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
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ABSTRACT |
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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 Ca2+ 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. The chord conductance at 40 mV was ~43% of the full conductance, whereas it was ~28% at a holding potential of +40 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 +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ~1.5. The IpTxa binding site was calculated to be located at ~23% of the voltage drop from the cytosolic side. IpTxa induced substates in
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 Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.
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INTRODUCTION |
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Ca2+ release channels (CRCs),1 also known as ryanodine receptors (RyRs), mediate the release of Ca2+
from an intracellular membrane compartment, the sarcoplasmic reticulum (SR), into the cytoplasm in striated muscle cells. The CRC has been purified as a 30 S
protein complex and shown to be composed of four
large RyR polypeptides of ~5,000 amino acid residues and four immunophilins (FK506 binding protein) of
~100 amino acid residues each. Three tissue-specific
isoforms of RyR have been identified. The conductances and pharmacological properties of the three isoforms are, to a large extent, similar (for reviews, see
Meissner, 1994; Sutko and Airey, 1996
).
Traditionally, venom from many poisonous snakes,
lizards, and scorpions has been a rich source of peptide
toxins that target various voltage- and ligand-gated
channels including the CRCs (Furukawa et al., 1994;
Morrissette et al., 1995
, 1996
). Recently, from the
venom of the African scorpion Pandinus imperator, two factors, imperatoxin activator (IpTxa) and imperatoxin
inhibitor (IpTxi), were isolated that selectively activated
and inhibited, respectively, the CRCs (Valdivia et al.,
1992
; El-Hayek et al., 1995
). IpTxa is a 33 amino acid
peptide that activated the skeletal CRC in [3H]ryanodine binding and single channel studies. It did not exert a significant effect on cardiac CRC (Valdivia et al.,
1992
; El-Hayek et al., 1995
; Zamudio et al., 1997a
).
IpTxi has been shown to be a heterodimeric protein
(subunits of 108 and 27 amino acid residues covalently
linked by a disulfide bond) with lipolytic action, and it
inhibited both skeletal and cardiac CRCs by generating a lipid product (Valdivia et al., 1992
; Zamudio et al.,
1997b
).
Here, we show that IpTxa induces voltage- and concentration-dependent subconductance states in both skeletal and cardiac CRCs when added to the cytosolic side. Analysis of voltage and concentration dependence and stochastic analysis of the events suggests that the induction of subconductance states corresponds to reversible, voltage-dependent binding and unbinding of IpTxa at a single, cytosolically accessible site located in the voltage drop across the channel. IpTxa also induced a subconductance state in the ryanodine-modified skeletal muscle CRC and affected [3H]ryanodine binding to skeletal and cardiac SR vesicles depending on the level of channel activation. These results provide evidence for the binding of IpTxa to a channel site different from that of ryanodine.
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MATERIALS AND METHODS |
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Phospholipids were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). All other chemicals were of analytical grade.
Preparation of SR Vesicles, and Purification and Reconstitution of Ca2+ Release Channels
SR vesicle fractions enriched in [3H]ryanodine binding and Ca2+
release channel activities were prepared in the presence of protease inhibitors from rabbit skeletal and canine cardiac muscle as
described (Meissner, 1984; Meissner and Henderson, 1987
). The
CHAPS (3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate)-solubilized skeletal or cardiac muscle 30 S Ca2+ release
channel complex was purified and reconstituted into proteoliposomes by removal of CHAPS by dialysis (Lee et al., 1994
).
Purification and Synthesis of IpTxa
IpTxa was purified from P. imperator scorpion venom in three
chromatographic steps as described (Valdivia et al., 1992; El-Hayek et al., 1995
; Zamudio et al., 1997a
). Synthetic IpTxa was
prepared as described (Zamudio et al., 1997a
).
