From the Institute for Molecular Medicine and
Department of Anatomy and Cell Biology, Uniformed Services University
School of Medicine (USUHS), Bethesda, Maryland 20814, the
§ Department of Physiology and Biophysics, Case-Western
Reserve University, Cleveland, Ohio 44106, and the ¶ Section on
Bioorganic Chemistry, Laboratory of Chemistry, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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8-Cyclopentyl-1,3-dipropylxanthine (CPX) and
1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine
antagonists which activate chloride efflux from cells expressing either
wild-type or mutant (F508) cystic fibrosis transmembrane conductance
regulator (CFTR). These drugs are active in extremely low
concentrations, suggesting their possible therapeutic uses in treating
cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also
generate a profound activation of CFTR channels incorporated into
planar lipid bilayers. The process of activation involves a pronounced
increase in the total conductive time of the incorporated CFTR
channels. The mechanism involves an increase in the frequency and
duration of channel opening events. Thus, activation by these drugs of
chloride efflux in cells very likely involves direct interaction of the
drugs with the CFTR protein. We anticipate that this new information
will contribute fundamentally to the rational development of these and
related compounds for cystic fibrosis therapy.
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INTRODUCTION |
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Cystic fibrosis (CF)1 is
the most common, fatal, autosomal recessive disease in the United
States, affecting nearly one birth in 2000 (1, 2). The responsible gene
has been identified as CFTR (cystic fibrosis
transmembrane conductance regulator), and the
vast majority of CF patients carry a deletion of phenylalanine from
position 508 (F508; Refs. 3-5). The physiological function of CFTR
includes cAMP-activated chloride channel activity (6-9), and the
mutation compromises the ability of CFTR to traffic out of the
endoplasmic reticulum to its appropriate location on the apical plasma
membrane (10). Repair of the mutation can be effected by transferring
wild-type CFTR into the mutant cell (11), and the long term strategy
for treatment of cystic fibrosis has thus become focused on gene
therapy at the level of the whole organism. However, it has also been
observed that the
F508 CFTR does have intrinsic channel activity
(12, 13). Furthermore, incubation of some mutant cells at low
temperature (14), or in chemical chaperones such as 1 M
glycerol (15), do permit the mutant CFTR to traffic out of the
endoplasmic reticulum to the plasma membrane. Once at the membrane, the
mutant CFTR exhibits cAMP-activated chloride channel activity, thereby
permitting functional repair. These data have thus been interpreted to
indicate that the
F508-CFTR is intrinsically active, and more recent
data have shown that in some cells a small but measurable portion of
the mutant CFTR actually spontaneously traffics to the vicinity of the
plasma membrane (16, 17). These data therefore suggest that an
alternative way to overcome the reduced chloride transport in CF cells
might be to find compounds which further activate the small portion of
mutant CFTR chloride channels which have trafficked to the cell
membrane.
The adenosine A1-receptor antagonist CPX
(8-cyclopentyl-1,3- dipropylxanthine) has been shown to stimulate
36[Cl] efflux from pancreatic CFPAC-1 cells
(18) in the concentration range of 20-100 nM. These cells
are homozygous for the
F508 genotype common to most cases of cystic
fibrosis. Similar results were obtained with the CF tracheal epithelial
cell line IB3-1 expressing the
F508 allele, and with recombinant
mouse fibroblast NIH 3T3 cells (19). Schweibert et al. (20)
have also shown that CPX (50 nM) activates outward chloride
currents in whole cell patch studies of primary explants of nasal
epithelial cells from homozygous
F508 CF patients as well as
wild-type CFTR control cells. More recently, Haws et al.
(17) showed that CPX could activate iodide efflux from recombinant
cells expressing
F508 CFTR. Thus wherever the CFTR mutant has been
expressed, or in some favorable cases the wild-type CFTR, an effect of
CPX on chloride efflux can be demonstrated.
In considering how CPX activates wild-type CFTR or repairs mutant CFTR, it is possible that CPX action might either be directly on the CFTR molecule, or be secondary to binding of CPX to another protein (21). As indicated above, CPX is best known as an adenosine A1-receptor antagonist (22). However, Northern blot analysis and structure-activity relationship ("SAR") studies with 26 different compounds indicate that the A1-receptor is not responsible for CPX action in CFPAC cells (23). As one useful example, 1,3-diallyl-8-cyclohexylxanthine (DAX), a poor A1 antagonist, is also highly potent and efficacious in stimulating chloride efflux from CFPAC-1 cells (23). Thus the site of action of these xanthines in stimulating chloride efflux appears to represent a novel site of action, possibly involving direct interaction with the CFTR molecule. Additional information consistent with this latter possibility are data indicating that radiolabeled CPX binds with high affinity to the recombinant first nucleotide-binding fold (NBF-1) of CFTR (24) and to a subdomain within NBF-1 (25).
