1. 0 1x10
5
2x10
5
3x10
5
4x10
5
5x10
5
0.0
0.2
0.4
0.6
0.8
1.0
A*
Ca
2+
[uM]
FractionalrCasq2boundtoTb3+
Ca2+ [μM]
Kd = 3.0 ± 2.1 mM
luminal Mg2+
3 mM luminal Mg2+0 mM luminal Mg2+
C
This study was supported by NIH R01-HL-084487
grant. The authors thank Dr. Jingui Zhu for cloning and
expressing ECFP Casq2 and LRET experiments.
500
290
240
160
117
97
66
55
40
all-proteins stain
anti Casq2
purified dog Casq2 mouse cardiac SR vesicles
3 2 1 -/- -/-+/+ +/+ -/- -/-+/+ +/+
Casq2
MW
rCasq2-YFP
100
75
50
150
250
37
25
MW
Western blot for Anti tetra-HIS
Ab before (b) and after (a) HIS
tag removal with Ac TEV enzyme
b a
100
75
50
150
250
37
25
rCasq2-YFP
MW
Silver stained PAGE
1 2 3
Tb3+
trans [M]
Po
Kd ≈ 600 nM
nH = 4
At 2 µM cytosolic [Ca2+], luminal Tb3+ increases the open probability (Po) of
single RyR2s isolated from dog heart regardless of the holding potential.
The increase of Po occurs without significant change in single channel
current; i.e. Tb3+ activation of RyR2 is independent of the feed through
mechanism.
C
10 pA
100 ms
[Tb3+]trans
0 nM
100 nM
300 nM
500 nM
1000 nM
Recombinant Dog Casq2 Native Dog Casq2
Cloning Strategy for Casq2
LUMINAL REGULATION OF SINGLE RYR2 CHANNELS BY CARDIAC CALSEQUESTRIN
Marcia Cortes-Gutierrez1, Heather R. Orrell1, Simon Williams2, Patricio Velez1, Ariel L. Escobar1
1UC Merced, Merced, CA, USA, 2TTUHSC, Lubbock, TX, USA.
CONCLUSIONS
INTRODUCTION
METHODS
HYPOTHESIS
We examined the hypothesis that calsequestrin (Casq2) can
regulate the RyR2 activity using a combination of single channel
electrophysiology and lanthanide resonance energy transfer (LRET).
Under steady-state, open probability (Po) of RyR2 of cardiac SR
fractions from dog is modulated by luminal [Ca2+]. Po increased when
luminal [Ca2+] was increased from 6 µM to 2 mM at a fixed cytosolic
[Ca2+] of 2 µM. This effect on RyR2 appears to be mediated by
luminal sites. Interestingly, RyR2 Po also increases when 2 mM Mg2+
was added to the luminal side. To gain mechanistic insights on the
Casq2-mediated luminal regulation, we used the binding of Casq2 to
Tb3+ as a functional assay. Luminescence produced by LRET between
the tryptophan’s of purified dog Casq2 and the lanthanide increased
as function of [Tb3+]. This fluorescence was reduced as [Ca2+]
increased suggesting that Ca2+ binds to purified Casq2 by displacing
tightly-bound Tb3+ from a common binding site. To further explore
the specificity of this regulation, we expressed recombinant dog
Casq2 in E. coli. The specificity of this interaction was assessed by
LRET lifetime measurements. Interestingly, we found that the
displacement of Tb3+ by Ca2+ was not significantly different than
the displacement of Tb3+ by Mg2+ (Kd ~ 2.5 mM) suggesting that Ca2+
and Mg2+ share a common binding site. Finally, we explore the
hypothesis that Tb3+ bound to Casq2 was able to modulate the RyR2
activity. At fixed cytosolic [Ca2+] of 2 µM, Po of single RyR2
increased as a function of luminal [Tb3+] (Kd ~ 500 nM, Hill coeff.
~4). These results are consistent with the idea that a multimeric
form of Casq2 acts as luminal divalent cation sensor and translates
it into changes in RyR2 gating.
Casq2 acts as a luminal Ca2+ sensor that controls
RyR2 open probability as a function of [Ca2+]
inside the sarcoplasmic reticulum.