Single Channel Measurements and Analysis
Single channel recordings of purified rabbit skeletal muscle or
canine cardiac muscle CRCs incorporated into planar lipid bilayers were carried out as described (Tripathy et al., 1995; Xu et al.,
1996
). Preliminary experiments suggested that the rate of IpTxa-induced subconductance state formation depends on channel open probability (Po) (see Fig. 4 for a quantitative description). Therefore, experiments were conducted under "Po-clamp" conditions with Po values kept in the range of 0.75-1.0. For the skeletal CRC, in most cases 1-2 mM ATP was added to the cis bilayer
chamber that also contained ~5 µM free Ca2+ to bring the Po in
the required range. For the cardiac CRC, where no determination of the kinetic constants was attempted, one set of experiments was conducted using cytosolic contaminant Ca2+ (~4 µM)
(Po = 0.3-0.5). In a second set, conducted under optimally activating conditions (10 µM cytosolic Ca2+), the Po was in the range
of 0.8-1. Channel activities were recorded and analyzed using
commercially available instruments and a software package (Axopatch 1D, Digidata 1200, and "pClamp 6.0.3;" Axon Instruments,
Burlingame, CA). Recordings were filtered at 2 or 4 kHz through
an eight-pole low pass Bessel filter (Frequency Devices Inc., Haverhill, MA) and digitized at 10 or 20 kHz. The current values for
the full and subconductance states at different holding potentials
were obtained by constructing all point amplitude histograms
and subsequent Gaussian fits of data from at least 2 min of channel recording. The probability of substate occurrence (Psubstate)
was calculated as time spent in substate divided by the total recording time. Another parameter CRCsubstate/CRCfull state was calculated as time spent in substate divided by the time in full conductance state. The durations of the bursts (when the channel
was in normal gating mode between the closed and full conductance levels) and subconductance states were obtained by manual positioning of the cursors.
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[3H]Ryanodine Binding
[3H]Ryanodine binding experiments were carried out with rabbit
skeletal and canine SR vesicles (protein: 150-250 µg/ml) in 0.25 M KCl, 20 mM imidazole, pH 7.0, 0.2 mM Pefabloc, 20 µM leupeptin, 0.1 mM EGTA, and [CaCl2] necessary to set the free
[Ca2+] in the range of 0.1 µM to 10 mM. After incubation for 24 h
at 24°C, bound radioactivity was determined by a filter assay as described (Tripathy et al., 1995). Nonspecific binding was determined with a 1,000-fold excess of unlabeled ryanodine.
Other Data Analysis and Conventions
Results are given as means ± SD with the number of experiments in parentheses. The SD is included within the figure symbol or indicated by error bars if it is larger. Significance of differences was analyzed with Student's paired t test. Differences were considered to be significant when P < 0.05.
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RESULTS |
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IpTxa Induces Subconductance States in Skeletal and Cardiac Muscle CRCs
The skeletal and cardiac CRCs have been shown to conduct monovalent ions more efficiently than Ca2+ and to
be essentially impermeant to Cl. In 250 mM KCl solution, the single channel conductances of both isoforms
are ~770 pS (Meissner, 1994
; and this study). When 15 nM native IpTxa was added to the cytosolic side (cis bilayer chamber) of a single skeletal CRC, the channel
frequently entered a subconductance state (Fig. 1 A, left
and right). In contrast to the linear current-voltage relationship of the unmodified CRC, the IpTxa-modified
channel showed a slightly rectifying behavior (Fig. 1 B).
The chord conductance at
40 mV holding potential
was 344 ± 37 pS (~43% of the full conductance state)
and that at +40 mV was 221 ± 17 pS (~28% of the full
conductance state) (n = 8). Addition of up to 100 nM IpTxa to the lumenal side (trans bilayer chamber) had
no effect (data not shown). This suggested that IpTxa
induced subconductance states by interacting with the
channel at a site accessible from the cytosolic side of
the skeletal CRC. Occasionally, the CRC entered into
conductance levels smaller than the main subconductance state. However, because of their very infrequent
occurrences, these were not analyzed.
