Amcheslavsky, Anna

Cahalan, Michael D.

Hu, Junjie

Yeromin, Andriy V.

Zhang, Shenyuan L.

Zheng, Hongying

Elsevier - Publisher Connector 

Activation of Orai1 Channels by Mutation of a Conserved Glycine Residue in TM1 

182a Monday, March 7, 2011Orai3-like gating characteristics, in a strongly cooperative manner. In conclu-
sion Orai subtype-specific gating requires a cooperative interplay of all three
cytosolic domains.
1000-MiniSymp
Molecular Determinants of CRAC Channel Gating
Beth McNally, Megumi Yamashita, Murali Prakriya.
Northwestern University, Chicago, IL, USA.
Store-operated Ca2þ release-activated Ca2þ (CRAC) channels regulate numer-
ous cellular functions including transcription, motility, and proliferation in
many cell types. The discoveries of Orai1, the CRAC channel pore subunit,
and STIM1, the ER Ca2þ sensor, have produced rapid progress in our under-
standing of the molecular features of the CRAC channel pore and the cellular
events involved in channel activation. It is now known that following depletion
of intracellular Ca2þ stores, STIM1 activates CRAC channels by interacting
with the cytoplasmic N- and C-terminal domains of Orai1. Orai1 and Orai3
CRAC channels can additionally be activated in a store-independent fashion
by the compound 2-APB. However, the molecular and structural mechanisms
of STIM1- and 2-APB-mediated gating remain poorly understood. Using
a combination of site-directed mutagenesis and cysteine accessibility analysis,
we are probing the structural alterations in the pore that occur during STIM1- or
2-APB-mediated activation of Orai1 and Orai3 channels. Our data indicate that
the pore of the CRAC channel changes significantly as it transitions from the
closed to the open state. Specifically, STIM1 binding causes large modification
of the pore architecture, giving rise to features classically associated with
CRAC channels such as its narrow pore and high Ca2þ selectivity. Thus, in
addition to serving as the ER Ca2þ sensor and activator of the CRAC channel,
STIM1 appears to function as a channel subunit, modifying the structural fea-
tures of the pore to confer its unique permeation profile in the active state.
1001-MiniSymp
Activation of Orai1 Channels byMutation of a Conserved Glycine Residue
in TM1
Shenyuan L. Zhang1,2, Andriy V. Yeromin1, Junjie Hu3,
Anna Amcheslavsky1, Hongying Zheng2, Michael D. Cahalan1.
1University of California, Irvine, Irvine, CA, USA, 2Texas A&M Health
Science Center, Temple, TX, USA, 3Nankai University, Tianjin, China.
Stim and Orai proteins comprise the molecular machinery of Ca2þ release-acti-
vated Ca2þ (CRAC) channels. In T-lymphocytes, Orai1 is the pore-forming sub-
unit and STIM1 serves as the ER Ca2þ-sensor that opens Orai1 following ER
Ca2þ-store depletion. We are investigating mutations within Orai1 TM1 that
alter channel activity in transfected HEK cells. At the extracellular side of
TM1, we and others had previously shown that E106 confers Ca2þ selectivity
on the STIM1-operated Orai1 current. We now show that R91, the site of the
R91W mutation that causes severe combined immune deficiency, forms a very
narrow part of the conducting pore at the cytosolic side of TM1. Orai1 R91C
when co-expressedwith STIM1was activated normally byCa2þ store depletion.
However, treatment with diamide, a thiol-oxidizing agent, induced formation of
disulfide bonds between R91C residues in adjacent Orai1 subunits. Moreover,
diamide rapidly blocked STIM1-operated Ca2þ current, and current recovered
during treatmentwith a reducing agent. In themiddle of TM1,mutation of a con-
served glycine residue to alanine (G98A) prevented STIM1-induced channel
activity. Interestingly, mutation to aspartate (G98D) caused constitutive channel
activation in a STIM1-independentmanner to form a non-selectiveCa2þ-perme-
able conductance that was relatively resistant to block by Gd3þ (310 nMKd vs 7
nM inwild-typeOrai1).Moreover, the doublemutantR91WG98Dwas also con-
stitutively active, overcoming the normal inhibition of channel activity by tryp-
tophan at the 91 position; and the double mutant R91CG98D was resistant to
diamide block.We tentatively propose that R91 forms the gate of theOrai1 chan-
nel at the narrow inner mouth of the channel, G98 functions as a gating-hinge,
and E106 promotes selective conduction of Ca2þ. The channel pore is widened
and ion selectivity perturbed by a negative charge at the G98 site.PLATFORMW: Imaging&OpticalMicroscopy I
1002-Plat
Circular Scanning Fluorescence Correlation Microscopy on Membranes
Zdenek Petrasek, Susan Derenko, Petra Schwille.
TU Dresden, Dresden, Germany.
In circular scanning Fluorescence Correlation Spectroscopy (FCS) the mea-
surement volume is scanned in a circle with a sub-micron radius, allowing de-
termination of the diffusion coefficient and concentration without any a priory
knowledge of the size of the detection volume. This feature is particularly im-
portant in measurements on two-dimensional surfaces, where the volume size,and therefore the quantitative outcome of the experiment, is determined by the
relative position of the surface and the objective focus, a parameter difficult to
