(Received for publication, January 11, 1996)
From the
Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic
reticulum (SR) calcium release. To elucidate the mechanism of
inhibition, the effects of neomycin on the binding of
[H]ryanodine to the Ca
release
channel and on its channel activity when reconstituted into planar
lipid bilayer were examined. Equilibrium binding of
[
H]ryanodine was partially inhibited by neomycin.
Inhibition was incomplete at high neomycin concentrations, indicating
noncompetitive inhibition rather than direct competitive inhibition.
Neomycin and [
H]ryanodine can bind to the channel
simultaneously and, if [
H]ryanodine is bound
first, the addition of neomycin will slow the dissociation of
[
H]ryanodine from the high affinity site.
Neomycin also slows the association of
[
H]ryanodine with the high affinity binding site.
The neomycin binding site, therefore, appears to be distinct from the
ryanodine binding site. Dissociation of
[
H]ryanodine from trypsin-treated membranes or
from a solubilized 14 S complex is also slowed by neomycin. This
complex is composed of polypeptides derived from the carboxyl terminus
of the Ca
release channel after Arg-4475 (Callaway,
C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C.,
Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L.(1994) J.
Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex
isolated with ryanodine bound produces a channel upon reconstitution
into planar lipid bilayers, and its activity is inhibited by neomycin.
Our data are consistent with a model in which the ryanodine binding
sites, the neomycin binding sites, and the channel-forming portion of
the Ca
release channel are located between Arg-4475
and the carboxyl terminus.
The Ca release channel in the terminal
cisternae of skeletal muscle allows the movement of Ca
from the lumen of the sarcoplasmic reticulum (SR) (
)to
the cytoplasm in response to a signal from the transverse
tubule(2) . The protein that forms this channel can be
activated by the binding of the plant alkaloid ryanodine at a high
affinity site(3, 4, 5) . The apparent
affinity of [
H]ryanodine for binding to the
Ca
release channel is dependent upon the functional
state of the channel, and changes in binding of this ligand can be used
to analyze and monitor the effects of modulators of Ca
release channel function(6, 7) . High affinity
ryanodine binding sites are located in the protein between Arg-4475 and
the carboxyl terminus (1) .
Neomycin, a polycationic,
aminoglycoside antibiotic(8) , inhibits Ca release and blocks [
H]ryanodine binding to
the SR
membranes(9, 10, 11, 12, 13) .
Wyskovsky et al.(13) reported that neomycin only
blocks the fast component of the release while ruthenium red completely
blocks the Ca
efflux from SR vesicles, suggesting
that inhibition by neomycin has a mechanism different from ruthenium
red. The exact mechanism by which neomycin inhibits channel activity is
unclear. Based on the effects of neomycin on the binding of
[
H]ryanodine to SR membranes and on
Ca
fluxes, Mack et al.(10) concluded that neomycin was competitive with ryanodine for
high affinity binding sites. Furthermore, they suggested that neomycin
at high concentrations slows the dissociation of
[
H]ryanodine from the high affinity site by an
allosteric mechanism. In the present work, we examine the effects of
neomycin on [
H]ryanodine binding and on the
behavior of the Ca
release channel incorporated into
planar lipid bilayers to demonstrate that ryanodine and neomycin bind
noncompetitively to the channel.
Dissociation of
[H]ryanodine from proteolyzed SR membranes was
performed as follows: SR membranes were incubated with 40 nM [
H]ryanodine in Buffer I (0.3 M KCl, 100 µM Ca
, and 20 mM MOPS, pH 7.4) at 37 °C for 2 h, then proteolyzed by trypsin
(1:500 trypsin:SR membranes) for 1 h at 37 °C. The membranes were
solubilized in 2% digitonin, and the 14 S complex was isolated using
sucrose gradient centrifugation. Aliquots (440 µl) of the 14 S
complex were diluted into 10 ml of Buffer I in the presence and absence
of 20 µM neomycin. Dissociation was performed at room
temperature, and aliquots (0.4 ml) of diluted sample were added to 200
µl of ice-cold Buffer I containing rabbit
-globulin (5 mg/ml),
BSA (5 mg/ml), and 10% polyethylene glycol (PEG) 8000. After 15 min of
incubation at 4 °C, the samples were filtered and washed with 5
5 ml of ice-cold wash buffer containing 10% polyethylene
glycol.
where B = bound ligand at time t, t = time after addition of
[
H]ryanodine, n = number of
components, A
= amount of ligand bound to
component i, a
=
1/k
, n
3.
where B = bound ligand at time t, n = number of components, A
= amount of bound ligand to component i at t = 0, a
=
1/k
where k
is the
corresponding rate constant.