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Fig. 2 shows similar experiments carried out with the cardiac CRC. Cytosolic addition of 15 nM IpTxa induced a substate (Fig. 2 A) with a magnitude and rectifying current-voltage relationship (Fig. 2 B) very similar to that of skeletal CRC. As was the case with skeletal CRC, addition of IpTxa to the lumenal side of cardiac CRC was without effect.
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With symmetrical 250 mM KCl and lumenal 25 mM Ca2+, the current carrier through the CRC is mostly Ca2+ at a holding potential of 0 mV. Under such recording conditions, IpTxa-induced subconductance states were observed for both the skeletal and cardiac muscle CRCs (data not shown). The substate conductances were ~30% of the control full conductance values in both cases. Thus, induction of subconductance states in the CRCs by IpTxa does not depend upon the current carrier species. From hereon, we shall describe experiments carried out on the skeletal CRC. Channel conductances and IpTxa binding constants (see below) of the cardiac CRC are summarized and compared with the skeletal CRC in Table I.
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Voltage, Concentration, and Po Dependence of IpTxa-induced Substates
The appearance of IpTxa-induced substates was highly
voltage dependent. The probability of substate occurrence (Psubstate) was ~0.05 at a holding potential of 60
mV. As the holding potential was made less negative,
Psubstate increased and reached a value close to 1 at positive holding potentials (40-60 mV) (data not shown).
The concentration dependence of IpTxa effects on
skeletal CRC was investigated at a holding potential of
20 mV by adding increasing concentrations of native
IpTxa (from 6 to 100 nM) to the cis bilayer chamber. As
shown in Fig. 3, Psubstate reached a value of 0.8 and greater
as IpTxa concentration was raised to 100 nM. A Lineweaver-Burk plot (Fig. 3, inset) indicated that the data
could be described by Michaelis-Menten-type kinetics with a Psubstate, max close to 1 and a Km of 20 nM. Under
the same recording conditions, a lower Km value (~2
nM) was observed when experiments were carried out
on skeletal CRC using synthetic IpTxa (data not shown).
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The rate of IpTxa-induced substate formation was dependent on channel open probability. Fig. 4 examines this question quantitatively by displaying the rates of subconductance state appearance and disappearance (see next section for the determination of IpTxa association and dissociation rates) as a function of Po. The subconductance states were rarely observed when the channel was closed, and the rate of their appearance increased linearly with Po. In contrast, IpTxa dissociation rate from the CRC was independent of Po.
A Kinetic Scheme of IpTxa and CRC Interaction, and Testing Its Predictions
The concentration, voltage, and Po dependence of IpTxa
action suggested that IpTxa binds to the open channel
at a single site located in the voltage drop across the
channel. Following published procedures (Strecker
and Jackson, 1989; Moczydlowski, 1992
; Tinker et al.,
1992a
), we modeled the interaction of IpTxa with CRC
according to Scheme I.
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In Scheme I, V refers to voltage across the membrane, von (V) and kon (V) are the observed microscopic
voltage-dependent association rate and rate constant of
IpTxa binding to the channel, respectively. The velocity
of IpTxa dissociation from the subconductance state, voff (V) is a unimolecular microscopic rate constant, koff,
and is also voltage dependent. Assuming a Boltzmann
distribution between the full and subconductance states,
we can write CRCsubstate/CRCfull state = exp [(zFV
G0)/
RT], where CRCsubstate/CRCfull state refers to time spent
in the subconductance state divided by the time spent
in the full conductance state of the CRC.
The explicit dependence of kon and koff on voltage
can be expressed as follows: kon(V) = kon(0)exp(z1FV/
RT), and koff(V) = koff(0)exp(
z
2FV/RT), where z is
the valence of the blocking particle,
(=
1 +
2) is the
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
presence of two different concentrations (15 and 100 nM) of native and one concentration (15 nM) of synthetic IpTxa at various holding potentials. Ln (CRCsubstate/
CRCfull state) values from one experiment in each condition are plotted against holding potential in Fig. 5. As
predicted, the data followed Boltzmann distribution.
