid from the American Heart As- sociation (to H.H. Valdivia) and NIH grants AR-18687 and HL-27430 (to G. Meissner). Original version received 3 September 1997 and accepted version received 10 March 1998. references

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690 Imperatoxin Activator Induces Subconductance States in Ryanodine Receptors