Model 1. Competitive interaction between neomycin and ryanodine at the high affinity binding site.
Model 2. Noncompetitive binding of neomycin allosterically alters the high affinity ryanodine binding site.
Inhibition of [H]ryanodine equilibrium binding
by neomycin was analyzed by nonlinear least squares fitting to the
following equation:
where B is the observed binding in the
presence of the inhibitor concentration I, B
is the bound [
H]ryanodine concentration at
maximal inhibition, A is the inhibitable binding and is equal
to B
- B
, and B
is the bound [
H]ryanodine
at zero inhibitor concentration. In Model 1, for competitive binding, B
equals nonspecific binding, and
and L = free [H]ryanodine
concentration.
From the cyclic model (2) of allosteric
inhibition, B and B
reflect the binding of [
H]ryanodine in the
absence and presence of inhibitor, respectively.
where L is the [H]ryanodine
concentration and K
is the equilibrium
dissociation constant for [
H]ryanodine and B
is the total binding site concentration.
Likewise
Thus, from the values of B, B
, and K
(determined
by curve-fitting), and the known values for K
and L, the binding constant for [
H]ryanodine
in the presence of inhibitor, K
, can be derived.
These equations will also yield the values for K
and K
, the dissociation constants of the
inhibitor in the absence and presence of
[
H]ryanodine, respectively.
Neomycin is a potent inhibitor of
[H]ryanodine binding and of Ca
release channel
activity(10, 11, 12, 13) . It has
been proposed that neomycin blocks channel activation by ryanodine by
competitively binding at the same site as
ryanodine(10, 11) . To investigate this mechanism more
closely, the effects of neomycin on [
H]ryanodine
binding were examined in detail. The [
H]ryanodine
concentrations were kept low (1-20 nM) in these
experiments to maintain binding exclusively at the high affinity
ryanodine binding site. This avoids contributions from binding to low
affinity sites. The inhibition of [
H]ryanodine
binding by varying concentrations of neomycin was determined at four
concentrations of [
H]ryanodine (Fig. 1A). At each concentration, inhibition was
incomplete and reached a plateau ranging from 43% to 74% of the
displaceable binding. The inhibition by neomycin is inconsistent with
simple competitive inhibition at the [
H]ryanodine
binding site. Competitive inhibition requires complete displacement of
[
H]ryanodine to nonspecific levels, and this is
not observed. For comparison, inhibition of
[
H]ryanodine binding by ryanodine at the same
conditions is shown in Fig. 1B. In all cases, ryanodine
completely displaces the binding of [
H]ryanodine,
consistent with competitive inhibition. Binding isotherms of
[
H]ryanodine in the absence or in the presence of
two concentrations of neomycin is shown in Fig. 1C as
Scatchard plots. In this experiment, neomycin increases the apparent K
for [
H]ryanodine from 14
nM in the absence of neomycin to 23 nM in 125 nM neomycin and to 46 nM in 10 µM neomycin.
Figure 1:
A, inhibition of
[H]ryanodine binding by neomycin. SR membranes
were incubated with 1.7 (
), 5 (
), 10 (
), or 20
(
) nM [
H]ryanodine in the presence
of 1 nM to 1 mM neomycin. B, inhibition of
[
H]ryanodine binding to rabbit skeletal muscle SR
membranes by ryanodine. SR membranes were incubated with 1.7 (
), 5
(
), 10 (
), or 20 nM (
)
[
H]ryanodine plus the indicated concentrations of
unlabeled ryanodine. The free Ca
concentration was
100 µM. Equilibrium [
H]ryanodine
binding was determined as described under ``Experimental
Procedures.'' C, Scatchard analysis of
[
H]ryanodine binding to SR membranes in the
presence and absence of neomycin. Equilibrium binding of
[
H]ryanodine at varying concentrations was
performed as described in the absence (
, K
= 14 nM), control, or presence of 125 nM neomycin (
, K
= 23
nM) or in the presence of 10 µM neomycin (
, K
= 46
nM).