The mean z values at 15 and 100 nM native IpTxa were
1.44 ± 0.28 (n = 4) and 1.42 ± 0.28 (n = 3), respectively. The mean z
from two experiments at 15 nM synthetic IpTxa was 1.66. When all the data were pooled,
the mean z
was 1.49 ± 0.25 (n = 9) (Table I).
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The voltage dependence of kon and koff was investigated for the skeletal CRC at 100 nM native and 15 nM
synthetic IpTxa at holding potentials varying from 60
to
20 mV. Dwell times corresponding to the substate
and the burst mode were calculated from single channel
records containing 30-150 events for each state. Following the predictions of Scheme I, the distribution of dwell
times in each state followed a single exponential (Fig. 6).
Plots of the natural logarithms of kon and koff (obtained from exponential fits of dwell-time data) from
four experiments against the holding potential followed Boltzmann distribution (not shown). The mean
z1 and z
2 from four experiments (two at 100 nM native and two at 15 nM synthetic IpTxa) were 0.48 ± 0.04 and 1.06 ± 0.21, respectively, with a mean z
of 1.54 ± 0.21. This latter value is almost identical to the mean z
value of 1.49 ± 0.25, which was obtained from the plots
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 dependent as the binding rate. The mean kon increased e-fold
per +53.3 (±5.6 mV, n = 4) of applied holding potential. The mean koff and mean Kd decreased e-fold per
+24.7 (±4.9 mV, n = 4) and +17.4 (±2.3 mV, n = 4)
of applied holding potential, respectively.
Next, we tested the predictions pertaining to the IpTxa
concentration dependence of von and voff (Fig. 7). Skeletal CRC channel activities were recorded at a holding
potential of 20 mV at five different native IpTxa concentrations varying from 6 to 100 nM. The regression
lines drawn through the mean von and voff data points
have coefficients of determination of 0.99 and 0.02, respectively, thus supporting the prediction that von is linearly dependent on IpTxa concentration and voff is independent of it. From the data, a kon (von/[IpTxa])
value of 1.7 × 107 M
1 s
1 and a koff value of 0.31 s
1
were obtained, which gave a Kd (koff/kon) of 18.2 nM.
This Kd value is very close to the Km value of 20 nM that
was obtained from the Michaelis-Menten analysis of
Psubstate vs. native [IpTxa] curve under identical recording conditions (
20 mV holding potential and 250 mM
KCl medium) (Fig. 3 and Table I).
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IpTxa Modifies [3H]Ryanodine Binding to Skeletal and Cardiac SR Vesicles
Previous studies had suggested that IpTxa preferentially
binds to and activates the skeletal CRC and that cardiac
CRC is a poor target of IpTxa (Valdivia et al., 1992; El-Hayek et al., 1995
; Zamudio et al., 1997a
). Since we observed that IpTxa induces subconductance states in
both the skeletal and cardiac CRCs, we reexamined the
effects of IpTxa in [3H]ryanodine binding experiments
using skeletal and cardiac SR vesicles. Fig. 8 shows the
Ca2+ activation profiles of CRCs as measured by [3H]ryanodine binding to skeletal and cardiac SR vesicles in
the absence and presence of 30 nM synthetic IpTxa. As observed previously (Valdivia et al., 1992
; El-Hayek et al.,
1995
; Zamudio et al., 1997a
), IpTxa increased [3H]ryanodine binding to skeletal SR vesicles. Activation occurred at all tested Ca2+ concentrations with the possible exception of 10 mM, where [3H]ryanodine binding levels
were close to background. In contrast to its effect on
skeletal SR vesicles, IpTxa decreased [3H]ryanodine
binding to the cardiac SR vesicles up to twofold at a
Ca2+ concentration of 10 µM-1 mM. At low (<1 µM)
and high (>1 mM) Ca2+ concentration, it was without
a noticeable effect.
