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J Biol Chem, Vol. 275, Issue 9, 6047-6050, March 3, 2000
From the Departments of Medicine and Physiology, Cardiovascular
Research Institute, University of California, San Francisco California
94143 and the We report the application of a targetable green
fluorescent protein-based cellular halide indicator. Fluorescence
titrations of the purified recombinant yellow fluorescent protein
YFP-H148Q indicated a pKa of 7.14 in the absence of
Cl Transport of Cl The existing Cl Recently a mutant form of GFP was introduced, the "yellow fluorescent
protein" (YFP), which contains four points mutations and has
red-shifted excitation and emission spectra compared with GFP (20, 21).
Analysis of YFP crystallographic structure indicated several cavities
in the vicinity of the chromophore and that the substitution of His148
to Gly permitted solvent access to the chromophore (22). It was found
that YFP fluorescence was sensitive not only to pH, but also to various
anions (23). The purpose of this study was to establish the mechanism
of YFP Cl Expression and Purification of YFP-H148Q Protein--
The H148Q
mutant of YFP was prepared using the polymerase chain reaction-based
QuickChangeTM site-directed mutagenesis kit (Strategene)
using plasmid pRSETB and a GFP template containing the mutations
T203Y/S65G/V68L/S72A (22). YFP-H148Q protein was expressed in
Escherichia coli BL21(DE3) and purified by nickel-affinity chromatography.
Spectroscopic Measurements--
Absorbance measurements
were carried out on a HP8452 photodiode array spectrophotometer
(Hewlett-Packard). Steady-state fluorescence measurements were done on
an SLM 8000C spectrofluorimeter (SLM Instruments, Urbana, IL).
Titrations of YFP-H148Q fluorescence versus pH and
[Cl Stopped-flow Kinetic Measurements--
The time course of
YFP-H148Q fluorescence in response to changes in pH and
[Cl Photobleaching Recovery Measurements--
Spot photobleaching
measurements were carried out on an apparatus with microsecond time
resolution as described previously (25). YFP-H148Q fluorescence was
excited by an argon ion laser (488 nm) and detected by a gated
photomultiplier using 530 ± 15 nm interference and 515 nm cut-on
filters. Measurements were performed by epifluorescence microscopy (× 20 or × 40 objectives) on 5-µm-thick layers of solution between
silica coverslips.
Cell Culture and Transfection--
Swiss 3T3 fibroblasts (ATCC
CCL 92) and Swiss 3T3 fibroblasts expressing CFTR were grown on 18-mm
diameter round glass coverslips at 37 °C (95% air, 5%
CO2) in Dulbecco's modified Eagle's medium H21 medium
supplemented with 10% fetal bovine serum. The YFP-H148Q coding
sequence was subcloned into mammalian expression vector pcDNA3.1
(Invitrogen). Cells were transfected 2-3 days after plating on
collagen-coated glass coverslips (at ~80% confluence) with 1 µl of
plasmid DNA and 8 µl of LipofectAMINE reagent (Life Technologies, Inc.) in a 0.2-ml volume of Opti-MEM (Life Technologies, Inc.). The
transfection medium was replaced at 5 h with 1 ml of culture medium. Cells were used at 2-3 days after transfection.
In Vivo Calibrations and Transport Measurements--
Coverslips
containing transfected cells were perfused in a laminar-flow chamber at
3-6 ml/min (37 °C) as described previously (12). Cell fluorescence
was measured continuously using an inverted epifluorescence microscope
(Nikon Diaphot) with an air objective (Nikon Plan-Apo × 20, N.A.
