* Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242-1109; Institute for Human Gene
Therapy and Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104; § Institute for Human Gene Therapy and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania
19104;
Department of Physiology and Medicine Divisions of Pediatrics and Nephrology, and Center for Medical Genetics, Johns
Hopkins University School of Medicine and Johns Hopkins Hospital, Baltimore, Maryland 21205; and ¶ Department of
Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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Abstract |
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The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus
oocyte cRNA expression system, we have evaluated the
molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient
change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl that was similar to that
reported in airway surface fluid. Mutagenesis of CFTR
demonstrated that Cl
conductance and ATP release
regulatory properties could be dissociated to different
regions of the CFTR protein. Despite the lack of a need
for Cl
conductance through CFTR to modulate ATP
release, alterations in channel pore residues R347 and
R334 caused changes in the relative ability of different
halides to activate ATP efflux (wtCFTR, Cl >> Br;
R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may
contribute a Cl
binding site within the CFTR channel
pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl
. In summary,
these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to
changes in the extracellular chloride concentration by
modulating the activity of an unidentified ATP efflux
pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway
through purinergic regulation of epithelial cells. Insight
into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.
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Introduction |
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CFTR is a member of the ATP-binding cassette
(ABC)1 superfamily that includes over thirty proteins that share extensive sequence similarity and
domain organization (Higgins et al., 1990; Ames et al.,
1986; Hyde et al., 1990
). Electrophysiologic analyses of the
CFTR gene product has demonstrated that it is a cAMP-activated chloride channel with a conductance of 7-10 pS
(Drumm et al., 1990
; Gregory et al., 1990
; Kartner et al.,
1990; Anderson et al., 1991
; Bear et al., 1992
). Although
altered Cl
permeability through CFTR has generally
been accepted to play a role in the pathogenesis of cystic
fibrosis (CF), it remains unclear whether this defect alone
can explain the complex pathophysiology of associated
lung disease. Recent studies have indicated that in addition to its well-defined Cl
channel activity, CFTR is also a
regulator of other epithelial ion channels, including an
outwardly rectifying Cl
channel (ORCC; Egan et al.,
1992
; Gabriel et al., 1993
; Schwiebert et al., 1995
; Schwiebert et al., 1998
; Jovov et al., 1995
), the epithelial Na+
channel (ENaC; Grubb et al., 1994
; Chinet et al., 1994
;
Johnson et al., 1995
; Hyde et al., 1993
; Ismailov et al., 1996
;
Stutts et al., 1997
; Kunzelmann et al., 1997
), and a kidney
K+ channel (ROMK; McNicholas et al., 1996
; McNicholas
et al., 1997
; Ho, 1998
). However, the mechanisms by which
these regulatory functions contribute to the maintenance
of airway electrolyte and fluid balance remain obscure.
Cantiello and colleagues first suggested that the multidrug resistance protein, a member of the ABC superfamily, might conduct ATP (Abraham et al., 1993). Several
groups have provided evidence that CFTR also plays a
role in ATP release (Reisin et al., 1994
; Schwiebert et al.,
1995
; Prat et al., 1996
; Pasyk and Foskett, 1997
; Sugita et al.,
1998
, Cantiello et al., 1998
). Guggino and colleagues suggested that CFTR stimulates the ORCC via a P2U purinergic receptor-dependent signal transduction pathway by
modulating ATP efflux (Schwiebert et al., 1995
). Similar
mechanisms of ion channel regulation have been proposed
for other ABC transporters that secrete ATP (Al-Awqati,
1995
). However, several laboratories have failed to observe an association of CFTR with ATP permeability (Reddy et al., 1996
; Li et al., 1996
; Grygorczyk et al., 1996
). In model systems that have been able to demonstrate
CFTR-modulated ATP release, it is presently unclear
whether CFTR itself, another unknown cofactor, or a
CFTR-linked biologic process such as exocytosis, represents the ATP permeation/release pathway. It is also unknown if dysregulation of this process contributes to CF
lung disease progression. Most recently, Foskett and coworkers demonstrated that ATP channels exhibiting slow
gating kinetics were associated within a subpopulation of
CFTR Cl
channels in MDCK cells (Sugita et al., 1998
).
They concluded that phosphorylation- and nucleotide hydrolysis-dependent CFTR gating is directly involved in
gating an associated ATP channel, but that the permeation
pathways for Cl
and ATP are distinct. Because the ATP
conduction pathway did not appear to be obligatorily associated with CFTR expression, they suggested the existence
of a cofactor that may mediate CFTR-regulated ATP conductance. However, a recent report reconstituting purified CFTR into lipid bilayers has suggested that CFTR enables
permeation of both Cl
and ATP (Cantiello et al., 1998
).
In the present study, we used a Xenopus oocyte cRNA
expression system coupled with a sensitive luciferin-
luciferase bioluminescence assay to explore the mechanisms that control CFTR-modulated ATP release. Our
results suggest that expression of CFTR can confer a Cl-sensitive ATP permeability to injected oocytes. Mutational analysis suggests that the interaction of extracellular
Cl
at arginine residues R334 and R347 within the channel
pore controls the ability of CFTR to modulate ATP release. These results suggest a novel mechanism by which
changes in the extracellular Cl
concentration participate
in the activation of CFTR-modulated ATP release.
