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J. Biol. Chem., Vol. 277, Issue 2, 1349-1353, January 11, 2002
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From the Wadsworth Center, New York State Department of Health,
Albany, New York 12201-0509 and the Department of Biomedical
Sciences, State University of New York,
Albany, New York 12201-0509
Received for publication, September 24, 2001, and in revised form, October 30, 2001
Calmodulin (CaM) binds to the ryanodine
receptor/calcium release channel of skeletal muscle (RyR1), both in the
absence and presence of Ca2+, and regulates the
activity of the channel activity by activating and inhibiting it,
respectively. Using cryo-electron microscopy and
three-dimensional reconstruction, we found that one apoCaM binds
per RyR1 subunit along the sides of the cytoplasmic assembly of the
receptor. This location is distinct from but close to the location
found for Ca2+-CaM, providing a structural basis for
efficient switching of CaM between these two positions with the
oscillating intracellular Ca2+ concentration that generates
muscle relaxation/contraction cycles. The locations of apoCaM and
Ca2+-CaM at a critical region for RYR1-dihydropyridine
receptor interaction are suggestive of a direct role for CaM in the
mechanism of excitation-contraction coupling.
In skeletal muscle, the calcium release
channel/ryanodine receptor
(RyR1)1 functions as one of
the main regulators of cytoplasmic Ca2+ concentration, the
signal for muscle contraction, by allowing rapid translocation
of Ca2+ from the sarcoplasmic reticulum to the
cytoplasm (1-3). Some of the RyR1s are thought to contact directly and
to be under the control of dihydropyridine receptors (DHPRs), voltage
sensors situated on the cell membrane infoldings (T-tubules) that are juxtaposed to the sarcoplasmic reticulum. The DHPR senses the membrane
depolarization wave associated with a neuronal impulse and undergoes a
conformational change and intramembrane charge movement (4). This is
apparently necessary and sufficient for RyR1 activation, which results
in a global intracellular Ca2+ increase from above pCa 7 to
pCa 6-4, a level of Ca2+ that triggers contraction of the
muscle fiber (5). RyR1 itself is regulated by Ca2+ in a
biphasic fashion with an activation peak at pCa ~5.5 and inhibition
below pCa 3 or above 7 (6). In addition to its vital function in
skeletal muscle, RyR1 is an abundant RyR isoform in cerebellar neurons
(7) where it also appears to communicate with the plasma membrane DHPR
(8). In the brain, RyRs have been hypothesized to play a role in
synaptic plasticity and apoptosis (9).
As has been reported for some other Ca2+ channels (10),
RyR1 is further regulated by CaM, the main mediator of Ca2+
signaling in eukaryotes. Modulation of RyR1 by CaM is complex. At pCa
4, Ca2+-saturated CaM (Ca2+-CaM) binds directly
to the RyR1 with a stoichiometry of one mole of apoCaM per RyR1 subunit
(i.e. four CaMs per tetrameric RyR1 complex) (11, 12) and
partially inhibits channel activity (6, 13), but at pCa 7, Ca2+-depleted CaM (apoCaM) becomes an activator (14-17).
In both cases CaM reduces the direct effect produced by
Ca2+ alone acting on the RyR1. CaM also mediates RyR1's
redox sensitivity to nitric oxide and oxygen species (18, 19).
Other proteins are also modulated by Ca2+-CaM and apoCaM.
Among these, either CaM is bound to the same sequence at both
high and low [Ca2+], or the effector protein has two or
more binding sites with different requirements of [Ca2+]
for CaM binding (20). For RyR1, the number of CaM-binding sites as well
as whether the two forms of CaM share common binding sites has been
subject of study (12, 14, 15). Within RyR1's ~5000 amino acid
residues, several putative CaM binding motifs have been proposed that
could account for both Ca2+-dependent and
-independent CaM binding (16, 21-23). Two recent independent reports
suggest that there are two binding sites per subunit (one for apoCaM
and one for Ca2+-CaM), both contained in a short segment
(~30 residues) of the RyR1 sequence (24, 25).