Electrophysiology:
Planar lipid bilayers were formed across a 150 μm diameter
opening in the wall of a Delrin cup. Initial solutions were 400 mM
CsCH3SO3 with 10 mM HEPES (pH 7.4) in the cis (cytosolic)
compartment and 20 mM CsCH3SO3 with 10 mM HEPES in the
trans (SR) compartment. After incorporation of RyR2, the trans
compartment was adjusted to 400 mM CsCH3SO3 with 10 mM
HEPES (pH 7.4). A patch-clamp amplifier (Axopatch 200 B, Axon
Instruments, Ca) was used to measures currents through ionic
channels. The recordings were filtered at 10 kHz and sampled at
200 kHz with 12 bit analog to digital converter .
Biochemistry of Microsomes:
Lanthanide Spectroscopy:
Steady state spectroscopic experiment were performed on a
spectrofluorometer (PTI, Canada). Lanthanide lifetime
measurements were performed using a lifetime fluorometer for
lanthanides (Easylife L, PTI, Canada). Series of 500 UV pulses
(1ms) at a frequency of 250 Hz to the recombinant protein in
solution. The solution containing the recombinant Calsequestrine
was placed in a 180 ml quartz cuvette. The tryptophan's in the
protein were excited at 289 nm and the emitted light was
recorded at 540 nm for EYFP and at 560 nm for Tb3+. All
spectroscopic experiments were performed at room temperature.
How does Casq2 regulate RyR2?
Native Canine RyR2
Steady State Lanthanide Spectroscopy
Effect of Luminal Tb3+ on RyR2 Po
Casq2 wild type dogs
Is Mg2+ competing with Ca2+ for a binding site on Casq2?
Effect of Luminal Magnesium on RyR2 activity
RESULTS
0 500 1000 1500 2000 2500 3000 3500
0.0
0.2
0.4
0.6
0.8
1.0
time [us]
lanthanidefluorescencedecay
NormalizedLuminescence
[Ca2+]
Time [µs]
7F0
7F3
7F4
7F5
7F6
6D0
Triplet
Singlet
291 nm
500 ns
Terbium Emitting
State
luminescence
Energy transfer
Initial Plasmid pEyFP-CSQ2 w/
mammalian promoter
PCR-Recombination TEV site,His tag
pENTR/TEV/D-TOPO
Recombination to bacterial
T7promotor pDEST17
Plasmid Extraction
Sequencing w/ gene specific primers
Transformation BL21
Amplicillin Resistance
Induction to protein synthesis w/
L-Arabinose
Protein Purification Native Conditions,
Using His-tag Neutral pH and RT
AcTEV enzyme digestion to remove His
tag
Centrifuge homogenate
4,000g
Polytron Pellet
Centrifuge at 4,000g
Polytron Supernatant
Centrifuge at 12,000g
Polytron Supernatant
Centrifuge at 100,000g
Recover Pellet
homogenize
Aliquot sample into
cuvettes
Flash freeze sample
Store in -80 C
Dog heart was harvested and homogenized.
Homogenate was centrifuged at 4000 g for
20 minutes. The recovered pellet then
underwent homogenization and
centrifugation. Resulting supernatant was
centrifuged at 12000 g for 20 minutes.
Supernatant was retrieved from
centrifugation and centrifuged at 100000 g
for 1 hour. Supernatant was discarded and
pellet was recovered and homogenized.
350 400 450 500 550 600 650
350 400 450 500 550 600 650
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
10 pA
100 ms
2 µM 2 mM
2 µM luminal Ca2+
2 mM luminal Ca2+
luminal [Ca2+]
C
FractionalAmplitudeOpenProbability
N=4
At 2 µM cytosolic [Ca2+], an
increase of luminal [Ca2+] from 2 µM
to 2 mM produces a significant
increase in open probability (Po) and
reduction of single channel current
(pA) of RyR2 obtained from Native
Canine RyR2.
Lifetime Lanthanide Spectroscopy
Binding of Tb3+ to
recombinant dog Casq2
(rCasq2) results in an
increase of fluorescence
(λex=295 nm, λem=544
nm). The apparent Kd of
the process, obtained by
subtracting fluorescence
due to Tb3+ from the
fluorescence in the
presence of 1 µM rCasq2,
was 9.4 µM.