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Materials and Methods |
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Xenopus Oocyte Isolation and Maintenance
Adult female Xenopus laevis were obtained from NASCO (Fort Atkinson, WI) and Xenopus-1 (Ann Arbor, Michigan). Immediately before surgery, frogs were anaesthetized by immersion in 0.17% (wt/vol) tricaine (3-aminobenzoic acid ethyl ester methanesulphonate) for 5 min, and were then cooled by adding an equal volume of ice to the Tricaine solution for 10 min. Ovaries were surgically removed from anaesthetized frogs through a small incision in the lower abdomen. Individual oocytes were isolated and defolliculated by collagenase treatment. In brief, isolated oocytes were rinsed in Ca2+-free SOS (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM Hepes-NaOH, pH 7.6) and incubated with Type IV collagenase (2.0 mg/ml; Sigma Chemical Co., St. Louis, MO) in the same media for 45 min with gentle shaking. After rinsing several times with Ca2+-free SOS, defolliculated oocytes were kept in SOS (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH 7.6) supplemented with 2.5 mM sodium pyruvate and 50 µg/ml gentamycin at 16°C until used.
During the course of this study, we found that only ~10% of the frogs
had oocytes that displayed CFTR-modulated ATP release, despite their
ability to generate CFTR-dependent cAMP-inducible Cl currents. We
therefore screened all frogs to identify those whose oocytes gave ATP responses. Responder frogs were placed in a separate tank and reused at intervals of no less than 5 wk. Oocytes from responders continued to demonstrate CFTR-modulated ATP release at a similar frequency and
magnitude during later harvests (up to five harvests have been performed
on responder frogs without a decline in ATP responses).
Complementary RNA Synthesis and Injection into Oocytes
Synthesis of complementary RNA (cRNA) from CFTR and mutant cDNAs was performed using the Megascript cRNA synthesis kit purchased from Ambion (Austin, TX). Linearized cDNA plasmids were transcribed at 37°C for 4 h in a reaction mixture supplemented with excess ribonucleotide solution that allows an increased rate and duration of cRNA synthesis. RNA cap analogue, m7G(5')ppp(5')G (New England Biolabs Inc., Beverly, MA) was used to cap and protect the 5' end of the cRNA. The final reaction mixture contained 2-4 µg of a linearized cDNA, 1× transcription buffer provided by the kit, 7.5 mM each of ATP, CTP, and UTP, 1.5 mM of GTP, 6 mM of the cap analogue, and 4 µl of the enzyme mix in a total volume of 40 µl. Xenopus oocytes were injected 24 h after isolation with 50 nl per oocyte of either DEPC water (mock-injected) or appropriate cRNA (50 ng) using a nanoliter injector (World Precision Instruments, Inc., Sarasota, FL). Typically, oocytes were analyzed for either ATP responses or electrophysiologic measurements 48 h after injection.
Measurement of the ATP Efflux in Single Xenopus Oocytes
Measurements of the ATP efflux from single oocytes were performed using a luciferin-luciferase assay in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA) under dim light. A single oocyte was rinsed twice in
an appropriate buffer, and was then immersed in 100 µl of the same buffer
containing 3.125 mg/ml luciferin-luciferase (Sigma Chemical Co.) in a 0.5-ml
Eppendorf tube. Most experiments were done with either Hepes phosphate-buffered Ringers (HPBR; 10 mM Hepes, pH, 7.4, 140 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM CaGluconate, 2.4 mM K2HPO4, and
0.4 mM KH2PO4) or standard oocyte solution (SOS; 5 mM Hepes, pH 7.6, 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2). When Cl-free buffers were used, NaCl was replaced by equivalent amounts of sodium
gluconate. The intracellular Cl
concentration of oocytes was calculated
from the averaged reversal potentials of CFTR-injected oocytes in the presence of IBMX (100 µM) and forskolin (10 µM) using the Nernst equation:
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The optimal protocol for eliciting CFTR-modulated ATP release required
the following series of sequential buffer changes that altered the external
Cl concentration: (a) Cl
-free; (b) Cl
-free + cAMP/forskolin; and (c)
Cl
(100-140 mM) + cAMP/forskolin. Luminescence readings were recorded by a computer data link for 10 readings of 10 s each in the luminometer with the sensitivity level set to 89%. When indicated, 8-cpt-cAMP/forskolin cocktail was added to the experimental buffer to achieve
a final concentration of 200 µM 8-cpt-cAMP and 2.5 µM forskolin. In
some experiments, ionomycin (10 µM; Calbiochem-Novabiochem Corp.,
La Jolla, CA) replaced 8-cpt-cAMP/forskolin in the assay buffer. Buffer
exchange was accomplished by rinsing the oocyte once in the new buffer;
the luminescence assay was then continued for the same amount of time
as described above. Care was taken never to touch the oocyte with pipette
tips. The buffer rinses typically took ~45 s, and account for the absence of
data values during solution switches. To determine the absolute amount
of ATP efflux from an oocyte, standard concentrations of ATP in appropriate buffer solutions were measured to establish a calibration curve. In
studies that evaluated the anion dependence of the activation of CFTR-modulated ATP release, the effects of different anions (gluconate, Cl
,
Br
, I
, F
, NO3, and thiocyanate) on the activity of luciferin-luciferase were evaluated in preliminary experiments. ATP calibration curves obtained in each buffer revealed that I
, F
, NO3, and thiocyanate profoundly inhibited luciferin-luciferase activity. The Cl
channel blocker
diphenylamine-2-carboxylic acid (DPC) also inhibited luciferin-luciferase
activity. These reagents were therefore not used in the studies. In contrast,
the ATP activity calibration curves were similar in the presence of gluconate, Cl
, or Br
.
CFTR-modulated ATP release in Xenopus oocytes using this luciferin- luciferase assay was successfully reproduced at both the University of Pennsylvania and Johns Hopkins University.
Two-electrode Voltage Clamp Recording
Whole-cell (Wc) currents were measured using the two-electrode voltage
clamp (TEV) method. Single oocytes were placed in a 1-ml chamber containing modified ND96 (96 mM NaCl, 1 mM KCl, 0.2 mM CaCl2, 5.8 mM
MgCl2, 10 mM Hepes, pH 7.2 by NaOH) connected to a reference bath
electrode by a 3 M KCl-agar bridge (Katayama and Widdicombe, 1991).