While the data of Fig. 1, A and C, are
inconsistent with simple competitive binding, the data can be analyzed
in terms of a cyclic model for allosteric, noncompetitive inhibition
(see ``Experimental Procedures''). The data of Fig. 1A were well fit by as shown by the
solid lines. From the values of B, B
, and K
obtained by
nonlinear least squares fitting and the affinity of
[
H]ryanodine (14 nM) and using the
equations described under ``Experimental Procedures,'' the
binding affinity for neomycin was calculated to be 300 nM and
1100 nM in the absence and presence of ryanodine, respectively (Table 1). Likewise, the affinity of
[
H]ryanodine in the presence of high
concentrations of neomycin was calculated to be 50 nM,
consistent with the value of 46 nM determined by direct
binding isotherm in the presence of 10 µM neomycin in Fig. 1C.
To further explore the interaction between
ryanodine binding and neomycin binding, the kinetics of association and
dissociation in the presence of [H]ryanodine were
examined. If there is no effect on association rate, then the binding
cannot be competitive. Changes in the rate of association can, however,
occur with either a competitive or a noncompetitive mechanism. The
association of [
H]ryanodine (Fig. 2) is
characterized by a single component (k
=
0.0025 min
M
). Neomycin
decreases the apparent rate of association to 0.00041, a 6.1-fold
effect. The predicted dissociation rate constant from the k
plot is 0.011 min
and does
not appear to change significantly in the presence of neomycin.
Figure 2:
Effect of neomycin on the association of
[H]ryanodine to rabbit skeletal muscle SR
membranes. SR membranes (0.1 mg) were added to 10 ml of 0.3 M KCl, 20 mM MOPS, pH 7.4, 100 µM Ca
, 2.5-20 nM [
H]ryanodine, 0.1 mg/ml BSA, 1 mM AMP-PCP, and protease inhibitors in the absence (
) and
presence (
) of 100 nM neomycin. At various times at room
temperature, 20-µl aliquots were filtered, and bound
[
H]ryanodine was determined as described under
``Experimental Procedures.'' Data were analyzed for a single
component as described under ``Experimental Procedures,'' and
the k
was plotted versus [
H]ryanodine concentration. Data were fit to
straight lines, and the slopes were used to determine the association
rate constants, k
= 0.0025
min
M
in the
absence of neomycin, and k
= 0.00041
min
M
in the
presence of neomycin.
Neomycin also slows the dissociation rate of bound
[H]ryanodine from the site (Fig. 3), a
finding consistent only with a noncompetitive interaction between the
neomycin and the ryanodine binding sites. In the absence of neomycin,
the dissociation data were fit with 3 descending exponential components (k
= 0.013 min
, k
= 0.0026 min
,
and k
= 0.00045
min
). The relative amounts of each of these
components varied only slightly among membrane preparations. In 12
preparations, the fast component constituted 25.0 ± 4.2% (mean
± S.E.), the intermediate 70.1 ± 3.6%, and the slow
component 5.7 ± 1.8% of the dissociation. There were no
statistically significant changes in the relative amounts of these
components with different initial occupancies by
[
H]ryanodine (data not shown).
Figure 3:
Effect of neomycin on the rate of
dissociation of [H]ryanodine from SR membranes.
SR membranes (0.4-0.5 mg of protein) were equilibrated with 40
nM [
H]ryanodine under conditions
described under ``Experimental Procedures.'' Dissociation was
initiated by diluting more than 400-fold in binding buffer plus
(
) or minus (
) 10 µM neomycin. Dissociation
experiments were performed at room temperature. After subtraction of
nonspecific binding, the data were fit to a three-component exponential
decay (solid lines) with rate constants indicated in the text. B is the specific counts/min bound at time t.
The data
obtained from dissociation experiments in the presence of 10 µM neomycin were also fit with the same exponential rates in 8
separate experiments. The fast component was 2.6 ± 1.0%, the
intermediate 25.7 ± 9.1%, and the slow component was 71.8
± 8.7% of the binding. Each value was significantly different
from the corresponding control value. The presence of neomycin in the
dissociation buffer decreases the relative amounts of the fast and
intermediate components of the dissociation and increases the amount of
slow component. The rate constant for the fast component is similar in
magnitude to that predicted from the association kinetics (k plot, Fig. 2). The effect of neomycin
concentration on the relative amounts of the three components is shown
in Fig. 4. At low concentrations of neomycin, the fast component
appears to be converted to the intermediate component, and, at higher
neomycin concentrations, the intermediate is converted to the slow
component.