0.75), HQ filter set (480 ± 20 nm excitation, 495 nm dichroic,
510 ± 20 nm emission) and photomultiplier detector. Gluconate/Cl Fig. 1A shows
steady-state fluorescence pH titrations of purified YFP-H148Q in
aqueous solutions containing specified [Cl Fig. 1B shows the decrease in YFP-H148Q fluorescence with
increasing [Cl
ACCELERATED PUBLICATION
Mechanism and Cellular Applications of a Green Fluorescent
Protein-based Halide Sensor*
,, and Institute of Molecular Biology and
Department of Physics, University of Oregon, Eugene, Oregon 97403
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, which increased to 7.86 at 150 mM
Cl. At pH 7.5, YFP-H148Q fluorescence decreased maximally
by ~2-fold with a KD of 100 mM
Cl. YFP-H148Q had a fluorescence lifetime of 3.1 ns that
was independent of pH and [Cl]. Circular dichroism and
absorption spectroscopy revealed distinct Cl-dependent spectral changes indicating
Cl/YFP binding. Stopped-flow kinetic analysis showed a
biexponential time course of YFP-H148Q fluorescence (time constants
<100 ms) in response to changes in pH or [Cl],
establishing a 1:1 YFP-H148Q/Cl binding mechanism.
Photobleaching analysis revealed a millisecond triplet state relaxation
process that was insensitive to anions and aqueous-phase quenchers. The
anion selectivity sequence for YFP-H148Q quenching
(ClO4 ~ I > SCN > NO3 > Cl > Br > formate > acetate)
indicated strong binding of weakly hydrated chaotropic ions. The
biophysical data suggest that YFP-H148Q anion sensitivity involves
ground state anion binding to a site close to the tri-amino acid
chromophore. YFP-H148Q transfected mammalian cells were brightly
fluorescent with cytoplasmic/nuclear staining. Ionophore calibrations
indicated similar YFP-H148Q pH and anion sensitivities in cells and
aqueous solutions. Cyclic AMP-regulated Cl transport
through plasma membrane cystic fibrosis transmembrane conductance
regulator Cl channels was assayed with excellent
sensitivity from the time course of YFP-H148Q fluorescence in response
to extracellular Cl/I exchange. The green
fluorescent protein-based halide sensor described here should have
numerous applications, such as anion channel cloning by screening of
mammalian expression libraries and discovery of compounds that correct
the cystic fibrosis phenotype by screening of combinatorial libraries.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
across cell plasma membranes is
required in many cellular processes such as cell volume regulation and
epithelial fluid absorption and secretion. Defective Cl
transport occurs in hereditary and acquired human diseases such as
cystic fibrosis (CFTR1
deficiency), nephrolithiasis (ClC5 deficiency), and cholera. Cl transport across intracellular vesicle membranes in
the endosomal and secretory compartments has been proposed to
facilitate vesicular acidification and regulate trafficking events.
However, there have been no measurements of Cl
concentration or transport in subcellular organelles in living cells.
Cl concentration in cytoplasm and Cl
transport across cell plasma membranes has been measured using Cl sensitive fluorescent indicators. Our laboratory
introduced quinolinium-type Cl indicators whose
fluorescence is quenched by Cl by a collisional mechanism
(1, 2). These indicators have been used widely to study
Cl transport mechanisms in mammalian cells, including
cells expressing mutant CFTR Cl channels and cells
obtained from human subjects in CFTR gene therapy trials (1). Various
cell-permeable (3), ratiometric (4), and long wavelength (5, 6) halide
indicators have been synthesized to facilitate cell measurements.
/halide indicators require cell loading,
are not retained perfectly within cells, and cannot be targeted easily to subcellular organelles. An alternative to exogenously added indicators is the use of an endogenously expressed chromophore such as
green fluorescent protein (GFP). Using appropriate targeting sequences,
GFPs have been targeted selectively to numerous intracellular sites
(7-12). The intrinsic pH sensitivity of various GFP mutants has been
exploited to study pH regulation in cytoplasm and intracellular organelles (12-14). Various tailored GFP-based indicators have been
introduced for measurement of calcium concentration (15, 16), membrane
potential (17), and protease activity (18, 19).
sensitivity by spectroscopic measurements on
purified protein and to examine the utility of YFP as an intracellular
Cl/halide sensor. The mutant YFP-H148Q was studied
because of its relatively high Cl sensitivity at
cytoplasmic pH. Steady-state titrations, time-resolved fluorescence,
circular dichroism, photobleaching, and stopped-flow kinetic analysis
indicated a ground-state Cl/YFP-H148Q binding mechanism.