Conventional TEV was performed (Stühmer, 1992) at room temperature
using an OC-725C amplifier (Warner Instrument Corp., Hamden, CT)
connected to a PowerMac 7100 via an ITC-16 interface (Instrutech Corp.,
Great Neck, NY). Pulse + PulseFit software (HEKA, Port Washington,
NY) was used to ramp the applied transmembrane potential (Vm) at 10-s
intervals from
60 mV to 20 mV at a rate of 16 mV/s. Vm was clamped at
the prestimulation reversal potential between voltage ramps. Transmembrane current (I) and Vm were digitized at 200 Hz during the voltage
ramps, and were written directly onto hard disk. During the course of TEV
recording, the major sources of equipment noise were the 60-Hz spikes induced by the power supply. Therefore, the TEV current-applied voltage
data were fitted with an empirical fifth order polynomial to provide a
smoothed approximation of the TEV data and eliminate the 60-Hz spikes.
The membrane conductance of the oocyte was then calculated as dI/dV
from this approximation. In all TEV experiments performed, a fifth order
polynomial fit very well the observed current-voltage data, which had a
simple monotonically increasing relations within the applied voltage
range. Halide selectivity experiments were performed by replacing NaBr
with NaCl in ND96 solutions. CFTR activation was accomplished by perfusing the oocyte with appropriate buffers containing 100 µM IBMX and 10 µM forskolin (Sigma Chemical Co.). In all experiments, CFTR Cl
conductance was defined as the difference in conductance measured in the
presence and in the absence of 100 µM IBMX and 10 µM forskolin. The
cAMP-stimulated Cl
and Br
conductances of the R347E mutant (5.3 ± 2.1 µs and 12.15 ± 3.3 µs, respectively) were much lower than that of the
wild type. In control experiments, the conductance changes of mock-injected oocytes in response to IBMX/forskolin treatment were -0.61 ± 0.62 µs in
the presence of Cl
and 0.11 ± 0.39 µs in the presence of Br
. These background non-CFTR conductance changes were subtracted from the measured changes of CFTR and mutants. In the case of mutant R347E, this
correction caused only a small change in the calculated GBr/GCl value
from 2.60 (without background correction) to 2.36 (with background correction). The Wc conductance (at
20 mV) and reversal potential (Erev)
after agonist stimulation of CFTR channels (wild type or mutants) were
evaluated from the slope and x intercept, respectively, of the background-corrected I-Vm curve using Igor Pro software (WaveMetrics, Lake Oswego, OR). After each buffer perfusion (1 min), conductance was measured over 10 min to ensure equilibrium was reached. GBr/GCl is the ratio
of the Wc conductances of stimulated CFTR channels in Br
vs. Cl
solution. The permeability ratio (PBr/PCl) was calculated from the reversal potentials Erev,Br and Erev,Cl in Br
and Cl
solutions, respectively, using
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which is based on the Goldman-Hodgkin-Katz equation, with [Br] and
[Cl
] being the concentrations of Br
and Cl
in the solutions, and R, T,
and F having their conventional meanings.
Preparation of CFTR Mutants
Five CFTR mutants were analyzed for their ability to modulate ATP release using the luciferin-luciferase assay as well as their capacity to conduct Cl and Br
using voltage clamping. For mutant R347, a cDNA segment was cut out from the PTM1-R347E CFTR construct (kindly
provided by Dr. M. Welsh, University of Iowa) by restriction enzymes
MroI and Bst1107I, and was subcloned into the pBQ-CFTR plasmid between the same restriction sites. Mutants R334W and R347P were constructed by replacing a SpH I-Xba I segment in the PSP-CFTR with a corresponding segment cut out from mutants PTM-R334W and PTM-R347P
(provided by Dr. M.J. Welsh; Sheppard et al., 1993
), respectively. Successful transfer of the mutated sequences was confirmed by sequencing. The
COOH-terminal truncation mutant, TMD1 CFTR, was constructed by introducing a stop codon at K370X followed by an EcoRV restriction site
using the mutagenic oligonucleotide 5'-GCAATAAACTAAATACAGGATATCTTAC-3'. The NH2-terminal truncation mutant
259-M265V
CFTR was constructed as previously described (Piazza Carroll et al.,
1995).
Immunoprecipitation of CFTR and Mutants from cRNA-injected Xenopus Oocytes
Synthesis of 35S-labeled wild-type and mutant CFTR proteins in Xenopus
oocytes was achieved by coinjection of appropriate cRNAs with [35S]methionine. The injection mixtures were made by adding 50 µCi of 35S-methionine (ICN) to 2.0 µl of transcripts. Each oocyte was injected with 50 nl
of a mixture containing 50 ng of cRNA and 0.5 µCi 35S. After incubating
for 3 h at 18°C in MBS, oocytes were collected, quickly frozen on dry ice,
and stored at 80°C until use. For immunoprecipitation, oocytes in
groups of five were thawed and homogenized on ice in 30 µl of 50 mM
Tris (pH 7.5), 0.25 M sucrose, 50 mM KCl, 5 mM MgCl2, and 1 mM DTT
followed by solubilization with 100 µl of 1% SDS and 0.1 M Tris (pH 8.0).