Figure 4:
The effect of neomycin on the 3 components
of [H]ryanodine dissociation. Dissociation
kinetics of [
H]ryanodine was performed as
described in Fig. 3at the indicated concentrations of neomycin
in the dilution buffer. Each set of dissociation data were fit to a
three-component exponential decay as described in Fig. 3and
under ``Experimental Procedures.'' The counts/min
corresponding to the fast (
), intermediate (
), and slow
(
) components at each neomycin concentration are
shown.
To investigate the mechanism by which neomycin blocks the
channel, we examined its effects on single channels reconstituted into
planar lipid bilayers. The effect of neomycin on the Ca release channel and the ryanodine-modified Ca
release channel reconstituted into planar lipid bilayers is shown
in Fig. 5. In the absence of ryanodine, neomycin decreases the
open probability of the channel from 0.20 ± 0.13 to 0.017
± 0.004 (n = 3, p < 0.01). The
addition of ryanodine after neomycin has no effect on the channel over
a 15-min time course (data not shown). The addition of neomycin to a
ryanodine-modified channel decreases the mean channel open time from
285.3 ± 79.1 ms to 31.1 ± 12.1 ms (n =
3). The channel opens only to the ryanodine-modified conductance level
in the presence of both ryanodine and neomycin (Fig. 5, c-e), supporting a model in which the two ligands can
bind simultaneously to the channel. These data again support a
noncompetitive interaction between neomycin and ryanodine.
Figure 5:
Effect of neomycin on the activity of
Ca release channel reconstituted into planar lipid
bilayer. SR membranes were added to the cis chamber, as
described under ``Experimental Procedures.'' The channel
modulators were also added to the cis chamber. The traces are
representative recordings of one of three independent experiments. Traces a and b, effect of neomycin on Ca
release channel; a control; b, 5 µM neomycin (P
decreases in this
experiment from 0.32 to 0.013). Traces c-e, effect of
neomycin on ryanodine-modified Ca
release channel; c, control; d, 2 µM ryanodine; e, 2 µM ryanodine, 10 µM neomycin. Upward deflections represent channel
openings.
Previous
work from this laboratory has shown that both high and low affinity
ryanodine binding sites are located in a 14 S proteolytic complex
composed only of polypeptides derived from the carboxyl terminus after
Arg-4475 in the primary sequence of the Ca release
channel(1) . In SR membranes (not solubilized), proteolysis
with trypsin under the conditions which generate the 14 S complex does
not significantly alter the rate of dissociation of
[
H]ryanodine (bound prior to proteolysis) nor
does it alter the ability of neomycin to slow dissociation (data not
shown), suggesting that the neomycin and ryanodine binding sites are
still capable of interacting after trypsin treatment. However,
proteolyzed SR membranes may also contain other proteolytic fragments
that contribute to the neomycin effect on ryanodine dissociation.
To
determine whether the neomycin binding site is on the 14 S complex, we
generated the 30 S (intact) and the 14 S (proteolyzed) solubilized and
purified forms of the protein, each containing bound
[H]ryanodine. The sucrose gradient profile is
shown in Fig. 6. We then determined the effect of neomycin on
the rate of dissociation of [
H]ryanodine from the
purified 30 S and 14 S complexes (Fig. 7, A and B). Similar to the results obtained from SR membranes, 20
µM neomycin slows [
H]ryanodine
dissociation from both the 30 S intact Ca
release
channel (Fig. 7A) and 14 S complex (Fig. 7B). Dissociation of
[
H]ryanodine from the detergent-solubilized and
gradient-purified ryanodine receptor is characterized by two components (k
= 0.016 min
, k
= 0.0063 min
),
both of which are faster than those observed in membranes (Fig. 3). Dissociation from 14 S is even faster and is best fit
by 3 components (k
= 0.026
min
, k
= 0.011
min
, k
= 0.0004
min
).
Figure 6:
Sedimentation of
[H]ryanodine-labeled proteins solubilized from
nontrypsinized and trypsinized SR membranes. SR membranes (6 mg) were
incubated with 50 nM [
H]ryanodine for 2
h in binding buffer at 37 °C. The membranes were proteolyzed by
trypsin (trypsin/protein ratio is 1:500) at 37 °C for 1 h.
Proteolysis was stopped with a 10-fold excess soybean trypsin
inhibitor. Both proteolyzed and nonproteolyzed SR membranes were
solubilized in 2% digitonin, 0.3 M KCl, 100 µM Ca
, 20 mM MOPS, pH 7.4, for 30 min at 4
°C, then sedimented on a 5-20% sucrose gradient as described
under ``Experimental Procedures.''