Although the interactions of CaM with short peptides corresponding to
the CaM-binding sites in several proteins have been characterized at
atomic detail (26-30), a structural model that includes the complete
regulated protein is lacking but is necessary to understand the
mechanism of action of CaM, particularly in those proteins that bind
both apoCaM and Ca2+-CaM. Cryo-electron microscopy and
image processing have been used to generate three-dimensional
reconstructions of native isolated RyR1 (31). In this study, we have
used these techniques to obtain three-dimensional reconstructions of
RyR1 in the presence and absence of apoCaM. Difference mapping shows
the locations where apoCaM binds to the RyR1, and incidentally,
provides an independent means to the biochemical assays used previously
to assess the stoichiometry of binding. The binding site for apoCaM
found in this study is distinct from but close to the known location
for Ca2+-CaM (11, 32). We propose a model for CaM activity
whereby this molecule acts as a mobile Ca2+-sensing subunit
of RyR1. Furthermore, the locations of RyR1-bound CaM occur in a region
implicated in interactions of RyR1 with DHPR, and on this basis we
propose that CaM could be involved in the mechanism of
excitation-contraction (E-C) coupling in skeletal muscle. Part of this
study has been published previously in abstract form (33).
Preparation of RyR1·apoCaM Complexes--
RyR1 was isolated as
described previously (11). Phosphatidylcholine (PC) was purchased from
Avanti Polar Lipids, Inc.; brain CaM and all other chemicals were
purchased from Sigma. RyR1·apoCaM complexes were prepared by mixing 1 volume of CaM-free RyR1 (in 10 mM Na-PIPES, pH 7.4, 0.45 M NaCl, 25 µM EGTA, 20 µM
leupeptin, 1% CHAPS, 0.5% PC, 0.5 mM fresh
dithiothreitol) and 1 volume of CaM in buffer C (20 mM
Tris-HCl, pH 7.4, 0.15 M KCl, 2 mM EGTA, 20 µM leupeptin) to achieve a CaM:RyR1 molar ratio of 18. The RyR1 control (no apoCaM) was also diluted with 1 volume of buffer C, and all mixtures were incubated for 20-40 min at room temperature prior to cryo-plunging.
Cryo-electron Microscopy--
PC and CHAPS concentrations were
diluted to 0.09 and 0.2%, respectively, so as to allow sufficient
image contrast, by adding buffer C during sample application to the
electron microscopy grid (34). Grids were blotted with Whatman filter
paper No. 540. Cryo-electron microscopy was performed on a
Philips EM 420 transmission electron microscope operated at 100 kV
under low dose conditions at a magnification of ×52,000, and
micrographs were taken at 0°, 30°, and 50° tilt. High quality
micrographs (71 for RyR1 control and 41 for RyR1·apoCaM) were
digitized on a Hi-Scan (Eurocore) densitometer at a pixel size of 3.8 Å, which was binned down to 7.7 Å prior to three-dimensional
reconstruction. Individual particles (7814 for RyR1 control and 4887 for RyR1·apoCaM) were selected from the micrographs and processed
using the SPIDER/WEB software package (35) as described previously
(36). 4-fold two-dimensional averages were generated using 900 particles. Three-dimensional reconstructions were computed using
projection-matching algorithms (37) with an existing three-dimensional
reconstruction of RyR1 (38) as a reference. The contribution of 4-fold
views was reduced with final numbers of particles of 2798 for RyR1
control and 2079 for RyR1·apoCaM. Two-dimensional averages and
three-dimensional reconstructions were filtered to their limiting
resolution as calculated according to the Fourier ring and shell
correlation criteria, respectively, with a cutoff value of 0.5 (39).
Three-dimensional reconstructions were displayed with isosurface
representations at a threshold that corresponds to the midpoint of the
density profile of the reconstruction boundary.
Two-dimensional Cryo-electron Microscopy--
Electron micrographs
of frozen hydrated specimens show the fine structure that is indicative
of well preserved tetrameric RyR1s (Fig.