Samples were incubated for 30 min at 37°C, diluted in 1 ml of TX SWB
buffer (0.1 M NaCl, 1% Triton X-100, 2 mM EDTA, 0.1 mM PMSF and
0.1 M Tris, pH 8.0) on ice, and further incubated at 4°C for 2 h. Before immunoprecipitation, samples were centrifuged at 16,000 g for 15 min, and
supernatants were collected. Immunoprecipitation of CFTR was initiated
by adding 1 µl of an epitope-specific rabbit antisera raised against a synthetic peptide corresponding to residues 45-56 in the CFTR NH2 terminus and mixing at 4°C for 1.5 h. 5 µl of Affigel protein A (Bio-Rad Laboratories, Hercules, CA) was added, and the reaction mixture was further incubated at 4°C for another 1.5 h. After washing five times in TX SWB, protein A beads were pelleted by brief centrifugation in a microcentrifuge
and resuspended in 1X sample loading buffer. Samples were denatured at
37°C for 30 min and analyzed on 7.5% SDS PAGE. After electrophoresis,
gels were fixed in 35% methanol and 10% HAc, dried, and exposed to
film for autoradiography.
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Results |
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ATP Release from CFTR-expressing Xenopus Oocytes
ATP release from Xenopus oocytes was studied using a
single oocyte luciferin-luciferase luminometric assay. Initial experiments compared water and CFTR cRNA-
injected oocytes under conditions that promote Cl flux
through CFTR. In the presence of Cl
-free Ringers containing 200 µM 8-(4-chlorophenylthio)-adenosine 3':5'-
cyclicmonophosphate (8-cpt-cAMP)/2.5 µM forskolin, no
activation of ATP efflux was observed for up to 10 min
(Fig. 1 and data not shown). Similarly, no ATP efflux was
observed when either CFTR- or water-injected oocytes
were incubated in the presence of high (140 mM) extracellular Cl
and cAMP/forskolin for 10 min (data not shown).
However, prestimulation of oocytes with cAMP/forskolin
in Cl
-free Ringers, followed by an acute exposure to 140 mM Cl
Ringers, was associated with a significant and immediate ATP efflux in a subset (~50%, N = 41) of CFTR-injected oocytes (Fig. 1, A and B). Activation of ATP efflux in oocytes was dependent on CFTR expression, and
was never observed in water-injected oocytes (N = 41).
CFTR activation was required to elicit ATP efflux since it
was not seen when similar protocols were carried out in
the absence of cAMP agonists (data not shown). The latency as well as the initial rate of ATP efflux during the
first 45 s could not be assessed because of the time required for buffer washes. However, steady-state rates of
ATP efflux were quantitated by calculating the slope of
relative light units vs. time for each individual oocyte, and
correlating the per minute light unit change with standards
of known ATP concentration. In a select number of experiments, steady-state rates of ATP efflux in CFTR-injected oocytes remained constant for periods extending up to 3 min, which was the longest time course analyzed. However, in CFTR-injected oocytes that gave very high levels
of ATP efflux, slight time-dependent declines in the
steady-state rates could be seen, and may represent transient depletions of available intracellular ATP pools. The cumulative data depicted in Fig. 1 C demonstrate a mean
ATP efflux rate of 1.99 ± 0.55 pmoles ATP/min (N = 41)
for CFTR cRNA-injected oocytes, compared with
0.005 ± 0.004 pmoles ATP/min (N = 41) for water-injected oocytes. The various treatment conditions and their effects
on CFTR-modulated ATP release are summarized in Table I.
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Because these studies used mammalian Ringer solutions, additional experiments were performed to compare
responses directly using SOS. In these studies, the ATP efflux rate (2.0 ± 1.1 pmoles ATP/min) in SOS buffer (100 mM
NaCl) was threefold lower than that seen in the mammalian buffer (140 mM NaCl; 6.3 ± 2.2 pmoles ATP/min).
However, activation of CFTR-modulated ATP release
was still dependent on cAMP activation and the switch
from low to high extracellular Cl. These findings suggest
that buffer osmolarity does not significantly alter the profile of CFTR-modulated ATP release. There were no significant changes in buffer osmolarity after anion replacement for either the HPBR or the SOS buffer series (Table
II). Therefore, it is unlikely that the pattern of ATP release seen after changes in extracellular chloride is due to
osmolarity changes in assay buffers. As addressed later,
the lower ATP efflux rates seen in SOS are likely due to
the dependence of CFTR-modulated ATP release on extracellular Cl
concentrations.
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ATP Release Cannot be Explained by CFTR-facilitated Changes in Membrane Potential or Cell Volume
Not all CFTR cRNA-injected oocytes that expressed
CFTR, as demonstrated by high whole cell (Wc) cAMP-activated Cl conductances, released ATP after a shift
from low to high Cl
extracellular medium in the presence
of cAMP agonists (Fig. 1 D). Thus, CFTR expression was
necessary, but not sufficient, to confer the ATP permeability. Nevertheless, we considered that elevation of extracellular Cl
might hyperpolarize the cell membrane and drive
ATP anions from the cell through unspecified pathways.
To determine whether changes in membrane potential
during the experimental protocol contributed to the
CFTR-associated ATP effluxes, we evaluated the effects of stimulating CFTR-independent Cl
channels in our
ATP release assay. Ionomycin (10 µM), which activates endogenous Cl
channels in oocytes by releasing Ca2+
from intracellular stores (Yoshida and Plant, 1992
; Liu and
Harmann, 1978
; Boton et al., 1990), failed to evoke ATP
efflux from mock-injected oocytes (Fig. 2, A and B). In
contrast, CFTR-injected oocytes from the same batches of
oocytes demonstrated substantial ATP effluxes in response to CFTR activation (Fig. 2, A and B). The lack of
ATP efflux in ionomycin-treated oocytes was not due to inhibitory effects of ionomycin on luciferin-luciferase activity, as determined by performing the standard ATP concentration calibration in the presence of ionomycin (Fig. 2
C). Treatment of uninjected oocytes with 10 µM ionomycin elicited Cl
conductances (104 ± 28 µs) of comparable
magnitude to those in CFTR cRNA-injected oocytes (121 ± 35 µs) stimulated with IBMX/forskolin. Preincubation of
oocytes for 5 min in a Cl
-free solution before TEV did
not significantly lower the intracellular Cl
concentration
(42.54 ± 1.65 mM), as assessed by reversal potential determination. These data therefore demonstrate that activation of a Cl
permeability per se is incapable of stimulating the ATP efflux, and suggest that the efflux observed
during activation of CFTR cannot be accounted for by
changes in electrical driving forces.