Anion transport measurements in mammalian cells expressing YFP-H148Q
established the utility of YFP-H148Q as a cellular halide indicator.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
] were performed by cuvette fluorimetry. Purified
YFP-H148Q (3-5 µg/ml) was dissolved in 10 mM MES, 10 mM MOPS containing specified [Cl] (0-400
mM) (counter ion sodium) and titrated to specified pH with
H2SO4/NaOH. The pH titration data were fitted
to the equation, F = A + B [1 + 10(pKa 
pH)nH]1,
where nH (Hill coefficient) and
pKa are fitted parameters. Titration data for
Cl and other anions were fitted to a single site binding
model, Y = a
[X]/(KD + [X]). Time-resolved fluorescence
measurements were carried out in the frequency domain using a Fourier
transform fluorimeter (48000 MHF, SLM instruments). YFP-H148Q
fluorescence was excited at 488 nm using an argon ion laser and
detected using a >520-nm cut-on filter. Lifetime measurements were
done using fluorescein in 0.1 N NaOH as reference, and
time-resolved anisotropy was measured using a rotatable analyzing
polarizer as described previously (24). Circular dichroism (CD) spectra
for YFP-H148Q (0.3 mg/ml in 2-mm path length quartz cuvette) were
recorded using a Jasco CD spectrometer. CD measurements were performed
in 4 mM
NaH2PO4/Na2HPO4 containing specified anion concentrations in which ionic strength was
maintained using Na2SO4 (since gluconate is asymmetric).
] was measured using a Hi-Tech SF51 stopped flow
apparatus. YFP-H148Q (0.3 mg/ml) in buffer A (10 mM MOPS)
at specified initial pH and [Cl] was mixed in <1 ms
with an equal volume of buffer B (10 mM MES at appropriate
pH and [Cl]) to give specified final (after mixing) pH
and [Cl]. Excitation was at 500 ± 20 nm, and
emission was filtered by a >530-nm cut-on filter and collected by a
photomultiplier. Fluorescence versus time data were fitted
to mono- and biexponential functions by nonlinear regression.
,
Cl/NO3 and
Cl/I exchange studies were performed by
perfusing the cells with PBS (137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 1.5 mM
KH2PO4, 8.1 mM
Na2HPO4, pH 7.4) and then with buffer in which
100 mM Cl was replaced by gluconate,
NO3 or I. For in
vivo calibration of pH, the perfusate contained 120 mM potassium gluconate, 20 mM sodium gluconate, 20 mM Hepes, 0.5 mM CaCl2, 0.5 mM MgSO4, 5 µM nigericin, 5 µM valinomycin, 5 µM CCCP, 10 µM tributyltinchloride, and 20 µM forskolin
with the pH adjusted to 6.5-8.0 (in 0.5 pH unit intervals).
Calibration of in vivo Cl sensitivity was
performed using a high K+ buffer containing 100 mM K+, 38 mM Na+,
indicated Cl concentrations (remaining anion gluconate),
5 µM nigericin, 5 µM valinomycin, 5 µM CCCP, 10 µM tributyltinchloride, and 20 µM forskolin, pH 7.4.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
]. In the
absence of Cl, the data fitted well to a single site
titration model (Hill coefficient 0.90) with pKa
7.14 ± 0.02. Apparent pKa values increased by
nearly one unit with increasing [Cl] with Hill
coefficients remaining near unity. The increased pKa with Cl indicates stabilization of the protonated form of
YFP-H148Q.
View larger version (34K):
[in a new window]
Fig. 1.
Sensitivity of YFP-H148Q spectral properties
to Cl . A, dependence of YFP-H148Q
fluorescence on pH at indicated [Cl]. B,
dependence of relative YFP-H148Q fluorescence on [Cl]
or [I] at pH 7.5. C, influence of
Cl on YFP-H148Q absorption spectrum at pH 6.4. D, influence of fluoride on YFP-H148Q CD spectrum.
