Ahuja, Malini

Jha, Archana

Muallem, Shmuel

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570a Wednesday, February 11, 2015constants (t) of calcium-transient decay among cell neighborhoods at baseline
and during stimulation of either beta-adrenergic receptors or cholinergic
receptors.
Beta-adrenergic receptor stimulation (isoproterenol) not only decreased cycle
length (CL) and average t vs. baseline, but also decreased the standard devia-
tion (SD) of all t’s across all neighborhoods, suggesting a shift into a less
heterogeneous, i.e. more synchronized calcium pumping throughout SANC.
Conversely, cholinergic receptor stimulation (carbachol) not only increased
CL and average t vs. baseline, but also increased the local SD(t), suggesting
a shift into a more heterogeneous, i.e. less synchronized calcium pumping
within SANC. Furthermore, on a beat-to-beat basis, the relationship between
either t or SD(t) and CL was linear under both baseline conditions and auto-
nomic stimulation.
Conclusions: The degree of heterogeneity of local calcium pumping is a new
universal factor that affects the CL and insures effective rate and rhythm
regulation of the coupled-clock pacemaker system via autonomic modulation.
More synchronized and faster calcium pumping is presumably achieved via
phospholamban phosphorylation and allows cell neighborhoods to reach
the calcium release threshold quicker and more synchronously, thereby syn-
chronizing LCRs and amplifying their ensemble diastolic signal, accelerating
pacemaker rate.
2884-Pos Board B314
Excitation-Metabolism Coupling in Mouse Heart
Andrew P. Wescott, W.J. Lederer, George S.B. Williams.
BioMET, University of Maryland Baltimore, Baltimore, MD, USA.
A beating heart must balance energy consumption with production. This
balance requires cellular ATP production to respond dynamically to changes
in cardiac output and energy demand. While it is well understood that mito-
chondrial oxidative phosphorylation serves as the primary pathway for ATP
production in heart, the mechanisms that govern the regulation of mitochon-
drial metabolism remain unclear. Calcium (Ca2þ), specifically Ca2þ in the
mitochondrial matrix ([Ca2þ]m), has been implicated as a likely signaling
pathway for this regulation. [Ca2þ]m has been suggested to regulate nearly
every stage of mitochondrial metabolism including the activity of the tricarbox-
ylic acid cycle (TCA) via Ca2þ-sensitive dehydrogenases (DHOs), critical
proteins in the electron transport chain (ETC), and even the F1/F0 ATP synthase
itself. Here we expand our local control mathematical model of excitation-
contraction coupling in mouse heart to investigate excitation-metabolism
coupling. The model features mechanistic initiation and termination of Ca2þ
sparks and cytosolic Ca2þ ([Ca2þ]i) transients in a system that maintains
sarcoplasmic reticulum (SR) Ca2þ pump/leak balance. Mitochondrial Ca2þ up-
take and export is simulated using experimentally constrained formulations
of the mitochondrial Ca2þ uniporter (MCU) and the mitochondrial sodium/
Ca2þ exchanger (NCLX). The model employs key mechanisms of ATP con-
sumption (i.e. SR Ca2þ ATPase and myosin ATPase) and buffering (phospho-
creatine) as well as a Ca2þ dependent model for mitochondrial ATP generation.
We investigated mitochondrial Ca2þ dynamics at physiological pacing fre-
quencies for mouse heart and show that changes in ATP consumption can be
translated to increased energy production through [Ca2þ]m signals. Our model
provides new insights into mitochondrial Ca2þ dynamics and how these
[Ca2þ]m signals may function to preserve energy homeostasis in the face of
increased demand.
2885-Pos Board B315
Similarities and Differences in Gating of the Two-Pore Channels TPC1
and TPC2
Archana Jha, Malini Ahuja, Shmuel Muallem.
nidcr,nih, rockville, MD, USA.
Endolysosomal Ca2þ homeostasis is implicated in several diseases and controls
many endolysosomal functions. A key to understanding endolysosomal Ca2þ
signaling to understand the function of the newly discovered endolysosomal
PI(3,5)P2-regulated two-pore channels (TPCs) and their potential activation
by NAADP. Our recent work concluded that the lysosomal TPC2 function as
a NAADP and PI(3,5)P2-activated channel regulated by cytoplasmic Mg
2þ.
The properties and gating of the mostly endosomal TPC1 are not known.
Recording whole-organelle currents of enlarged endosomes expressing GFP-
TPC1, we discovered that TPC1 is potently activated by PI(3,5)P2 and
NAADP. Moreover, PI(3,5)P2 facilitates TPC1 activation by NAADP. Unlike,
TPC2, the activity of TPC1 showed poor inhibition by Mg2þ. Most notably, the
concentration dependence of activation of TPC1 and TPC2 by NAADP are
remarkably different. While activation of TPC2 by NAADP followed normal
saturation dependence with no sign of inhibition by high NAADP concentra-
tion, activation of TPC1 by NAADP followed a bell shaped dependency,
