Bustamante, Mario A.

Eltit, Jose M.

Estrada, Manuel

Jaimovich, Enrique

Elsevier - Publisher Connector 

120a Sunday, March 1, 2009612-Pos Board B491
Chromophore Formation of Fluorescence Proteins and Its Application of
Developing Ca2þ Sensors
Jin Zou, Malcom Delgado, Yun Huang, Shen Tang, Yusheng Jiang,
Blein McIntyre, Anne Wang, Jenny J. Yang.
Georgia State University, Atlanta, GA, USA.
Ca2þ regulates numerous biological processes through spatio-temporal changes
of cytosolic Ca2þ concentration and subsequent interactions with Ca2þ binding
proteins in living cells. There is a strong need to developCa2þ sensors capable of
real-time quantitative Ca2þ measurements in specific subcellular environments
without using natural Ca2þ binding proteins participating in signaling transduc-
tion.1, 2 Taking advantage of fluorescence proteins (FPs) as a useful tool, we cre-
ated a series of Ca2þ sensors by engineering a sensitive location of single FPwith
different color.3 Both spectroscopic properties including extinction coefficient,
quantum yield, and pKa and metal binding properties of engineered FPs were
identified with different spectroscopic methods including absorbance, fluores-
cence, and circular dichroism. The engineeredCa2þ sensors exhibit a ratiometric
fluorescence and absorbance changes upon Ca2þ binding with affinities corre-
sponding to the Ca2þ concentration found in the ER (Kd values range from 0.4 -
2 mM). The developed Ca2þ sensors have applied to monitor Ca2þ changes oc-
curring in various subcellular compartments including ER and mitochondria of
various mammalian cells upon response of different agonists. These sensors
were engineered into virus transfection system for its application to monitor cel-
lular Ca2þ signaling during muscle contraction and neuron events.
References
1. Mank, M. Griesbeck, O., Chem Rev 2008, 108, (5), 1550–1564.
2. Rockefort, N.L. Konnerth, A. Nat Methods 2008, 5, (9), 761–762.
3. Zou, J. Hofer, A.M. Lurtz, M.M. Gadda, G. Ellis, A.L. Chen, N. Huang, Y.
Holder, A. Ye, T. Louis, C.F. Welshhans, K. Rehder, V. Yang, J.J.,
Biochemistry 2007, 46, (43), 12275–12288.
613-Pos Board B492
Orai1 Channel And Ca2þ-independent Phospholipase A2 Are The New
Determinants Of Proliferation AndMigration Of Vascular SmoothMuscle
Cells
Bo Yang, Victoria M. Bolotina.
Boston University School of Medicine, Boston, MA, USA.
Ca2þ entry is known to play an important role in proliferation and migration of
vascular smooth muscle cells (SMC), but the molecular mechanisms that me-
diate these processes are far from being understood. Here we introduce
Ca2þ-independent phospholipase A2b (iPLA2b) and Orai1-encoded plasma
membrane channel (that mediate store-operated Ca2þ entry (SOCE) in non-
excitable as well as excitable cells) as new molecular determinants of
proliferation and migration of aortic SMC. Patch-clamp, Ca2þ imaging, immu-
nocytochemistry and molecular approaches were used in this study. We dem-
onstrated that molecular knock down of either iPLA2b or Orai1 (but not
TRPC1) channel resulted in full inhibition of store-operated current and
Ca2þ entry in primary aortic SMC. Transfection of SMC with siRNA to
Orai1 impaired their proliferation: Orai1 knock down resulted in 6954% de-
crease in the number of BrDU positive cells and 6051% reduction in the
rate of their proliferation. Similar effects were observed in iPLA2b-deficient
cells (6553% and 7154% reductions, respectively). Interestingly, transfec-
tion of SMC with antisense to TRPC1 did not affect SOCE, but significantly
reduced the rate of cell proliferation (by 4854%). Orai1 and TRPC1 produced
additive effects on proliferation, and knock down of both channels resulted in
7658% reduction in the rate of SMC proliferation, consistent with different
mechanisms for Orai1and TRPC1-mediated Ca2þ entry. Molecular knock
down of iPLA2b, or Orai1 channel also impaired SMC ability to migrate in re-
sponse to 20% FBS, high glucose, or SERCA inhibition. These results suggest
that SOCE mediated by iPLA2b-dependent activation of Orai1 channel in SMC
is required for their proliferation and migration, and may be involved in vascu-
lar SMC responses and angiogenesis triggered by different physiological and
pathological conditions.
614-Pos Board B493
Complexity Of Relationship Between STIM1, iPLA2b And Orai1 Expres-
sion, Puncta Formation And SOCEActivation In Native AndHeterologous
Systems
Tomasz Gwozdz, Joanna Dutko-Gwozdz, Vladislav Zarayskiy, Victoria
M. Bolotina.
Boston University School of Medicine, Boston, MA, USA.
STIM1, Orai1 and iPLA2b have been identified as crucial elements of the store-
operated Ca2þ entry (SOCE) pathway, but the mechanism of their functional
interaction and molecular requirements for SOCE activation remain controver-
sial. Here we used high resolution imaging, patch clamp, molecular and phar-macological approaches to study functional behavior and mutual relationship
between STIM1, Orai1 and iPLA2b in native cells and heterologous systems
in which fluorescently tagged STIM1 and/or Orai1 were over-expressed. We