, gradient profile of
30 S complex (nonproteolyzed);
, gradient of 14 S complex
(proteolyzed). Fractions range from the bottom to the top of the
gradient.
Figure 7:
A,
dissociation of [H]ryanodine from isolated 30 S
(unproteolyzed) complex. The purified
[
H]ryanodine-labeled 30 S complex isolated from
the sucrose gradient was diluted into binding buffer at room
temperature to initiate [
H]ryanodine
dissociation. Aliquots were filtered at the indicated times as
described under ``Experimental Procedures.''
, control;
, 20 µM neomycin. B, dissociation from the
14 S complex. The [
H]ryanodine-labeled SR
membranes were trypsinized, and the binding protein was purified on a
5-20% sucrose gradient as described under ``Experimental
Procedures.'' The 14 S complex was diluted into Buffer I at room
temperature to initiate dissociation.
, control;
, 20
µM neomycin.
The question of whether the 14 S complex
still behaves as an ion channel was addressed by reconstitution of the
14 S complex into planar lipid bilayers. To obtain a higher purity for
the 14 S complex, the fraction purified by a sucrose gradient and the
DEAE-column was further purified on a second sucrose gradient. The
gradient profile is shown in Fig. 8A, and the
polypeptides from fractions 10 through 24 are shown on a silver-stained
gel in Fig. 8B. A Western blot with an antibody to the
last 9 amino acids is shown in Fig. 8C. The major
components of the 14 S complex (labeled a-e) have
previously been identified by Callaway et al.(1) as
the 76 kDa (4476-carboxyl terminus) (a), 66 kDa (a cleavage
product of the 76 kDa containing the carboxyl-terminal residues) (b), 56 kDa (calsequestrin) (c), 37 kDa (derived from
N terminus of 76 kDa and recognized by an antibody to residues
4670-4685) (d), and 27 kDa (beginning with 4756 and
containing the carboxyl terminus) (e). In this preparation,
the identity of the 76-kDa, the 56-kDa, and the 27-kDa peptides were
confirmed by amino-terminal sequencing. All other bands were identified
in Western blots as described previously. The 14 S complex isolated
with [H]ryanodine bound to the high affinity site
was reconstituted into planar lipid bilayers, and the channel activity
was monitored. In Fig. 9, 3 channels were incorporated, each
with a conductance of 570 pS in 225 mM KCl (Fig. 9A). Neomycin caused the channel to close more
frequently (Fig. 9B). Similar channels were seen in 6
different 14 S preparations and in a total of 8 trials.
Figure 8: Further purification of the 14 S complex and characterization of polypeptide composition. A, sucrose gradient profile from purification of the 14 S. The ryanodine-modified 14 S complex purified as described under ``Experimental Procedures'' was further purified on a 17-ml 5-20% sucrose gradient, and 50-µl fractions were counted. Fractions range from the bottom to the top of the gradient. B, polypeptide composition of sucrose gradient fractions: 20 µl of fractions 10-24 were electrophoresed, and the gel was silver-stained. C, the peak fraction of the ryanodine-modified 14 S complex was identified by silver staining of an SDS-polyacrylamide gel (lane 4) and Western blotting with an antibody to the last 9 amino acids at the carboxyl terminus (lane 3) or with an antibody to residues 4670-4685 (lane 2). Molecular weight standards are in lane 1.
Figure 9: Effect of neomycin on the channel activity of the 14 S complex. The purified and ryanodine-modified 14 S proteolytic complex was incorporated into planar lipid bilayer by addition to the cis chamber. The control recording (a) appears to have 3 channels. The addition of 50 µM neomycin increased channel closings (b). Upward deflections represent channel openings. 225 mM KCl was used in the bilayer solutions.
The data presented here demonstrate that the channel-forming
regions of the channel, the ryanodine binding sites, and the neomycin
binding sites are all located between Arg-4475 and the carboxyl
terminus of the Ca release channel, but that the
neomycin binding site is distinct from that of ryanodine. Neomycin
inhibits [
H]ryanodine binding and the activity of
both the intact channel and the ryanodine-modified channel in a manner
consistent with neomycin binding being noncompetitive with ryanodine
binding.