1A) and a high proportion of
4-fold-symmetric views, corresponding to RyR1s lying with their 4-fold
symmetry axes normal to the carbon support film. Two-dimensional
averages performed on such 4-fold views (resolution of 28 Å), prepared
in the presence and absence of apoCaM, display the typical square shape
with protruding corners (clamps) and a central cross (31, 38) (Fig. 1,
B and C). Direct comparison of the two
two-dimensional averages reveals that in the presence of apoCaM, a
small region in domain 3 (Fig. 1C, arrows) is
increased in density, and a small protuberance appears on its external
side. Subtraction of the RyR1 control average from that of the
RyR1·apoCaM yields a difference map showing four discrete positive
(white) densities (Fig. 1D) that are
statistically significant at the 99.9% confidence level according to
the t test (40). It is highly likely that these regions of
excess density correspond to apoCaM bound to RyR1.
Three-dimensional Localization of apoCaM on the RyR1--
The
three-dimensional reconstructions of RyR1 with and without (control)
apoCaM (Fig. 2, A and
B) with resolutions of 28 and 26 Å, respectively, show a
square prism-shaped cytoplasmic assembly with 10 distinguishable
domains and a differentiated smaller transmembrane region, as was
described previously (31, 38, 41). As anticipated from the
two-dimensional analysis, the two reconstructions display a major
difference at the outer face of domain 3: a concave region present in
the control RyR1 reconstruction becomes protuberant when apoCaM is
added (compare boxed areas in Fig. 2, A and
B). Subtraction of the two volumes yields four
ellipsoidal-shaped masses (Fig. 2C), each having its major
axis approximately perpendicular to the plane of the square defined by
the cytoplasmic assembly. We attribute each of these masses to an
apoCaM molecule (i.e. one apoCaM per RyR1 subunit).
Consistent with this interpretation, we found that an atomic model of a
peptide·apoCaM complex (42), following low pass filtration to the
resolution of our three-dimensional reconstructions, closely matches in
shape and size the ellipsoidal-shaped masses that form the difference
map (not shown). To check the internal consistency of our results, the
three-dimensional reconstructions and three-dimensional difference map
were projected onto a plane normal to the 4-fold symmetry axis of the
RyR1 to simulate the results of the two-dimensional analysis. These
computed projections (Fig. 1, E-G) reproduce the main
features of the two-dimensional analysis (Fig. 1, B-D).
An alternative but unlikely interpretation of the structural
differences between RyR1 and RyR1·apoCaM is that they are due to
conformational changes in RyR1 induced by apoCaM binding to RyR1.
However, conformational differences should result in comparable levels
of both positive and negative densities in difference images, whereas
the three-dimensional difference map obtained from the RyR1·apoCaM
and RyR1 three-dimensional reconstructions does not display any
significant negative density. Therefore, we conclude that the four
additional ellipsoidal masses arise from four molecules of apoCaM bound
to the surface of RyR1. This does not preclude the possibility of
conformational changes at a smaller scale, which might not produce
detectable differences at the level of resolution attained.
Relative Positioning of apoCaM and Ca2+-CaM on the
RyR1--
We compared the location of apoCaM to the location already
found for Ca2+-CaM (11). Both forms of CaM contact domain 3 of each of the four RyR1 subunits. However, depending on the
Ca2+ concentration, CaM locates at distinct sites on the
RyR1. ApoCaM is situated closer to the T-tubule face of RyR1 and
protrudes more from the lateral surface of the structure, whereas
Ca2+-CaM appears in each of the crevices formed by domains
3 and 6/8 (Fig. 3, A and
B). The major axes of the Ca2+-CaM and apoCaM
molecules form an angle of ~140° (Fig. 3C), and their
geometric centers are 33 ± 5 Å apart. If bound simultaneously, the volumes of Ca2+-CaM and apoCaM would interpenetrate
partially (Fig. 3D). Therefore, simultaneous occupation of
both sites seems unlikely.
Correlation with Biochemical Results--
ApoCaM and
Ca2+-CaM, which differ substantially in their structural
and physicochemical properties as well as in their biological effects,
have been spatially localized on the intact RyR1 tetrameric complex
(summarized in Fig. 3, A and B). The two forms of
CaM bind on the sides of the cytoplasmic assembly of RyR1 to distinct, overlapping locations with centers separated by 33 ± 5 Å. The two sites are sufficiently close, and the orientations of the bound
CaMs are such that simultaneous binding of apoCaM and
Ca2+-CaM would appear to be unlikely due to steric
interference. This is further evidence that only four CaM molecules
could be bound to the RyR1 at a given time, regardless of the
Ca2+ concentration.