] at constant pH. At a typical
cytoplasmic pH, a 10% change in fluorescence occurs for an increase in
[Cl] from 0 to 40 mM. The fluorescence
decrease was saturable with a maximal ~2-fold reduction in
fluorescence at high [Cl]. The fluorescence intensity
of YFP-H148Q was more sensitive to I with a >50%
decrease in fluorescence at 30 mM I. Similar
titrations at pH 7.5 indicated that YFP-H148Q fluorescence is sensitive
to many anions with relative potencies: F ~ ClO4 > I > SCN > NO3 > Br > Cl > formate > acetate (Table
I). YFP-H148Q fluorescence was not sensitive to sulfate, gluconate, isethionate, phosphate, and cations. The anion selectivity sequence correlates with the lyotropic series, ClO3 > I > SCN > NO3 > Br > Cl > acetate > sulfate > F (based on the ion
dehydration energies) (26), with the exception of fluoride, suggesting
that the bigger ions having lower dehydration energies bind more
strongly to the putative anion binding site. The anomalous strong
fluoride binding may result from steric effects.
Binding constants and maximal fluorescence decrease for YFP-H148Q
quenching by anions at pH 7.5
F is the maximum percentage
decrease in fluorescence at high [anion].
Time-resolved fluorescence measurements showed a lifetime of 3.1 ns and
apparent rotational correlation time of 13 ns, consistent with that of
~10 ns predicted for a 27,000-kDa spherical protein (YFP-H148Q
monomeric size). These values were independent of pH (6.0-8.0) and
[Cl] (0-400 mM). The rotational
correlation time increased with solution viscosity without evidence for
segmental depolarizing rotations, indicating that theYFP-H148Q
chromophore is rigidly fixed in its 
-barrel structure. These results
implicate a static Cl sensitivity mechanism probably
involving binding of Cl to site(s) on the YFP-H148Q protein.
Absorption and CD spectroscopy provided further support for a
Cl binding mechanism. Fig. 1C shows absorption
titrations of YFP-H148Q at pH 6.4 at different [Cl].
Two absorption peaks were observed, at 514 and 416 nm, corresponding to
the neutral and anionic forms of the YFP-H148Q chromophore. The
parallel [Cl]-dependent changes in
absorbance and fluorescence indicates that Cl affects
YFP-H148Q molar absorbance rather than quantum yield, as predicted by
the lifetime results. The absence of a distinct isosbestic point
indicates an equilibrium involving more than two absorbing species. The
10-20 nm blue shift in the absorption maximum at 416 nm suggests that
destabilization of the transition dipole of neutral form results from a
decrease in the polarity of the environment near the chromophore,
providing evidence for ground-state Cl/YFP-H148Q binding.
CD analysis in the visible region showed two negative peaks at 415 and
516 nm (Fig. 1D), corresponding to the anionic and neutral
forms of the chromophore, respectively. Cl or
F addition induced a blue shift of the peak at 415 nm and
a change in molar ellipticity at both wavelengths, indicating that
anion binding alters the symmetry of the chromophore environment
and destablizes the absorption transition dipole of the protonated form
of YFP-H148Q.
Stopped-flow fluorescence measurements were done to establish a kinetic
mechanism for the interactions of Cl with the protonated
and deprotonated forms of YFP-H148Q. Aqueous YFP-H148Q solutions were
subjected to rapid changes in pH or [Cl]. Fig.
2A shows the fluorescence
response to a 1.0 unit pH increase and decrease in the absence
(top) and presence (bottom) of 100 mM
Cl. The time course data for the pH change from 8 to 7 in
the absence of Cl was reversible and fitted well to a
monoexponential decay with a t1/2 of 7 ms. The time
course data in the presence of Cl was biexponential with
a fast component of t1/2 8-12 ms and a slow
component of t1/2 102-106 ms. The fast component
corresponded to the prototropic equilibrium of unbound YFP-H148Q. Fig.
2B shows the response at pH 8.1 and pH 6.4 to rapid
Cl addition and dilution at constant pH. At pH 8.0, Cl binding and dissociation were relatively slow
(t1/2 190 and 230 ms, respectively); at pH 6.4, the
t1/2 were 66 and 76 ms. A kinetic model which
incorporates all of these quantitative results is given in Fig.