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Although osmolarity was held constant in the protocols,
we evaluated the potential involvement of cell volume
perturbations that might arise from Cl fluxes during the
changes in extracellular Cl
concentration. The volumes
of oocytes injected with CFTR cRNA were assessed with
digital imaging as previously described (Takahashi et al.,
1996
; Jentsch, 1996
). Batches of CFTR cRNA-injected oocytes that demonstrated CFTR-modulated ATP release
exhibited undetectable changes (<2%) in cell volume (9.0 × 10
4 ± 0.11 × 10
4 cm3) after exposure to the series of solutions used to activate CFTR-modulated ATP release.
The data suggest that substantial changes in oocyte volume do not account for the observed ATP efflux.
Dissociation of Cl Conductance and ATP Efflux
Functions Associated with CFTR
Because the results indicated that CFTR was necessary
but not sufficient for ATP permeability, we attempted to
identify the important domains in CFTR by comparing
wild-type CFTR with two deletion mutants, TMD1 and
259-M265V. The TMD1 mutant encompassing the NH2-terminal portion of CFTR encoded the first 369 amino acids (first six transmembrane helices), while in the
259-M265V mutant the first 259 amino acids were deleted and
the methionine 265 was mutated to a valine. Oocytes expressing the TMD1 CFTR mutant generated ~30% of the
wild-type CFTR Cl
conductance. However, ATP efflux
was not associated with this mutant (Fig. 3). In contrast,
expression of the
259-M265V mutant was associated with
near wild-type rates of ATP efflux in the absence of detectable Cl
conductances (Fig. 3). These results demonstrate that the ability of the CFTR to modulate ATP release can be dissociated from its Cl
conducting activity.
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Activation of CFTR-modulated ATP Release Demonstrates Selectivity for Extracellular Halides
In addition to Cl, CFTR is permeable to other halides
and small anions, including Br
, I
, F
, and NO3
(Anderson et al., 1991
; Tabcharani et al., 1993
). Because changing the extracellular Cl
concentration appeared to be critical
for activating CFTR-modulated ATP release in oocytes,
we explored the anion dependence of this activation. As
indicated in Materials and Methods, all anions tested, with
the exception of Br
and Cl
, significantly inhibited the
luciferin-luciferase reaction (Fig. 4 B). The anion dependence experiments were therefore limited to Cl
and Br
.
Replacement of 140 mM extracellular Cl
with 140 mM
Br
caused a fourfold decrease in the rate of CFTR-
dependent ATP release (Fig. 4 A), which was reversed
when Br
was replaced with Cl
(Fig. 4 A). This halide
specificity was also seen in SOS buffers. No effect of
changing the extracellular halide was observed in water-injected oocytes (data not shown). Although the magnitude of CFTR-modulated ATP release was threefold
lower in the presence of 100 mM Cl
when compared with
140 mM Cl
, the Cl
:Br
halide dependence of ATP release was significantly greater in SOS buffers (Fig. 4 C).
The sensitivity of CFTR-modulated ATP release to the
external Cl
concentration and the apparent halide selectivity of this phenomenon suggested that a Cl
sensor may
exist within CFTR that responds to Cl
concentration
changes in the external environment by activating ATP efflux. To test this hypothesis, we examined the dependence of ATP release on the concentration of extracellular Cl
in CFTR cRNA-injected oocytes.
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Dependence of CFTR-modulated ATP on the External
Cl Concentration
The effect of extracellular Cl on the rate of ATP efflux in
CFTR-expressing oocytes was determined by increasing in
a step-wise fashion the extracellular concentration of Cl
from 0 to 140 mM in the presence of cAMP/forskolin. Osmolarity was kept constant throughout the experiments by
gluconate replacement. The cumulative results from two
independent batches of oocytes demonstrated a sharp
threshold for activation of CFTR-modulated ATP release
at extracellular concentrations of Cl
>100 mM (Fig. 5 A).
Results from two additional batches of oocytes narrowed
the range of Cl
necessary for activation of ATP release.
The cumulative results from four frogs demonstrated that
ATP efflux rates were dramatically increased between 110 and 120 mM extracellular Cl
(Fig. 5 B). In these studies,
maximum levels of ATP efflux were fivefold greater in the
presence of extracellular Cl
when compared with Br
. Interestingly, when the external Cl
concentration was decreased step-wise after a strong ATP response was induced with 140 mM NaCl, the rate of ATP efflux remained elevated at maximum levels at external Cl
concentrations
>100 mM, and only returned to baseline once more significant reductions in extracellular Cl
(<80 mM) were
imposed (data not shown). These results suggest that
CFTR-modulated ATP release may have differential requirements for extracellular Cl
depending on the direction of the gradient change and/or the state of prior activation. Together, these findings suggest that a chloride
sensor within CFTR may be capable of regulating ATP efflux near physiologic concentrations (85-130 mM) of Cl
seen in the airway (Joris et al., 1993
; Gilljam et al., 1989
; Smith et al., 1996
).