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[3H]ryanodine binding measurements have been
and are extensively used as indicators of the number of
channels and their activities (Meissner, 1994). We did
not observe an increase in Po of the skeletal CRC nor a
decrease in Po of cardiac CRC in bilayer experiments after addition of IpTxa (data not shown). Therefore, we
were surprised to find that IpTxa affected the [3H]ryanodine binding to skeletal and cardiac CRCs so differently. To gain further insights, we performed Scatchard
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 maximum binding (Bmax) (from 13.5 to 17.5 pmol/mg) was
also observed after IpTxa treatment. In contrast, 30 nM
IpTxa increased the Kd of [3H]ryanodine binding to
cardiac SR ~3.5-fold without an appreciable effect on
the Bmax value. The above experiments were carried out at 100 µM free Ca2+. The cardiac CRC is nearly fully active at this [Ca2+] (Xu et al., 1996
), whereas in the absence of other activating ligands, 100 µM Ca2+ cannot
fully activate the skeletal CRC. To test if the different effects of IpTxa on skeletal and cardiac SR vesicles were
due to the different activation levels of CRCs, we performed Scatchard analysis of [3H]ryanodine binding
using maximally activating conditions (100 µM Ca2+
and 5 mM AMP-PCP) for both CRC isoforms. Under
these conditions, IpTxa affected the [3H]ryanodine
binding to the skeletal and cardiac SR vesicles in a similar manner. The Kd of [3H]ryanodine binding to the
skeletal SR vesicles increased ~1.3-fold and that for
the cardiac SR vesicles ~4.5-fold without a change in
the Bmax values in both cases (Table II).
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IpTxa Induces Substates in Ryanodine-modified Skeletal Muscle CRC
In Fig. 9, we explored the cointeraction of IpTxa and
ryanodine with the skeletal CRC at the single channel
level. After addition of 100 nM IpTxa to the cis bilayer
chamber, the channel current fluctuated between the
full conductance and IpTxa-induced subconductance states (Fig. 9 A, top). After further addition of 5 µM ryanodine to the cis chamber, the channel current fluctuated between two new levels (Fig. 9 A, bottom). The current-voltage relationships in presence of both IpTxa
and ryanodine are shown in Fig. 9 B. The higher subconductance state had linear current-voltage characteristics and was identified as the ryanodine-modified
subconductance state of the CRC (Lai et al., 1989). The
less conducting IpTxa-induced substate of the ryanodine-modified CRC had a rectifying current-voltage characteristic. For comparison, the control and the IpTxa-induced substate current-voltage curves (in the absence
of ryanodine) from Fig. 1 B are redrawn as a dashed and
dotted line, respectively, in Fig. 9 B. The new substate
conductance in the presence of both IpTxa and ryanodine was significantly smaller than the IpTxa-induced substate (in the absence of ryanodine) at all applied
holding potentials.
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DISCUSSION |
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The present studies explored the effects of IpTxa on purified CRCs in single channel experiments, and on SR vesicles in [3H]ryanodine binding experiments. In these studies, we provide the first evidence that IpTxa directly interacts with both skeletal and cardiac CRCs to induce voltage- and concentration-dependent subconductance states in both isoforms by binding at a single site, located in the voltage drop across the channel. Second, we show that IpTxa affects the [3H]ryanodine binding characteristics of the two isoforms depending on the level of channel activation.