Although nine potential binding sites for CaM have been
identified within the sequence of the RyR1 polypeptide (21-23, 43), conflicting reports have appeared regarding the actual number of
CaM-binding sites (12, 14, 15). Our findings, based upon three-dimensional structural determination, agree with the results of
biochemical assays (12), which show that each RyR1 subunit binds only
one molecule of Ca2+-CaM or one of apoCaM.
Bound apoCaM or Ca2+-CaM protects the same two peptide
bonds (between residues 3630-3631 and 3637-3638) from proteolysis by trypsin (12), and further, a synthetic peptide containing these residues of the RyR1 subunit (amino acids 3614-3643) was found to be
capable of binding with high affinity either apoCaM or
Ca2+-CaM (24). Additional experiments employing other
related peptides, together with experiments using single point RyR1
mutants (25), were interpreted as supporting a model in which apoCaM
and Ca2+-CaM bind to distinct but overlapping sites on the
peptide. Our spatial localizations of the two forms of CaM are
compatible with such results, although the 33 ± 5 Å distance
between the centers of the sites that we found is somewhat greater than
might have been expected.
We propose several modes of CaM binding that could account for
the ~33-Å separation between the centers of apoCaM and
Ca2+-CaM while both forms of CaM still preserve
interactions with amino acid residues contained within a short segment
of the RyR1 sequence (Fig. 4). (i) For
instance, the apoCaM and Ca2+-CaM sites could be related by
a translation of the CaM along the linear sequence of the RyR1 such
that the "protected region" is bound by or in the immediate
neighborhood of one or the other of the two major domains of CaM (Fig.
4A). (ii) The domain of CaM not interacting with the
protected sequence could even bind to a non-contiguous region of the
RyR1 sequence (Fig. 4B), a situation that is reminiscent of
a recently characterized mode of CaM binding to a target protein (30).
(iii) The translation of CaM could involve a change in the polarity of
the amino and carboxyl domains of CaM with respect to the target
peptide (Fig. 4C). Although CaM interacts predominantly with
its target peptides in an antiparallel fashion with the amino
terminus of the peptide interacting with the C-lobe of
Ca2+-CaM, the opposite orientation has also been described
(29, 44). (iv) Alternatively, conformational differences between RyR1·apoCaM and RyR1·Ca2+-CaM could contribute to the
separation of CaM-binding sites at high and low [Ca2+]
(Fig. 4D).
RyR1 itself is sensitive to Ca2+, showing a bell-shaped
activation curve with a maximum at pCa 4. Interestingly, CaM bound to RyR1 counteracts this effect partially, inhibiting the channel at
millimolar Ca2+ concentrations and activating it at
submicromolar Ca2+ concentrations. Because both CaM and
RyR1 are affected by Ca2+, the RyR1-CaM interactions
need to be interpreted in this context. We explored the possibility of
a Ca2+-induced conformational change as a cause for the
different locations of CaM at high and low [Ca2+] by
comparing three-dimensional reconstructions of RyR1 without added CaM
at pCa >6 and at pCa 4 (11, 36). Although there are some minor
conformational differences in the vicinity of the CaM-binding regions,
the relatively large distance between apoCaM and Ca2+-CaM
is unlikely to be solely the result of a conformational change induced
by Ca2+.
The target surface for both forms of CaM is situated ~10 nm away from
the Ca2+-conducting pore contained in the transmembrane
region, suggesting that there is long range allosteric coupling between
domain 3 and the transmembrane domain. Similar results have been
obtained with other RyR1 modulators, such as FK-506 binding
protein and Imperatoxin A (IpTxa) (11, 36).