2C. The model contains four equilibria involving YFP-H148Q
protonation and Cl binding. The rate constants for each
process and the relative fluorescence for the four YFP-H148Q species
are given in the figure legend.
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It has been suggested that the chromophore in YFP-H148Q might be
exposed to the aqueous environment (21). Spot photobleaching measurements were done to examine the accessibility of the chromophore to exogenously added triplet-state quenchers. Fig. 2D
(top) shows irreversible photobleaching of YFP-H148Q giving
a diffusion coefficient of 1.1 × 106
cm2/s, similar to that of GFP (27). In addition, a fast
reversible fluorescence recovery process was identified with
exponential time constants in the range 3-5 ms (bottom,
left curve). Similar recovery processes in GFP-S65T and
fluorescein were shown to arise from triplet-state quenching processes
(27, 28). The efficiency (bleach depth) and recovery rates for
reversible fluorescein photobleaching were sensitive to triplet state
quenchers, including the small molecules oxygen, azide, and
Mn2+, whereas the reversible photobleaching of GFP-S65T and
YFP-H148Q (Fig. 2D, bottom curves) are not,
suggesting that the chromophore in YFP-H148Q is not exposed directly to
aqueous-phase quenchers. Together, the biophysical analysis indicates
that Cl modifies YFP-H148Q fluorescence by binding to a
site close to the chromophore where it alters the electrostatic
environment producing a change in apparent pKa.
YFP-H148Q was expressed in mammalian cells to test its utility as a
cellular Cl indicator. As reported for various GFPs,
YFP-H148Q was expressed in the cytoplasm and nucleus with an
aqueous-phase staining pattern (Fig.
3A). Titrations using
solutions containing high K+ and ionophores were done to
determine the sensitivity of intracellular YFP-H148Q fluorescence to pH
and [Cl]. Fig. 3B shows reversible changes
in YFP-H148Q fluorescence in response to pH changes at 0 Cl with an apparent pKa of 6.84, similar to that measured in aqueous solutions in the absence of
Cl. Fig. 3C shows reversible changes in
YFP-H148Q fluorescence at pH 7.4 in response to changes in
[Cl], with 50% decrease in fluorescence at ~140
mM Cl, similar to that measured in aqueous
solutions.
|
The dependence of intracellular YFP-H148Q fluorescence on
[Cl] suggests the utility of YFP-H148Q as a cellular
Cl/halide indicator. An important application of
fluorescent halide indicators has been in the functional analysis of
CFTR and cystic fibrosis-causing CFTR mutations. CFTR functions as a
cAMP-activated channel that effectively conducts Cl,
NO3 and I, with lesser
conductance for gluconate (29). In cells expressing CFTR and bathed in
forskolin-containing solutions to activate CFTR, replacement of
solution Cl by gluconate (to drive Cl
efflux) produced a small (~12%) increase in cellular fluorescence (Fig. 4A, top). The
increase was not observed in control cells not expressing CFTR (data
not shown). In response to
Cl/NO3 exchange,
cellular fluorescence was slightly decreased (Fig. 4A,
middle), as predicted, since YFP-H148Q fluorescence is more sensitive to NO3 than to
Cl. The small change in cellular YFP-H148Q fluorescence
is in agreement with that predicted for a ~25 mM change
in cytoplasmic [Cl] produced by
Cl/NO3 exchange in cells
with a normal interior negative potential.
|
To increase the signal size for assays of anion transporters like CFTR,
we exploited the observations that YFP-H148Q fluorescence is
substantially more sensitive to I than to
Cl (Fig. 1B) and that anion channels conduct
I efficiently. Cl/I exchange
is the protocol generally used in fluorescence assays of CFTR
conductance (1). Fig. 4A (bottom) shows large
changes in fluorescence with Cl/I exchange.