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Mutations in the CFTR Channel Pore Alter the Halide Dependence of ATP Release
To explore the mechanisms by which changes in the extracellular Cl concentration affect activation of ATP release, we examined the CFTR mutants R334W, R347P,
and R347E. We hypothesized that positively charged residues within the channel pore might bind Cl
and elicit
structural changes in CFTR necessary for modulating ATP release. Immunoprecipitation studies using a rabbit
antisera raised against a synthetic peptide corresponding
to residues 45-65 in the CFTR NH2 terminus demonstrated similar levels of protein expression in wtCFTR,
R334W, R347P, and R347E cRNA-injected oocytes (Fig.
6 A). No CFTR protein was detected in water-injected oocytes.
|
The electrophysiologic properties of wtCFTR and each
of the mutants were analyzed in cRNA-injected oocytes
by two-electrode voltage clamp. WtCFTR-injected oocytes demonstrated a large increase in slope conductance
(GcAMP = 121 ± 35 µs) in response to 10 µM forskolin
and 100 µM IBMX (Fig. 6 and Table II). Of note, the conditions used in the ATP efflux experiments (200 µM 8-cpt-cAMP/2.5 µM forskolin) produced approximately tenfold
lower conductance changes (data not shown). No cAMP-activated currents were seen in water-injected oocytes under either condition. We therefore used maximum levels
of stimulation to facilitate detection of subtle changes in
halide selectivities of low-conducting mutants. The stimulated Cl
conductance of R347P was comparable to that of
the wtCFTR (
GcAMP = 125 ± 28 µs). In contrast, the
stimulated Cl
conductances were 4- and 20-fold lower in
the R334W and R347E mutants, respectively (Fig. 6 and
Table II). The I/V relationships for unstimulated and
cAMP-stimulated oocytes in the presence of Cl
and Br
are shown in Fig. 6 and summarized in Table II. The mutants R347P and R334W demonstrated slight alterations in
their conductance ratios (GBr/GCl; Br
> Cl
) as compared with wtCFTR (Cl
Br). In contrast, both GBr/GCl
and PBr/PCl were significantly altered by the R347E mutation (Table II). These data support previous findings that
arginine 347 is important in determining the halide selectivity of the CFTR channel pore (Tabcharani et al., 1993
;
Anderson et al., 1991
).
Rates of CFTR-modulated ATP release for the mutant
CFTRs are summarized in Fig. 7. These studies were designed to enable comparison of ATP efflux rates in response to extracellular changes in Cl and Br
within a
single oocyte. Frogs that had previously demonstrated CFTR-modulated ATP release in >25% of oocytes were
used. As previously demonstrated, exposure of wtCFTR-expressing oocytes to Cl
-free buffers was necessary for
activation of ATP efflux following a concentration change
to high extracellular Cl
(JATP = 8.7 ± 3.4 pmoles/min).
This represents a greater than 400-fold increase in ATP efflux when compared with side-by-side controls in water-
injected control oocytes (JATP = 0.021 ± 0.001 pmoles/
min). The typical ATP response curves obtained for
wtCFTR demonstrated a halide dependence of Cl
>>
Br
. In the presence of extracellular Br
, JATP was approximately fourfold lower (1.9 ± 0.5 pmoles/min) compared
with the rate in the presence of Cl
. Again, this inhibition
was reversed by replacing extracellular Cl
(Fig. 7 A).
|
The halide dependence of CFTR-modulated ATP release was similarly analyzed in R334W, R347P, and R347E
cRNA-injected oocytes. WtCFTR cRNA-injected oocytes
were also examined at the same time to assure the responsiveness of the oocytes. The halide dependence of ATP
release for R347P CFTR was similar to that of wtCFTR
(Cl >> Br
); JATP in the presence of Cl
(2.0 ± 0.98 pmoles/min) was eightfold greater than that in the presence of Br
(0.26 ± 0.17 pmoles/min; Fig. 7 and Table II).
These results correlate with the similar anion conductance
and permeability characteristics of this mutant and
wtCFTR. In contrast, the R334W mutant had no halide dependence in the activation of ATP release (JATP[Cl] = 3.4 ± 1.3 pmoles/min, JATP [Br] = 3.7 ± 1.5 pmoles/min; Fig. 7).
Most importantly, the halide dependence in the R347E mutant was reversed to Br
>> Cl
; JATP in the presence
of Cl
(1.5 ± 0.49 pmoles/min) was 7.3-fold lower than
that in the presence of Br
(11 ± 4.8 pmoles/min; Fig. 7
and Table II). Interestingly, exposure of R347E-expressing oocytes to extracellular Br
had a lasting affect on
JATP, even after extracellular Br
was replaced with Cl
. In
summary, these results suggest that residues R334 and
R347 may participate in the extracellular halide dependence of CFTR-modulated ATP release. We hypothesize
that these positively charged residues which have been
suggested to contribute to the Cl
selectivity of the CFTR
pore may also contribute to a chloride sensor at which Cl
binding facilitates structural changes in CFTR necessary
to modulate ATP release.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results from this study indicate that CFTR can modulate ATP release in Xenopus oocytes, provided that several requirements are met. First, activation of ATP release
requires CFTR expression. Second, CFTR must be activated by cAMP. Finally, CFTR-modulated ATP release
was dependent on an incremental increase in the extracellular Cl concentration. These findings suggest a novel
Cl
-dependent mechanism by which CFTR regulates ATP
release. The complexities associated with activation of this
CFTR-mediated phenomenon may in part explain the
present controversies concerning CFTR-modulated ATP
release.
Possible Mechanisms of CFTR-modulated ATP Release
We evaluated the possibility that activation of Cl conductance per se, combined with a change of extracellular
Cl
concentration, could account for ATP release as a
consequence of hyperpolarizing the membrane potential.