A Switching Mechanism for CaM--
Tripathy et al. (15)
found that the kinetics of apoCaM and Ca2+-CaM binding to
RyR1-enriched vesicles were sufficiently slow so as to preclude a
mechanism of modulation by CaM in which it dissociates from one site
and reassociates with the other with each cycle of contraction. To
account for this result, they postulated that one of the CaM-binding
sites present on RyR1 was occupied by both apoCaM and
Ca2+-CaM. Our findings, as well as those of Rodney et
al. (24) and Yamaguchi et al. (25), argue strongly for
two distinct sites for apoCaM and Ca2+-CaM. Thus, it would
appear that if the results of the kinetic studies and the evidence
showing distinct sites for the two forms of CaM apply to the in
vivo situation, then a mechanism for switching between the two
sites must be non-dissociative in nature.
Based on the relatively close positioning of apoCaM and
Ca2+-CaM reported in this study, we propose a model whereby
CaM switches between these two positions with the oscillating
[Ca2+] responsible for the cycles of
contraction-relaxation in skeletal muscle without dissociation from the
RyR1. The necessity to capture another CaM molecule from the
cytoplasmic pool would be avoided, allowing a fast response as has been
reported (13). Given the intracellular CaM concentration (2 µM in skeletal muscle (45) and 50 µM in
brain (1)) and the high affinity of RyR1 for apoCaM and
Ca2+-CaM (Kd values both in the
nanomolar range (14, 15)), RyR1 could exist primarily as a complex with
CaM, even at resting Ca2+ levels. Tripathy et
al. (15) also suggested this scenario based upon the slow kinetics
with which CaM dissociates from RyR1 relative to contraction/relaxation
cycles. Recent evidence supports the preassociation of apoCaM with
voltage-gated Ca2+ channels that are regulated by
Ca2+-CaM, including the L-type channel (DHPR) that
interacts with RyR1 at triad junctions (47). We propose that for
pCa>6, apoCaM is bound to the apoCaM-binding site of RyR1.
After Ca2+ release, CaM would lose affinity for the apoCaM
site and increase affinity for the neighboring Ca2+-CaM
site of RyR1. Several characteristics of CaM are consistent with a
non-dissociative movement of a single CaM molecule between the two CaM
binding sites: two structural and functional modules capable of binding
to their targets independently and with different affinities (46), the
ability to form stable complexes with only one domain bound (28), and
the high flexibility of the tether region that connects the two modules
(26). These properties could contribute to a scenario whereby the
Ca2+-induced translocation of CaM (e.g. such as
in the models depicted in Fig. 4 A-C) is achieved by a
two-step process with movement of one domain of CaM at a time. Thus,
CaM could act as a mobile Ca2+-sensing subunit of RyR1
producing two alternative, opposite modulatory effects. Such
coordinated CaM interactions could have a parallel in other proteins
that bind apoCaM and Ca2+-CaM (20, 47, 48).
Does CaM Have a Role in E-C Coupling?--
The currently favored
mechanism of E-C coupling in skeletal muscle, first proposed by
Schneider and Chandler (49), posits a "mechanical coupling" between
RyR1 and the voltage sensor, identified as the DHPR (4). According to
this mechanism, the DHPR undergoes a voltage-dependent
conformational change that in turn induces a conformational change in
RyR1 through a physical coupling of the two receptors (for reviews, see
Refs. 50-52). A cytoplasmic region of the DHPR known as the II-III
loop has been strongly implicated in the interaction with the RyR1 that
mediates E-C coupling (53). Previously we mapped the binding location
on RyR1 of IpTxa, a peptide that mimics a portion of the
II-III loop of DHPR but binds with much higher affinity to RyR1 than
the II-III loop itself (54). IpTxa was found to bind within
the crevices formed by RyR1's domains 3 and 6/8 (36), which are near
to and perhaps overlapping the binding location Ca2+-CaM
(Fig. 3, E and F). Most likely, additional
regions of the RyR1, particularly within domain 3 and the clamps
(domains 5-10), are involved in the interaction with DHPR (31, 52, 55,
56). The existence of two closely spaced but distinct sites for apoCaM and Ca2+-CaM on RyR1, together with the proximity of these
sites to a region of RyR1 that potentially interacts with DHPR, leads
us to speculate that CaM might play a more direct role in the molecular mechanism of E-C coupling than previously considered (6, 13, 15,
57).