resembling the NAADP-mediated Ca2þ release in intact cells. These findingsprovide the first direct evidence for gating of TPC1 by NAADP and indicate
that endosomal, rather than lysosomal, Ca2þ release is the key trigger Ca2þ
pool that sensitizes the IP3-mediated Ca
2þ release from the ER. The interaction
between TPC1-expressing endosomes and the ER are being examined.
2886-Pos Board B316
Mitochondrial Calcium and Bioenergetics Controlled by Tight Coordina-
tion of MCU and NCLX
Ming-Feng Tsai, Chuck Phillips, Christopher Miller.
Brandeis University, waltham, MA, USA.
Mitochondrial Ca2þ is regulated by Ca2þ transport proteins in the mitochon-
drial inner membrane. Among these are the mitochondrial calcium uniporter
(MCU), a Ca2þ channel responsible for most Ca2þ uptake in mitochondria,
and NCLX, a Naþ/Ca2þ exchanger, mediating Ca2þ release to the cytosol.
After more than five decades of intensive study, the MCU gene was finally
cloned in 2010. Strikingly, MCU-KO mouse, whose mitochondria lose
>90% Ca2þ uptake capacity, was born normally with very mild phenotype
and only slight reduction of mitochondrial Ca2þ. How does the animal maintain
normal physiology without MCU, which is highly conserved and tightly regu-
lated in most eukaryotes?
To address this issue, we use an MCU-KO HEK293T line as our model system.
Consistent with animal experiments, these cells have normal mitochondrial
morphology, matrix [Ca2þ], and ATP production via oxidative phosphoryla-
tion. Interestingly, we observed a slow Ca2þ uptake in MCU-KOmitochondria,
operating at 5-10% of the MCU capacity, resistant to a potent MCU inhibitor
Ru360. Furthermore, these mitochondria lose Naþ-dependent Ca2þ release,
due to decreased expression of the NCLX protein. We therefore propose that
mitochondria compensate the loss of MCU by invoking a backup Ca2þ uptake
mechanism, and by suppressing Ca2þ release. Supporting this hypothesis, we
show that expressing NCLX in MCU-free mitochondria drastically reduces
matrix [Ca2þ], and abolishes >80% ATP output. Moreover, overexpression
of WT-NCLX, but not a loss-of-function mutant S468A-NCLX, induces rapid
death of MCU-KO cells.
We’re now investigating how mitochondria invoke these compensatory
mechanisms. Naþ/Ca2þ exchange can be restored by introducing human
MCU gene into MCU-KO cells, but not by expressing transport-capable
MCU homologues from A. thaliana or D. Discoideum, implicating that
mitochondria might balance Ca2þ uptake and release through an MCU-
NCLX interaction that stabilizes inner-membrane expression of NCLX.
Nucleo-Cytoplasmic Transport
2887-Pos Board B317
Observing Signal Transduction Directly at the Single-Molecule Level in
Live Eukaryotic Cells
Adam J.M. Wollman1, Sviatlana Shashkova2, Erik Hedlund1,
Stefan Hohmann2, Mark C. Leake1.
1Departments of Physics and Biology, University of York, York, United
Kingdom, 2Department of Chemistry & Molecular Biology, University of
Gothenburg, Gothenburg, Sweden.
All cells dynamically sense their environment through signal transduction
mechanisms, allowing them to respond to environmental changes at the genetic
level. The glucose sensory mechanism in Sacchoromyces cerevisiae is a model
system of signal transduction. It uses the multi-copy inhibitor of the GAL gene
expression Mig1 protein to repress unwanted genes in the presence of elevated
level of extracellular glucose. In wild-type cell strains, and in genetically
and biochemically impaired signal transduction phenotypes, we have fluores-
cently labelled the Mig1 protein with the green fluorescent protein GFP via
chromosomal integration at native levels of expression, in addition to the
RNAP nuclear reporter protein Nrd1 with the red fluorescent protein mCherry.
[1] Using millisecond dual-color fluorescence excitation with Slimfield
microscopy, [2] we track the fate of single molecules of Mig1 in live yeast cells
at millisecond timescales. We present data showing a consistent difference be-
tween confined, high stoichiometry assemblages of bound Mig1 in the nucleus
and freely diffusing single Mig1 molecules across the entire cell as well as more
complex behavior in Mig1 traversing the nuclear membrane. The mobility and
stoichiometry distribution of Mig1 assemblages we observe is functionally
dependent on the signal transduction pathway and the concentration of extra-
cellular glucose, allowing us to directly observe gene regulatory events.
1. Bendrioua, L. et al. Yeast AMP-activated Protein Kinase Monitors Glucose
Concentration Changes and Absolute Glucose Levels. J. Biol. Chem. 289,
12863-75 (2014).
2. Reyes-Lamothe, R., Sherratt, D. J. & Leake, M. C. Stoichiometry and archi-
tecture of active DNA replication machinery in Escherichia coli. Science 328,
498-501 (2010).


Similarities and Differences in Gating of the Two-Pore Channels TPC1 and TPC2 

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Similarities and Differences in Gating of the Two-Pore Channels TPC1 and TPC2

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