However, the absence of ATP release in response to ionomycin, which activated a non-CFTR endogenous Ca2+-activated Cl
conductance, ruled out such a mechanism.
We also considered a second mechanism involving perturbations of cell volume. In mammalian cells it has been reported that the release of intracellular ATP can be observed under conditions that cause cell swelling (Greger et
al., 1993
, Koslowsky et al., 1994
). However, direct measurements failed to reveal any alteration of cell volume in
oocytes that demonstrated CFTR-modulated ATP release. Furthermore, for changes in cell volume to occur in
CFTR cRNA but not water-injected oocytes, one would
anticipate that Cl
flux through CFTR would be required
for movement of NaCl and water into oocytes. However,
the CFTR mutant
259-M265V, which failed to conduct
Cl
, also conferred near wild-type rates of ATP release. In
summary, the lack of correlation between CFTR-modulated ATP release with the magnitude of anion conductance suggests that salt and water movement and changes
in cell volume are likely not involved in activating ATP release in this system.
Several lines of evidence suggest that ATP and Cl permeation occur through distinct pathways in CFTR-
expressing oocytes. First, the CFTR mutant
259-M265V
demonstrated near wild-type rates of CFTR-modulated
ATP release despite its inability to conduct Cl
. In contrast, TMD1 mutants gave appreciable Cl
conductances
in the absence of activatible ATP release. Furthermore, the R347E had a greater than 20-fold reduced GBr as compared with wtCFTR, yet the magnitude of JATP in the presence of extracellular Br
was sixfold higher for R347E
when compared with wtCFTR cRNA-injected oocytes.
Taken together, these data demonstrate that the permeation pathway associated with CFTR-modulated ATP release is independent of the Cl
conductance pathway in
the channel pore. The observations that only a subset of
CFTR-expressing oocytes displayed an ATP permeability despite equivalent levels of cAMP inducible Cl
conductances also supports this conclusion. Of note, however, frogs that produced ATP responder oocytes continued to
produce oocytes with CFTR-modulated ATP release in
subsequent harvests (see Materials and Methods for more
detail). We therefore hypothesize that another factor(s) is
required in addition to activated CFTR to confer ATP release in Xenopus oocytes. This conclusion is similar to that
reached in MDCK cells, where CFTR-dependent ATP
conductances were seen at the single channel level in 30%
of patches containing active CFTR Cl
currents (Sugita
et al., 1998
). Despite the possible need of a cofactor, the
rate of CFTR-modulated ATP release correlated (r = 0.74) with the magnitude of the Cl
conductance as measured sequentially in the same oocyte (Fig. 1 D). These
data suggest that both the abundance of CFTR and some other factor(s) likely affect the overall magnitude of ATP
efflux.
CFTR-mediated ATP Release Depends on Changes in Extracellular Chloride Concentration
Unique to the present study evaluating CFTR-mediated
ATP release is the dependence of activation on changes in
the extracellular Cl concentration. Chloride-dependent
activation of CFTR-modulated ATP release occurred
within a very narrow range of Cl
concentrations (110-120
mM), although the range of chloride concentrations necessary to sustain maximal levels of ATP efflux was slightly broader (85-140 mM) once CFTR-modulated ATP release was activated. We hypothesize that the interaction of
Cl
with basic residues within extracellular domains or the
channel pore may mediate structural changes in CFTR
necessary to activate ATP release. To explore this idea,
we examined the halide dependence of ATP release and
compared it to the halide conductance properties of CFTR
channels. The halide dependence experiments were limited to Cl
and Br
, since other anions, including I
, F
,
NO3
, and SCN
, significantly inhibited luciferin-luciferase
activity. Our studies of Wc conductance and permeability
of wtCFTR in the presence of external Cl
and Br
were
similar to previous findings (GCl
GBr and PBr > PCl;
Anderson et al., 1991
). Interestingly, these ratios (GBr/GCl = 0.93 ± 0.01, PBr/PCl = 1.13 ± 0.02) were significantly larger
than the ratio of halide dependence of CFTR-modulated
ATP efflux (JATP[Cl] = 8.7 ± 3.4 pmoles ATP/min,
JATP[Br] = 1.9 ± 0.52 pmoles ATP/min). Thus, the activation of CFTR-modulated ATP release appears to have a
higher specificity for Cl
when compared with the permeability properties of the CFTR channel pore. We therefore
hypothesized that a chloride sensor exposed to the external aqueous environment might exist within a region(s) of the
CFTR protein that modulates ATP release by responding to changes in the extracellular concentration of Cl
.
A Chloride Sensor Within the CFTR Channel Pore Regulates ATP Release
Obvious candidate sites in the CFTR molecule that might
be capable of sensing the extracellular Cl concentration
are aqueous exposed basic residues within the CFTR channel pore. We therefore characterized the effects of
arginine mutations R334W, R347P, and R347E on CFTR-modulated ATP release. R347 has been implicated as a
predominant site for Cl
binding that at least in part determines the halide selectivity of the channel pore (Anderson
et al., 1991
). Mutations in R347 have also been shown to
alter the number of anions that can simultaneously occupy
the pore (Tabcharai et al., 1993). Comparison of halide conductance and permeability of CFTR to the rate of
CFTR-mediated ATP release for these mutants suggests
that the properties of anion binding in the channel pore
may directly affect the activation of ATP efflux. For example, the R347P mutant demonstrated minimal changes in
GCl(cAMP), GBr/GCl, and PBr/PCl when compared with wtCFTR (Table II). Although the rate of ATP release in
the presence of external Cl
was approximately fourfold
lower for this mutant when compared with the wild
type, the halide dependence ratio of ATP release rates
(JATP[Br]/JATP[Cl] = 0.13) was quite similar to that of wtCFTR (JATP[Br]/JATP[Cl] = 0.22). Thus, a mutation that
resulted in minimal alterations in the anion conductance
properties of the CFTR channel pore was associated with
minimal changes in the halide dependence of ATP release.