Fig. 4B shows a Cl/I exchange
protocol in which cells were initially perfused with a 100 mM iodide buffer and then with PBS. Addition of the cAMP agonist forskolin produced a substantial increase in fluorescence as
cytoplasmic I was replaced by Cl. This
large signal change is comparable with that observed for the best
chemical halide indicator developed to date, LZQ (6).
The biophysical and cellular studies establish the mechanism of
YFP-H148Q anion sensitivity and its utility as a sensitive fluorescent
halide indicator for quantitative transport measurements in living
cells. Changes in YFP-H148Q fluorescence are rapid, reversible, and
halide-specific for suitable experimental protocols as used here. The
excellent brightness and iodide sensitivity of YFP-H148Q, together with
its targetable cellular expression, make it extremely useful in a wide
variety of biological applications. The noninvasive loading and perfect
cell retention of YFP-H148Q make it preferable to chemical halide
indicators, such as SPQ and LZQ, for many studies. For example, a major
goal in cystic fibrosis research is the identification of compounds
that restore normal halide conductance in cells expressing
Phe508 CFTR, the most common mutation causing cystic
fibrosis. High throughput screening of compound libraries would be
efficiently carried out in cells stably expressing YFP-H148Q and
Phe508 CFTR. Similarly, mammalian expression cDNA
libraries would be efficiently screened for novel anion channels using
YFP-H148Q expressing cells as the host cell. Finally, YFP mutants
provide a possible approach to study halide concentrations and
transport in subcellular organelles.
| |
ACKNOWLEDGEMENT |
|---|
We thank Karla Gregg for cell culture and transfections.
| |
FOOTNOTES |
|---|
* This work was supported by Research Development Grant R613 from the National Cystic Fibrosis Foundation and Grants DK43840, DK35124, HL59198, and HL60288 from the National Institutes of Health (to A. S. V.) and Grant MCB9728162 from the National Science Foundation (to S. J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Cardiovascular Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; GFP, green fluorescent protein; YFP, yellow fluorescent protein; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; CCCP, carbonyl cyanide p-chlorophenylhydrazone.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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 A. Pera, S. Dossena, S. Rodighiero, M. Gandia, G. Botta, G. Meyer, F. Moreno, C. Nofziger, C. Hernandez-Chico, and M. Paulmichl Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred syndrome and nonsyndromic EVA PNAS, November 25, 2008; 105(47): 18608 - 18613. [Abstract] [Full Text] [PDF]
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 M. Mukhtarov, O. Markova, E. Real, Y. Jacob, S. Buldakova, and P. Bregestovski Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors Phil Trans R Soc A, October 13, 2008; 366(1880): 3445 - 3462. [Abstract] [Full Text] [PDF]
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 R. De La Fuente, W. Namkung, A. Mills, and A. S. Verkman Small-Molecule Screen Identifies Inhibitors of a Human Intestinal Calcium-Activated Chloride Channel Mol. Pharmacol., March 1, 2008; 73(3): 758 - 768. [Abstract] [Full Text] [PDF]
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 H. Miyazaki, A. Shiozaki, N. Niisato, and Y. Marunaka Physiological significance of hypotonicity-induced regulatory volume decrease: reduction in intracellular Cl- concentration acting as an intracellular signaling Am J Physiol Renal Physiol, May 1, 2007; 292(5): F1411 - F1417. [Abstract] [Full Text] [PDF]
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 N. Pedemonte, E. Caci, E. Sondo, A. Caputo, K. Rhoden, U. Pfeffer, M. Di Candia, R. Bandettini, R. Ravazzolo, O. Zegarra-Moran, et al. Thiocyanate Transport in Resting and IL-4-Stimulated Human Bronchial Epithelial Cells: Role of Pendrin and Anion Channels J. Immunol., April 15, 2007; 178(8): 5144 - 5153. [Abstract] [Full Text] [PDF]