530 535 540 545 550 555 560
0.0
0.2
0.4
0.6
0.8
1.0
[Ca2+]
Wavelength [nm]
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
0.0
0.2
0.4
0.6
0.8
1.0 no rCasq2
1 µM rCasq2
Kd1 = 6.96 µM
Kd2 = 3532 µM
e
dw i e
Tb3+
Ca2+
UV (295 nm)
Ca2+ [µM]
FractionalrCasq2boundtoTb3+
Kd = 10.6 ± 3.5 μM
Tb3+ [μM]
FractionalrCasq2boundtoTb3+
0 2x10
1
4x10
1
6x10
1
8x10
1
0.0
0.2
0.4
0.6
0.8
1.0
0 500 1000 1500 2000 2500 3000 3500
0.0
0.2
0.4
0.6
0.8
1.0
Time [µs]
[Tb3+]
NormalizedLuminescence
0 500 1000 1500 2000 2500 3000 3500
0.0
0.2
0.4
0.6
0.8
1.0
[Mg2+]
Time [µs]
NormalizedLuminescence
Displacement experiments
were performed by
displacing Tb3+ from
rCasq2. The concentration
of rCasq2 was 500 nM and
the free Tb3+ 10 M. Tb3+
was displaced from the
coordination site for
increasing concentrations
of Ca2+ or Mg2+.
Native Canine RyR2
Wavelength [nm]
10
1
10
2
10
3
10
4
10
5
10
6
0.0
0.2
0.4
0.6
0.8
1.0
Ca2+ [ M]
FractionalrCasq2boundtoTb3+
Kd1 = 2.5 ± 1.8 mM
Kd2 = 56.1 ± 17.6 mM
N=6
e
dw i e
Tb3+
Ca2+
UV (289 nm)
10
-4
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
0.0
0.2
0.4
0.6
0.8
1.0
Kd = 9.4 µM
Tb3+ [ M]
FractionalCasq2boundtoTb3+
e
d
w i
e
Tb3+
UV (295 nm)
10 pA
100 ms
e
dw i e
Tb3+
Mg2+
UV (289 nm)
10
1
10
2
10
3
10
4
10
5
10
6
0.0
0.2
0.4
0.6
0.8
1.0
Mg2+ [ M]
Kd1 = 3.5 ± 2.1 mM
Kd2 = 172.1 ± 92.9 mM
N=5
FractionalrCasq2boundtoTb3+
0.0
0.2
0.4
0.6
0.8
1.0
0 mM 3 mM
Fractional Amplitude
N = 4
0 mM 3 mM
0.0
0.2
0.4
0.6
0.8
1.0
luminal Mg2+
Open Probability
Tb3+ bound to rCasq2 can be displaced by Ca2+in steady state.
This displacement occurs with two Kd’s, 6.96 µM and 3532 µM.
Cloning and expression of dog Casq2-EYFP fusion protein based on
Gateway System (Invitrogen)
Casq2 sequence illustrating the charged amino acids (+blue, -orange) that possibly interact with the Ca2+ binding
sites and the tryptophans (w; in purple) in the protein.
At 2 µM cytosolic [Ca2+], an increase of luminal [Mg2+] from 0 mM to 3
mM produces a significant increase in open probability (Po) and reduction
of single channel current (pA) suggesting Mg2+ permeates the RyR2 pore.
Increasing the [Tb3+] increases the luminescence due to the energy
transfer between the Tryptophans in rCasq and the lantanide. Here we
illustrate the slow relaxation of this process and the displacement by Ca2+.
1. Dog rCasq2 has at least one binding site for Tb3+.Tb3+
can be displaced by Ca2+, suggesting a common binding
site.
2. Luminal nM Tb3+ resembles the effect of luminal mM
Ca2+ by activating dog RyR2 independently from the feed
through mechanism.
3. Mg2+ competes with the Ca2+ binding site on Casq2.
4. Luminal mM Mg2+ regulates RyR2 activity (Po) similar to
the effect of luminal Ca2+.
5. These observations are consistent with Casq2 acting as
a luminal sensor by detecting intra-SR [Ca2+] changes
and translating them into changes of RyR2 Po.
ACKNOWLEDGEMENTS