In contrast, the R347E mutation caused pronounced alterations of both electrophysiologic as well as ATP release
properties of CFTR. The significant increase in GBr/GCl
when compared with wtCFTR is in agreement with previous studies (Anderson et al., 1991
). Concomitantly, the
R347E mutation dramatically altered the halide dependence of ATP efflux (JATP[Br]/JATP[Cl] = 7.3) when compared with wtCFTR (JATP[Br]/JATP[Cl] = 0.22). These analyses of the R347P and R347E mutations suggest that
the molecular interactions of Cl
and Br
within the channel pore likely influence activation of ATP efflux. The mutant R334W demonstrated no halide dependence in the
ATP efflux rates (JATP[Br]/JATP[Cl] = 1.1) when compared
with wtCFTR, suggesting that multiple residues within the
CFTR channel pore may be involved in halide binding and
subsequent conformational changes necessary for activation of CFTR-modulated ATP release.
In summary, mutational analyses implicate basic residues R347 and R334 within the CFTR channel pore as important sites that contribute to the effects of extracellular
halides on CFTR-modulated ATP release. Because CFTR
is insufficient to promote ATP release, we hypothesize
that the properties of the Cl and Br
binding sites within
the channel pore reflect those of a chloride sensor that determines the extent of functional interaction between CFTR and a second cofactor necessary to mediate ATP
release (Fig. 8). In wtCFTR, this sensor has a dependence
of Cl > Br. We hypothesize that mutations within the
channel pore that alter the affinity of Cl
and/or Br
binding are also responsible for changes in the halide dependence of ATP efflux. An unexplained feature is the need
for both cAMP agonist stimulation and a change in extracellular Cl
concentration (from low to high external Cl
).
We believe that these conditions may be necessary to trap
CFTR in a particular conformation that is favorable for
activating the ATP release machinery (Fig. 8), and that
Cl
binding within the channel pore may facilitate attainment of this conformation. Why CFTR-modulated ATP
release is not seen in cAMP-stimulated oocytes bathed in
high external Cl
without prior exposure to Cl
-free solutions is currently unknown. However, this feature suggests that CFTR can sense changes of external Cl
concentration and respond by invoking structural changes necessary for interaction with an ATP release pathway (Fig. 8). The
magnitude of such concentration changes may be extremely small (10-15 mM Cl
) as indicated by the sharp
relationship between ATP efflux and extracellular Cl
concentration (Fig. 5).
|
Implications in CF Airways Disease
The mechanism(s) by which CFTR regulates ionic composition of surface airway fluid are only beginning to be understood. Several studies have demonstrated regulatory
affects of CFTR on other epithelial ion channels such as
ENaC and the ORCC (Egan et al., 1992; Gabriel et al., 1993
;
Schweibert et al., 1995; Jovov et al., 1995
; Grubb et al., 1994
;
Chinet et al., 1994
; Johnson et al., 1995
; Hyde et al., 1993
; Ismailov et al., 1996
). Defects in the functional properties of
both the ORCC and ENaC have been observed in CF airways disease (Guggino, 1993
; Gabriel et al., 1993
; Stutts et
al., 1995
; Kunzelmann et al., 1997
; Iwase et al., 1994). Previous studies have implicated CFTR-modulated ATP release as likely mechanisms by which CFTR regulates the
ORCC through P2U purinergic receptors pathways (Schwiebert et al., 1995
). The relevance of this dysregulation in CF
airways disease is currently unknown, but may reflect one
potential mechanism by which CFTR regulates Cl
transport in the airways. Findings from the present study suggest that CFTR can sense the extracellular Cl
concentration in the airways and respond by activating ATP release pathways, which may in turn be responsible for regulating
other channels in the apical membrane of epithelial cells.
Because the concentration of Cl
necessary for activation
of CFTR-modulated ATP release is within the physiologic
range of the known Cl
concentration in the airway (85-
130 mM; Smith et al., 1996
; Gilljam et al., 1989
; Joris et al.,
1993
), we speculate that this regulatory function of CFTR
may act as a salt concentration sensor important in maintaining airway electrolyte balance. Such mechanisms that
maintain the appropriate salt concentration in the airway have important consequences on the antibacterial activity
of surface airway fluid (Smith et al., 1996
).
|
![]() |
Footnotes |
---|
Received for publication 16 June 1998 and in revised form 4 September 1998.
Address all correspondence to Dr. John F. Engelhardt, Ph.D., Associate
Professor, University of Iowa, Department of Anatomy and Cell Biology,
1-111 BSB, 51 Newton Road, Iowa City, IA 52242-1109. Tel.: (319) 335-7753. Fax: (319) 335-6581. E-mail: john-engelhardt{at}uiowa.edu
We thank Dr. Welsh for his mutant CFTR cDNAs R334W, R347E, and R347P, and his thoughtful review of this manuscript.
We gratefully acknowledge the support of the Cystic Fibrosis Foundation for its fellowship support of Dr. Jiang, grant support to J.K. Foskett, W.B. Guggino, and J.F. Engelhardt (9720), and National Institutes of Health DK49136 (J.F. Engelhardt), DK48977 (W.B. Guggino), and HL47122 (W.B. Guggino).
![]() |
Abbreviations used in this paper |
---|
ABC, ATP-binding cassette;
CF, cystic
fibrosis;
ORCC, outwardly rectifying Cl channel;
TEV, two-electrode
voltage clamp;
Vm, transmembrane potential;
Wc, whole cell.
![]() |
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