Interaction of IpTxa with CRCs as Revealed by Single-Channel Experiments
We modeled the interaction of IpTxa with the CRC as outlined in Scheme I. Briefly, we envisaged that when the channel opened, an IpTxa molecule entered the conductance pathway from the cytosolic side to bind at an internal site. The model presented us with some testable predictions that were all satisfied. The distribution of dwell-times in each state (burst and subconductance) followed a single exponential. The reciprocal mean dwell-time in the burst state was linearly dependent on, and the reciprocal mean dwell-time in the subconductance state was independent of, IpTxa concentration. A Kd value of IpTxa binding to skeletal CRC could be calculated from the dwell-time analysis, and it was very close to that obtained from the Michaelis-Menten-type concentration-dependence curve under identical conditions.
The effective valence of the reaction leading to substate formation was determined to be ~1.5 for the skeletal CRC (the value for cardiac CRC was ~1.3). From
the known amino acid sequence of IpTxa (Zamudio et
al., 1997a) and using the program "Isoelectric" of the
GCG suite (Genetics Computer Group, Inc., Madison,
WI), we calculated a net charge of 6.5 at pH 7.3. Dividing the effective valence by this number gives an electrical distance of ~0.23 from the cytosolic side for the location of IpTxa binding site. This is based on the assumption that all the charges of IpTxa enter the CRC
conduction pathway. The unbinding reaction of IpTxa
from the CRC has a twofold higher voltage dependence than the binding reaction. Higher voltage dependence for the unbinding rate has also been observed in case of tetrabutyl ammonium (TBA+)-induced
substate in the cardiac CRC (Tinker et al., 1992a
) and curare-induced subconductance state in the acetylcholine receptor channel (Strecker and Jackson, 1989
).
IpTxa is a relatively small peptide of 3,765 D with
three pairs of cysteine residues that would stabilize the
three-dimensional (3-D) conformation and help it assume a compact globular form by forming disulfide
bridges (Zamudio et al., 1997a). Preliminary modeling
work suggests that IpTxa structure can be roughly approximated as a sphere of ~2.5 nm in diameter. The
selectivity filter of the skeletal CRC has a diameter of
~0.7 nm, as choline+, Tris+, and glucose can permeate
through the channel (Meissner, 1986
; Smith et al., 1988
),
while sucrose cannot (G. Meissner, unpublished observations). Therefore, IpTxa cannot enter the selectivity
filter of the CRC. On the other hand, studies with large
tetraalkyl ammonium ions (Tinker et al., 1992a
) and
charged local anesthetics (Tinker and Williams, 1993
)
have suggested that the sheep cardiac CRC has a relatively large vestibule facing the cytosolic side. Theoretical considerations have suggested that a significant proportion of the voltage drop may fall over the wide vestibule of a channel (Jordan, 1986
). Our studies showed
that the vestibule has to be at least 2-2.5-nm wide to accommodate IpTxa, and at least ~23% of the voltage
falls over the vestibule. Interestingly, 3-D cryo-electron micrographic reconstruction of the skeletal muscle
CRC (Radermacher et al., 1994
; Serysheva et al., 1995
)
shows a central opening of ~5 nm in the cytoplasmic
domain (facing the transverse tubule), and thus supports the presence of a wide vestibule facing the cytoplasmic side of the CRC.
Mechanism of Subconductance State Formation
We considered it unlikely that IpTxa would produce
subconductance states by a surface charge mechanism
(Green and Andersen, 1991). The estimated decrease
in local [K+] using the Guoy-Chapman double-layer
theory (McLaughlin, 1977
) was only approximately
fourfold (assuming zero net surface charge near the cytosolic face of the CRC and the IpTxa net positive charge of 6.5 spread out over a sphere of ~2.5-nm diameter). This decrease (from 250 to 62.5 mM) was not
sufficient to cause the subconductance state as the Kd
of K+ for the cardiac CRC has been shown to be ~20
mM (Tinker et al., 1992b
). However, if the positive
charges of IpTxa are clustered in a much smaller area
(leading to a higher charge density), the local [K+]
could be much lower, which may explain the formation
of subconductance state. The above possibility is also
not very likely as the ratios of subconductance to full
conductance values were similar, and there was no indication of any relief of block, when the [KCl] in the bilayer chambers was increased symmetrically from 250 to 500 mM (data not shown). However, we observed an
increase in the Kd of IpTxa binding to CRC when [KCl]
was raised from 250 to 500 mM (our unpublished studies). Similar effects of increasing [KCl] on Kd have
been observed by other investigators studying other toxins (Anderson et al., 1988
; Lucchesi and Moczydlowski, 1990
).