We thank J. Berkowitz for technical
assistance, and we are grateful for the Wadsworth Center's electron
microscopy core facilities.
*
This work was supported by Grants AR 40615 and RR01219-19
from the National Institutes of Health and by a grant from the Ministry of Science and Technology of Spain (to M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, November 2, 2001, DOI 10.1074/jbc.M109196200
The abbreviations used are:
RyR1, skeletal muscle isoform of the ryanodine receptor;
CaM, calmodulin;
DHPR, dihydropyridine receptor;
E-C, excitation-contraction;
IpTxa, Imperatoxin A;
PIPES, 1,4-piperazinediethanesulfonic
acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
Apocalmodulin and Ca2+-Calmodulin Bind to
Neighboring Locations on the Ryanodine Receptor*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Raw micrograph and two-dimensional study of
the 4-fold-symmetric views. A, unprocessed
cryo-electron micrograph showing RyR1s incubated with apoCaM.
Panels B-D, 4-fold symmetrized two-dimensional averages of
RyR1·apoCaM, RyR1 control, and difference map, respectively. One of
the four copies of domain 3 and of the clamp region
(cl) is highlighted. Arrowheads point
to the locations where there is extra mass in the presence of apoCaM.
Panels E-G, projections of the three-dimensional
reconstructions in the direction required to recreate a 4-fold view
directly comparable with panels B-D, respectively. The
bright white areas in the difference maps D and
G correspond to apoCaM. Scale bars, 20 nm.
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Fig. 2.
Three-dimensional differential mapping of
apoCaM bound to RyR1. Three-dimensional reconstructions of
RyR1·apoCaM (A) and RyR1 control (B).
Numerals indicate characteristic domains of the RyR1
three-dimensional reconstruction (31, 38). The boxed areas
highlight the region of difference created by the addition of apoCaM.
C, difference map corresponding to apoCaM (copper
color) superimposed on the RyR1 control three-dimensional
reconstruction.
View larger version (82K):
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Fig. 3.
Details and implications of the interactions
of CaM with RyR1. A and B, superposition of
the densities attributed to apoCaM (copper) and
Ca2+-CaM (yellow) on the RyR1 three-dimensional
reconstruction (semi-transparent green) as seen from the
side and from the T-tubule/plasma membrane face. C, apoCaM
and Ca2+-CaM oriented along the plane indicated
by the dotted line in panel B to show the angle
between their major axes. D, relative positions of apoCaM
and Ca2+-CaM (in semi-transparent display) to
reveal the region of overlap. E and F, RyR1
three-dimensional reconstructions showing the positioning of
IpTxa·streptavidin (36) (purple) relative to
apoCaM (copper) and Ca2+-CaM
(yellow). The asterisk indicates the point of
attachment of IpTxa to RyR1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
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Fig. 4.
Four schematic models for CaM switching
between the apoCaM and Ca2+-CaM locations.
The two distinct locations of apoCaM and Ca2+-CaM can be
accounted by either two distinct target sequences on the RyR1 with
differing affinities for the two forms of CaM, a
Ca2+-induced conformational change on the RyR1 that moves a
unique CaM-binding site, or a combination of both. The relative
positions for the two CaM domains and the target peptide at high and
low Ca2+ conditions are indicated for several models:
translation to contiguous (A) or non-contiguous
(B) sites, translation involving change in polarity of CaM
relative to the target peptide (C), and
Ca2+-induced conformational change of RyR1 (D). A
conformational change as depicted in panel D could accompany
the mechanisms shown in panels A-C. The vertical
bar indicates the RyR1 peptide(s) containing the targets for CaM.
The region containing two trypsin cleavage sites (residues 3630-3638),
which is protected from digestion by both forms of CaM (12), is shown
in lighter gray. The circles in different shades
of gray (one of them in semi-transparent display)
represent the two domains of CaM. In panel B, the second
vertical bar represents a region of RyR1 that is nearby
spatially (but non-contiguous in sequence) to the segment containing
residues 3630-3638.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Wadsworth
Center, Empire State Plaza, Albany, New York 12201-0509; Tel.:
1-518-4746516; Fax: 1-518-4747992; E-mail: samso@wadsworth.org.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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