found that STIM1 accumulated in puncta equally well in the presence or ab-
sence of Orai1, and STIM1 accumulation in puncta is not sufficient for Orai1
accumulation in the same areas. The normal ICRAC could be activated in
STIM1-deficient cells. We further found that the effects of C-terminus of
STIM1 may be profoundly different in native cells and in cells in which
Orai1 and/or full length STIM1 were over-expressed. Also, we found that while
inhibition of iPLA2b caused dramatic impairment of endogenous SOCE in na-
tive cells, its effects on SOCE in heterologous expressing systems may be less
prominent, and may require higher concentrations of inhibitors. Our new data
provide first indications of the potential differences in SOCE between native
cells and heterologous systems, and challenge the idea of a direct conforma-
tional coupling between STIM1 and Orai1 as a mechanism of puncta formation
and SOCE activation in native cells.
615-Pos Board B494
Membrane Depolarization of Skeletal Muscle Cells Induces IL6 and
SOCS3 mRNA Expression Through Calcium Dependent Stat3 Phosphor-
ylation
Mario A. Bustamante1, Jose M. Eltit1,2, Manuel Estrada1,
Enrique Jaimovich1.
1Centro de Estudios Moleculares de la Celula - Universidad de Chile,
Santiago, Chile, 2Harvard Medical School, Brigham and Women’s Hospital,
Boston, MA, USA.
Physical activity has been implicated as a major stimulus for Interleuquin 6
(IL6) expression and release from skeletal muscle in humans. However, the
pathways involved in these processes are not well established. Previously,
we have reported that potassium depolarization induces IL6 mRNA expression
in cultured rat myotubes, Ca2þ being an important second messenger for this
expression, possibly acting through CREB, NFkB and AP-1.
In other cells systems, STAT3 is a key protein in signaling mediated by IL6,
and it has been implicated in the IL6 autocrine up-regulation. Some recent re-
ports have shown a possible calcium regulation of STAT3 tyr 705 phosphory-
lation, and others have implied GSK3b as a regulator of STAT3 activation; in
any case, the mechanisms for STAT3 regulation have not been demonstrated.
We report calcium-dependent regulation of STAT3, induced by membrane de-
polarization as a result of electrical stimulation of cultured rat myotubes. Two
peaks of STAT3 phosphorylation (5 and 60 minutes) were identified, when an
external electrical field (400 1 ms pulses at 45 Hz) was applied. When myo-
tubes were stimulated with 40 mM Caffeine, a similar STAT3 activation was
observed, indicating that Ca2þ is necessary for STAT3 phosphorylation. On
the other side, overexpression of the Ca2þ chelating protein parvalbumin, not
only completely inhibited the activation of STAT3, but also diminished by
30% the basal levels of STAT3 phosphorylation.
Wealso showsomepreliminary results depicting inhibitionof STAT3phosphor-
ylation by lithium, a well known GSK3b inhibitor, suggesting that this pathway
could be playing a role in STAT3 activation by membrane depolarization.
FONDAP # 15010006
616-Pos Board B495
Isoproterenol-Enhanced Diastolic Sarcoplasmic Reticulum Ca Leak in
Ventricular Myocytes Requires Activation of Nitric Oxide Synthase
Jerry Curran1, Usama Ahmed1, Donald M. Bers2, Mark Ziolo3, Thomas
R. Shannon1.
1Rush University Medical, Chicago, IL, USA, 2University of California,
Davis, Davis, CA, USA, 3Ohio State University, Columbus, OH, USA.
We have previously shown increased cardiac ryanodine receptor (RyR)-depen-
dent diastolic SR Ca leak to be present in heart failure (HF) and in conditions
when beta-adrenergic (b-AR) tone is high. This SR Ca leak could contribute to
the cause of the observed decreased contractility in HF by limiting SR Ca load.
Simultaneously, it could also lead to arrhythmogenic Ca-dependent inward de-
polarizing current commonly seen in failing hearts. We recently demonstrated
that this leak increases in manner dependent on calcium-calmodulin-dependent
protein kinase II (CaMKII) and completely independent of either protein kinase
A (PKA) activation or an increase in bulk free Ca concentration ([Ca]i). Here
we investigate this PKA- and [Ca]i-independent activation of CaMKII. We
have found that while stimulating intact myocytes with the b-AR agonist iso-
proterenol (ISO, 250 nM) the CaMKII-dependent enhancement of SR Ca
leak is abolished by treatment of the myocytes with nitric oxide synthase
(NOS) inhibitor Nu-Nitro-L-arginine methyl ester (L-NAME, 100 mM).
When SR Ca load was matched in each group (156 mM), myocytes treated
with ISO alone had significantly higher leak (14.25 2.0 mM) vs. those treated
with ISO and L-NAME (3.8 5 1.4 mM) or those left completely untreated


Membrane Depolarization of Skeletal Muscle Cells Induces IL6 and SOCS3 mRNA Expression Through Calcium Dependent Stat3 Phosphorylation 

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Membrane Depolarization of Skeletal Muscle Cells Induces IL6 and SOCS3 mRNA Expression Through Calcium Dependent Stat3 Phosphorylation

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