In our experiments with the purified CRCs in bilayer experiments, substates of different amplitudes are sometimes observed even in control recordings. These substates show linear current-voltage relationships. Therefore, it is conceivable that IpTxa preferentially binds to and stabilizes a normally occurring CRC substate. However, we think this to be an unlikely mechanism as the IpTxa-induced substates had different conductances at positive and negative holding potentials (Figs. 1 and 2, and Table I).
How then does IpTxa reduce channel conductance?
Is it by partial occlusion of the channel pore or by a
conformational change? Tetrabutyl and tetrapentyl ammonium-induced substates in the cardiac CRC have
been explained in terms of partial occlusion of the conduction pathway (Tinker et al., 1992a). Convincing arguments have been put forward in favor of partial block
of acetylcholine receptor channel by curare (Strecker
and Jackson, 1989
) and complete block of a maxi Ca2+-activated K+ channel by charybdotoxin (Anderson et
al., 1988
; MacKinnon and Miller, 1988
). In the former
case, displacing curare from the agonist binding sites of
acetylcholine receptor channel had no effect on the kinetics of subconductance state occurrence. In the latter
case, enhancement of external toxin dissociation by internal K+ and competition for the toxin-binding site by
external tetraethylammonium ions gave strong support
to the occlusion model. On the other hand, Zn2+-induced
substates in canine cardiac sarcolemma sodium channel (Schild et al., 1991
) and proton and deuterium ion-induced substates in a voltage-dependent Ca2+ channel
(Prod'hom et al., 1987
; Pietrobon et al., 1989
) have been explained in terms of conformational changes; as
in the above two cases, voff was dependent on the
blocker concentration. Furthermore, in case of dendrotoxin-I (DTX-I)-induced substate formation in a
maxi Ca2+-activated K+ channel, a conformational
change mechanism was also favored as the toxin converted the linear control current-voltage relationship to a rectifying one, though in this case von and voff were
linearly dependent on and independent of the toxin
concentration (Lucchesi and Moczydlowski, 1990
). In
the present case, IpTxa binding converted an ohmic
CRC to a rectifying one. Furthermore, IpTxa-modified CRCs (both skeletal and cardiac) had higher Kds of
[3H]ryanodine binding under fully activating conditions (conditions similar to bilayer recording conditions) (see below for further discussion on this aspect).
These observations favor an IpTxa-induced conformational change in both the skeletal and cardiac CRCs as the mechanism causing the subconductance state. Alternatively, resultant conformational changes by the
membrane electric field may cause IpTxa to differentially occlude the CRC at positive and negative holding
potentials to produce substates of different conductances. Regardless of whether partial occlusion of the
pore or a conformational change is the mechanism
causing subconductance state formation, our studies
strongly suggest the existence of a cytosolically accessible IpTxa binding site in the conductance pathway of
both the skeletal and cardiac CRC.
Difference in Potency Between Native and Synthetic IpTxa
Most of our experiments were conducted using native, purified IpTxa. In some experiments carried out using synthetic IpTxa, we observed that the synthetic toxin was ~10-fold more effective in inducing substates in skeletal and cardiac CRCs than its native counterpart. The reasons for this difference are not clear. One possibility is that during purification the native toxin lost part of its biological activity.