control in practice. We have implemented this technique in a simple instrument
based on a 2D piezo scanner, and applied it to studies of molecular diffusion in
model systems, supported lipid bilayers and giant unilamellar vesicles, and in
living cells. The method is shown to be minimally sensitive to disturbing inter-
ferences due to fast kinetics (dye photophysics) and long-time effects (axial
membrane motion, depletion due to photobleaching), and yields reliable results
even for tilted or fluctuating membranes. The resulting robustness makes this
technique particularly suitable to applications in living organisms.
1003-Plat
Probing T-Tubular Electrophysiology by Random Access Two-Photon
Microscopy in Cardiac Myocytes
Leonardo Sacconi1, Raffaele Coppini2, Cecilia Ferrantini2, Jacopo Lotti1,
Chiara Tesi2, Ping Yan3, Leslie M. Loew3, Elisabetta Cerbai2,
Corrado Poggesi2, Francesco S. Pavone1.
1University of Florence, Sesto Fiorentino (FI), Italy, 2University of Florence,
Florence, Italy, 3University of Connecticut Health Center, Farmington,
CT, USA.
In cardiac cells many membrane channels are heterogeneously distributed be-
tween Surface and T-tubule membranes. Simultaneous recording of membrane
potential in the two sarcolemma domains can reveal potential peculiarities of
T-tubule electrophysiology. This is not feasible with traditional electrophysio-
logical techniques. Current optical techniques for recordingmembrane potential
can potentially overcome the microelectrode limitation. However, most ap-
proaches to the optical recording of membrane potential events lack the spatial
and temporal resolution needed for regional assessment of action potential (AP)
profile. Here, we developed an ultrafast random access two-photon microscope
capable of optically recording fast membrane potential transients inmultiple po-
sitions of the cell membrane with mm spatial resolution. The random access mi-
croscope, in combination with a novel voltage sensitive dye, was used to
simultaneously record AP in surface sarcolemma and T-tubules in isolated car-
diacmyocyteswith sub-millisecond time resolution.We found that inmyocytes,
paced at 0.2 Hz, the AP in the T-tubule has identical amplitude and kinetics as in
the surface sarcolemma, indicating that the tight electrical coupling between the
two membrane domains prevails over the inhomogeneous distribution of mem-
brane currents. Consistently, in myocytes that had been acutely detubulated by
formamide-induced osmotic shock, T-tubule AP was absent, indicating a com-
plete uncoupling from the surface sarcolemma. The electrophysiological
properties of t-tubules may be altered in pathological conditions, when detubu-
lation and T-tubule remodelling occur. To mimic a model of pathological detu-
bulation, myocytes were cultured for 24-36 hours, thus obtaining a significant
loss and disorganization of the T-tubular network.Membrane staining confirmed
the loss and morphological alterations of T-tubules; however, the electrical
activity in the remaining remodelled T-tubules was preserved, suggesting that
remodelled T-tubules were still coupled to the surface sarcolemma.
1004-Plat
Fluorescence Imaging of Influenza Virus H1N1 mRNA in Living Infected
Cells using Single Chromophore FIT-PNA
Susann Kummer, Andrea Knoll, Elke Socher, Lucas Bethge,
Andreas Herrmann, Oliver Seitz.
Humboldt University, Berlin, Germany.
Replication of the influenza virus involves, amongst other critical steps, the
synthesis of viral mRNAwhich is used for ribosomal synthesis of viral proteins.
Significant efforts have been devoted to the development of methods that allow
the imaging of specific mRNA sequences in living cells. In contrast to other ap-
proaches, fluorescent oligonucleotide probes enable the analysis of non-modi-
fied wild-type targets. For imaging the probes must recognize the target with
high specificity and deliver measurable signals that provide high signal-to-
background ratios. We have introduced single labelled peptide nucleic acid
(PNA) probes, so-called FIT-PNA probes, which contain a single thiazole or-
ange intercalator that serves as artificial fluorescent nucleobase. These probes
respond to changes of the local structure in the vicinity of the dye rather than
to the more global changes of conformation that are required for fluorescence
signalling by the dual labelled molecular beacons. FIT-probes are unique be-
cause a single fluorophore provides for both high sensitivity and high target
specificity at non-stringent hybridization conditions where both matched and
mismatched probe-target complexes coexist. We show that FIT-probes are ide-
ally suited for applications in live cell RNA imaging. We chose mRNA coding
for neuraminidase of influenza virus A/PR/8 as a target. FIT-PNA probes are
useful to detect the expression of viral mRNA in single living infected cells
with high specificity. In particular, FIT-PNA sensitivity was superior to that
of a molecular beacon. Our study suggests that FIT-PNAs are attractive tools


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Activation of Orai1 Channels by Mutation of a Conserved Glycine Residue in TM1

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