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 D. Mason, G. V. Mallo, M. R. Terebiznik, B. Payrastre, B. B. Finlay, J. H. Brumell, L. Rameh, and S. Grinstein Alteration of Epithelial Structure and Function Associated with PtdIns(4,5)P2 Degradation by a Bacterial Phosphatase J. Gen. Physiol., March 26, 2007; 129(4): 267 - 283. [Abstract] [Full Text] [PDF]
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 K. J. Rhoden, S. Cianchetta, V. Stivani, C. Portulano, L. J. V. Galietta, and G. Romeo Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L Am J Physiol Cell Physiol, February 1, 2007; 292(2): C814 - C823. [Abstract] [Full Text] [PDF]
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C. Muanprasat, N.D. Sonawane, D. Salinas, A. Taddei, L. J.V. Galietta, and A.S. Verkman Discovery of Glycine Hydrazide Pore-occluding CFTR Inhibitors: Mechanism, Structure-Activity Analysis, and In Vivo Efficacy J. Gen. Physiol., July 26, 2004; 124(2): 125 - 137. [Abstract] [Full Text] [PDF]
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 G. T. Hanson, R. Aggeler, D. Oglesbee, M. Cannon, R. A. Capaldi, R. Y. Tsien, and S. J. Remington Investigating Mitochondrial Redox Potential with Redox-sensitive Green Fluorescent Protein Indicators J. Biol. Chem., March 26, 2004; 279(13): 13044 - 13053. [Abstract] [Full Text] [PDF]
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A. S. Verkman Drug discovery in academia Am J Physiol Cell Physiol, March 1, 2004; 286(3): C465 - C474. [Abstract] [Full Text] [PDF]
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 S. Le Gall, A. Neuhof, and T. Rapoport The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules Mol. Biol. Cell, February 1, 2004; 15(2): 447 - 455. [Abstract] [Full Text] [PDF]
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D. P. Barondeau, C. D. Putnam, C. J. Kassmann, J. A. Tainer, and E. D. Getzoff Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures PNAS, October 14, 2003; 100(21): 12111 - 12116. [Abstract] [Full Text] [PDF]
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 K. Takemoto, T. Nagai, A. Miyawaki, and M. Miura Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects J. Cell Biol., January 21, 2003; 160(2): 235 - 243. [Abstract] [Full Text] [PDF]
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A. Rekas, J.-R. Alattia, T. Nagai, A. Miyawaki, and M. Ikura Crystal Structure of Venus, a Yellow Fluorescent Protein with Improved Maturation and Reduced Environmental Sensitivity J. Biol. Chem., December 20, 2002; 277(52): 50573 - 50578. [Abstract] [Full Text] [PDF]
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M. G. Espey, S. Xavier, D. D. Thomas, K. M. Miranda, and D. A. Wink Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms PNAS, March 19, 2002; 99(6): 3481 - 3486. [Abstract] [Full Text] [PDF]
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N. D. Sonawane, J. R. Thiagarajah, and A. S. Verkman Chloride Concentration in Endosomes Measured Using a Ratioable Fluorescent Cl- Indicator. EVIDENCE FOR CHLORIDE ACCUMULATION DURING ACIDIFICATION J. Biol. Chem., February 8, 2002; 277(7): 5506 - 5513. [Abstract] [Full Text] [PDF]
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L. V. J. Galietta, S. Jayaraman, and A. S. Verkman Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1734 - C1742. [Abstract] [Full Text] [PDF]
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 H.C. Rodgers and A.J. Knox Pharmacological treatment of the biochemical defect in cystic fibrosis airways Eur. Respir. J., June 1, 2001; 17(6): 1314 - 1321. [Abstract] [Full Text] [PDF]
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L. J. V. Galietta, M. F. Springsteel, M. Eda, E. J. Niedzinski, K. By, M. J. Haddadin, M. J. Kurth, M. H. Nantz, and A. S. Verkman Novel CFTR Chloride Channel Activators Identified by Screening of Combinatorial Libraries Based on Flavone and Benzoquinolizinium Lead Compounds J. Biol. Chem., June 1, 2001; 276(23): 19723 - 19728. [Abstract] [Full Text] [PDF]
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O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien Reducing the Environmental Sensitivity of Yellow Fluorescent Protein. MECHANISM AND APPLICATIONS J. Biol. Chem., July 27, 2001; 276(31): 29188 - 29194. [Abstract] [Full Text] [PDF]
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