Cointeraction of IpTxa and Ryanodine with CRCs
[3H]ryanodine binding correlates reasonably well with
CRC activation levels. Because of its simplicity and convenience, it is extensively used to explore the effects of
many drugs/toxins on CRC. But as this study shows, direct measurements of channel activity can provide insights that might not be detected by [3H]ryanodine
binding measurements. When the effects of IpTxa were assessed at 100 µM free Ca2+, an increase in [3H]ryanodine binding level for the skeletal but a decrease for the cardiac SR vesicles was observed (Fig. 8), though
the net effect is the formation of subconductance states
in both isoforms in bilayer experiments. Furthermore,
using purified skeletal CRC, we demonstrated that IpTxa
can still bind and induce substates of slightly smaller
conductances in the ryanodine-modified channel (Fig.
9, A and B). Similar paradoxical results were observed
in studies exploring the effects of polyamines on the
CRCs. Polyamines increased [3H]ryanodine binding to
skeletal SR vesicles approximately fivefold (Zarka and
Shoshan-Barmatz, 1992). However, in single-channel studies, it caused a blockade of the cardiac CRC, and
no increase in Po was observed (Uehara et al., 1996
).
Though [3H]ryanodine binding and single channel experiments were done on different CRC isoforms, the
above experiments and the present study call for caution in interpreting [3H]ryanodine binding results in
the presence of other drugs and toxins.
The apparently paradoxical effects of IpTxa on skeletal and cardiac SR vesicles in [3H]ryanodine binding
measurements led us to investigate its effects in detail.
At 100 µM Ca2+, IpTxa caused a decrease in Kd (with a
small increase in Bmax) for the skeletal SR vesicles, but
an increase in Kd with little or no change in Bmax for the
cardiac SR vesicles (Table II). The affinity of [3H]ryanodine binding to SR vesicles correlates with the CRC activation level, suggesting that ryanodine binds to an
open channel (Meissner, 1994). The IpTxa-induced
long-lived substates in the skeletal CRC would thus provide a favorable environment over the rapidly gating
control low channel activity (at 100 µM Ca2+) for ryanodine entry into the CRC interior and subsequent
binding. For the cardiac CRC, which is maximally active at 100 µM Ca2+, the substates provided no further
improvement. On the contrary a decrease was observed, probably because the IpTxa-modified channel may have reduced affinity for ryanodine. In favor of
this idea, we observed that under maximally activating
conditions for both CRC isoforms (100 µM Ca2+ and 5 mM AMP-PCP), IpTxa decreased the Kds of [3H]ryanodine binding to both skeletal and cardiac SR vesicles
(Table II). Furthermore, IpTxa increased [3H]ryanodine binding to cardiac SR vesicles under minimally activating conditions (~1 µM Ca2+) just as it increased
[3H]ryanodine binding to skeletal SR vesicles under
suboptimally activating conditions (100 µM Ca2+) (our
unpublished studies). Thus, our data show that the IpTxa-modified CRC (both skeletal and cardiac) has a Kd of
[3H]ryanodine binding intermediate between those of
the minimally and maximally activated channel.
In conclusion, the present work demonstrates a direct, high affinity interaction of IpTxa with both the skeletal and cardiac CRCs. The use of IpTxa should aid in the structural elucidation of the ion-conductance pathway of the skeletal and cardiac muscle CRCs. Furthermore, we show that experiments exploring effects of drugs/toxins on CRCs by [3H]ryanodine binding measurements must be interpreted with caution and be correlated with measurements of channel activity.
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FOOTNOTES |
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Address correspondence to Ashutosh Tripathy, Ph.D., Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260. FAX: 919-966-2852; E-mail: tripathy{at}med.unc.edu
Received for publication 3 September 1997 and accepted in revised form 10 March 1998.
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 Association (to H.H. Valdivia) and NIH grants AR-18687 and HL-27430 (to G. Meissner).
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Abbreviations used in this paper |
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CRC, Ca2+ release channel; IpTxa, imperatoxin activator; RyR, ryanodine receptor; SR, sarcoplasmic reticulum.
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