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UBC Theses and Dissertations
Ligand and modulator binding in the skeletal muscle ryanodine receptor Lobo, Paolo Antonio
Abstract
Calcium (Ca²⁺) is required in the cytoplasm as a potent second messenger for a variety of vital physiological events in the cell. Its supply to the cytoplasm can either be from extracellular sources, or from intracellular sarcoplasmic reticulum stores, predominantly through the ryanodine receptors (RyRs). The latter are large, ~2 MDa homotetrameric channels that sense initial Ca²⁺ blips due to voltage-‐gated calcium channel influx and respond by opening to result in physiologically significant concentration spikes. This action is known as calcium induced calcium release and is the major process by which an excitation signal is translated to a physical, muscular contraction. Cytoplasmic Ca²⁺ concentrations have to be very well regulated and must return to resting levels for subsequent contractions to occur. The importance of this with regard to the RyR can be seen in two different ways: Firstly, its large cytoplasmic bulk, a huge docking site for modulators, emphasizes the necessity for regulation, and secondly, mutations in the RyR can cause severe genetic diseases as a result of Ca²⁺ mishandling. Presented here, are preliminary structural and binding studies for several different RyR regulators, both physiological and pharmacological. Skeletal muscle RyR (RyR1) 1-‐617 that contains the drug dantrolene’s supposed binding site was crystallized and its structure determined. Isothermal Titration Calorimetry (ITC) and co-‐crystallization attempts have not confirmed the binding of dantrolene. The structure does however shed light on the physical involvement of phosphatases as modulators of the channel. Caffeine binding was detected successfully by ITC and attributed to RyR1 217-‐ 536. A co-‐crystal structure yielded a binding site in the construct that could not be knocked out by site-‐directed mutagenesis according to ITC. Three types of modulators were shown to bind RyR1 4071-‐4128 by ITC. 1) Ca²⁺, which affects the channel both positively and negatively, 2) magnesium ions, which inhibit the channel, and 3) an intrinsic ligand in RyR1: residues 4295-‐4325; a peptide that shows affinity for Calmodulin, yet another modulator of RyR. The results provide insight into allosteric reactions as a result of RyR ligand or modulator binding.
Item Metadata
| Title |
Ligand and modulator binding in the skeletal muscle ryanodine receptor
|
| Creator | |
| Publisher |
University of British Columbia
|
| Date Issued |
2012
|
| Description |
Calcium
(Ca²⁺)
is
required
in
the
cytoplasm
as
a
potent
second
messenger
for
a
variety
of
vital
physiological
events
in
the
cell.
Its
supply
to
the
cytoplasm
can
either
be
from
extracellular
sources,
or
from
intracellular
sarcoplasmic
reticulum
stores,
predominantly
through
the
ryanodine
receptors
(RyRs).
The
latter
are
large,
~2
MDa
homotetrameric
channels
that
sense
initial
Ca²⁺
blips
due
to
voltage-‐gated
calcium
channel
influx
and
respond
by
opening
to
result
in
physiologically
significant
concentration
spikes.
This
action
is
known
as
calcium
induced
calcium
release
and
is
the
major
process
by
which
an
excitation
signal
is
translated
to
a
physical,
muscular
contraction.
Cytoplasmic
Ca²⁺
concentrations
have
to
be
very
well
regulated
and
must
return
to
resting
levels
for
subsequent
contractions
to
occur.
The
importance
of
this
with
regard
to
the
RyR
can
be
seen
in
two
different
ways:
Firstly,
its
large
cytoplasmic
bulk,
a
huge
docking
site
for
modulators,
emphasizes
the
necessity
for
regulation,
and
secondly,
mutations
in
the
RyR
can
cause
severe
genetic
diseases
as
a
result
of
Ca²⁺
mishandling.
Presented
here,
are
preliminary
structural
and
binding
studies
for
several
different
RyR
regulators,
both
physiological
and
pharmacological.
Skeletal
muscle
RyR
(RyR1)
1-‐617
that
contains
the
drug
dantrolene’s
supposed
binding
site
was
crystallized
and
its
structure
determined.
Isothermal
Titration
Calorimetry
(ITC)
and
co-‐crystallization
attempts
have
not
confirmed
the
binding
of
dantrolene.
The
structure
does
however
shed
light
on
the
physical
involvement
of
phosphatases
as
modulators
of
the
channel.
Caffeine
binding
was
detected
successfully
by
ITC
and
attributed
to
RyR1
217-‐
536.
A
co-‐crystal
structure
yielded
a
binding
site
in
the
construct
that
could
not
be
knocked
out
by
site-‐directed
mutagenesis
according
to
ITC.
Three
types
of
modulators
were
shown
to
bind
RyR1
4071-‐4128
by
ITC.
1)
Ca²⁺,
which
affects
the
channel
both
positively
and
negatively,
2)
magnesium
ions,
which
inhibit
the
channel,
and
3)
an
intrinsic
ligand
in
RyR1:
residues
4295-‐4325;
a
peptide
that
shows
affinity
for
Calmodulin,
yet
another
modulator
of
RyR. The
results
provide
insight
into
allosteric
reactions
as
a
result
of
RyR
ligand
or
modulator
binding.
|
| Genre | |
| Type | |
| Language |
eng
|
| Date Available |
2013-12-31
|
| Provider |
Vancouver : University of British Columbia Library
|
| Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
|
| DOI |
10.14288/1.0073428
|
| URI | |
| Degree | |
| Program | |
| Affiliation | |
| Degree Grantor |
University of British Columbia
|
| Graduation Date |
2013-05
|
| Campus | |
| Scholarly Level |
Graduate
|
| Rights URI | |
| Aggregated Source Repository |
DSpace
|
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Attribution-NonCommercial-NoDerivatives 4.0 International