To demonstrate clearly the existence of a distinct binding site for neomycin, it was necessary to show that the binding was inconsistent with competitive binding to the ryanodine binding sites. Competitive inhibition can be distinguished from noncompetitive binding by analysis of equilibrium binding and by kinetic experiments. Since noncompetitive inhibition is mediated by allosteric effects, the result is a decrease in binding affinity with concomitant changes in the association rate, dissociation rate, or both. The change in affinity is saturable with inhibitor concentration as the inhibitor site becomes fully occupied. Competitive binding should reveal a change in apparent affinity that is not saturable with increasing inhibitor concentration and have no changes in the dissociation rate. Neither competitive nor noncompetitive inhibition will display a change in the number of binding sites.
The data in Fig. 1clearly show incomplete
inhibition of [H]ryanodine binding by neomycin,
which is inconsistent with competitive inhibition but fully consistent
with noncompetitive inhibition. Thus, the effect of neomycin is to
change the affinity of ryanodine about 4-fold while ryanodine has a
reciprocal 4-fold effect on the affinity of neomycin. The affinity of
neomycin for the ryanodine receptor is about 300 nM. The
kinetic data further support noncompetitive inhibition of
[
H]ryanodine binding by neomycin. At a high
concentration of 100 µM, neomycin decreases the
association rate of [
H]ryanodine dramatically.
(If neomycin was competitive with ryanodine, the association rate
should decrease proportionally with the predicted occupancy of
neomycin. With a K
= 300 nm the association
rate should have decreased about 300-fold but actually changed only
6.1-fold.) More definitive is the analysis of the dissociation rates. A
competitive inhibitor should not affect the dissociation rate under
conditions where there is no rebinding of ligand. However, neomycin
clearly inhibits dissociation of [
H]ryanodine and
alters the proportion of the three distinct rates of dissociation
toward the slower components. Slowing of both association and
dissociation rates is consistent with allosteric effects. Since the
effect on the association rate constant is stronger, the net result is
a decrease in ryanodine binding affinity at equilibrium.
Inhibition
of [H]ryanodine binding by neomycin was
interpreted by Mack et al.(10) as competitive.
However, their data are not discrepant with the data presented here and
are fully consistent with an allosteric model for noncompetitive
inhibition. They also display data demonstrating slower dissociation of
[
H]ryanodine in the presence of high
concentrations of neomycin, a result incompatible with a simple
competitive mechanism.
Further support for a model wherein neomycin
binds a site distinct from the ryanodine binding site is obtained from
functional assays of channel activity in planar lipid bilayers.
Ryanodine alone promotes long open states with a lower conductance than
seen in the absence of ryanodine. The further addition of neomycin
produces frequent fast closings: the mean open time is decreased.
However, the channel opens to the conductance level seen in the
presence of ryanodine, never to the higher conductance level
characteristic of the unbound channel. If neomycin acted by competitive
displacement of ryanodine, the channel would be expected to
occasionally reopen to the unmodified level and this is not observed.
Ryanodine, thus, appears to remain bound in the presence of neomycin
inhibition of channel activity. The effect of neomycin on the affinity
of [H]ryanodine does not directly account for
inhibition of the ryanodine-modified channel. The mechanism of neomycin
inhibition may be through stabilization of a closed conformation or by
direct channel block.
Neomycin slows the dissociation of
[H]ryanodine from the purified Ca
release channel (30 S) and a 14 S complex which we have
previously shown to be composed of peptides derived from the carboxyl
terminus after Arg-4475(1) . The ryanodine-modified 14 S
complex purified after trypsin digestion forms a channel in the
bilayer, and this activity is inhibited by neomycin. This is consistent
with the slowing of the dissociation of
[
H]ryanodine from its binding site on the 14 S
complex by neomycin. It is extremely difficult to eliminate the
possibility that a minor contaminant of this preparation is forming the
ion channels. However, these data taken together with the binding data
support a model in which the channel-forming portion of the protein is
localized in a complex of a 76-kDa peptide fragment which is the part
of the protein between amino acid 4476 and the carboxyl terminus of
Ca
release channel. This same region contains both
high and low affinity ryanodine binding sites (1) and part or
all of the putative transmembrane domains of the Ca
release channel.
In summary, neomycin inhibits Ca release and noncompetitively inhibits
[
H]ryanodine binding sites by an allosteric
mechanism. The neomycin binding sites as well as the high and low
affinity ryanodine binding sites are located in a peptide region
encompassing the amino acid sequence from Arg-4475 to the carboxyl
terminus.