To test this hypothesis directly at the single channel level, we have incorporated recombinant wild-type CFTR channels from HEK293 cell microsomal membranes into planar lipid bilayers, and tested whether CPX could activate chloride currents through CFTR channels. In addition, we tested whether DAX, another active CPX analogue, could also activate chloride currents through CFTR channels. We report here that both CPX and DAX potently activate cAMP-dependent CFTR chloride channels. Furthermore, both compounds affect CFTR channel kinetics differently, and in a manner predictable from their respective actions on chloride efflux from different cell types. Furthermore both CPX and DAX also bind with high affinity to the recombinant first nucleotide-binding fold domain (NBF-1) of CFTR (24). These data together indicate that the mechanism of CFTR channel activation very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy.
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MATERIALS AND METHODS |
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Expression of CFTR in HEK293 Cells-- Wild-type CFTR cDNA was subcloned into the eukaryotic expression vector pCEP4 (Invitrogen) between the NheI and XhoI restriction sites to create the recombinant vector, pCEP4(CFTR) (11). A human embryonic kidney cell line (HEK293-EBNA: Invitrogen) was used for the transfection and expression of CFTR protein (26-28). This cell line contains the vector pCMV-EBNA which constitutively expresses the Epstein-Barr virus EBNA-1 gene product which increases the transfection efficiency of Epstein-Barr virus-based vectors. The parent cell line was maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% glutamine. Geneticin (G418, 250 µg/ml) was added to the cell culture media to maintain selection of the cells containing pCMV-EBNA vector until after CFTR gene transfer. pCEP4(CFTR) was then introduced to the cell using Lipofectin reagent (Life Technologies), and 2 days after transfection, the cells were passaged and selected for hygromycin resistance (hygromycin B, 260 µg/ml). Three weeks after transfection, microsomal vesicles were isolated from transfected cells. The expression of CFTR protein was conformed by Western blot using an antibody against the R domain of CFTR (mAb 13-1, Genzyme; Ref. 28).
Isolation of Microsomal Membranes from Cultured
Cells--
Microsomal vesicles were isolated from HEK 293 cells
expressing wild-type CFTR protein using a modified protocol of
Gunderson and Kopito (29), as described previously (26, 30). Briefly, 12 × 75-cm2 flasks of HEK 293 cells transfected with
pCEP4(CFTR) vectors were harvested. The cell pellet was resuspended in
ice-cold hypotonic lysis buffer (10 mM HEPES/NaOH, pH 7.2, 1 mM EDTA, 5 µM diisopropyl fluorophosphate,
10 µg/ml pepstatin A, 10 µg/ml aprotinin, and 10 mg/ml benzamidine)
before lysis by 10 strokes in a tight-fitting Dounce glass homogenizer,
followed by 15 strokes after the addition of an equal volume of sucrose
buffer (500 mM sucrose, 10 mM HEPES/NaOH, pH
7.2). Microsomes were collected by centrifugation of a postnuclear supernatant (600 × g for 15 min) at 100,000 × g for 45 min, and resuspended in 1 ml of prephosphorylation
buffer (250 mM sucrose, 10 mM HEPES/NaOH, pH
7.2, 5 mM Mg-ATP, and 100 units/ml PKA catalytic subunit).
The membrane vesicles were stored at a protein concentration of 2-6
mg/ml at 75 °C until use.
Preparation of CPX and DAX-- CPX was synthesized according to GMP ("Good Manufacturing Practice") as part of our program for preparing CPX for clinical trials on cystic fibrosis patients. The CPX was solubilized at a concentration of 10 mM in dimethyl sulfoxide, and further diluted in dimethyl sulfoxide prior to dilution into the chamber solution. Prior to recording the effects of the drug, the contents of the chamber were mixed for 30 s using an internal mixing apparatus. The final concentration of dimethyl sulfoxide never exceeded 1%, and this amount of dimethyl sulfoxide alone was found to be entirely inactive, either on the naked bilayer or on incorporated CFTR channels. DAX was synthesized as described previously (23).
Planar Lipid Bilayer Technology-- Planar bilayers were formed by applying a suspension of palmitoyloleoyl phosphatidylethanolamine and palmitoyloleoyl phosphatidylserine, 1:1, 50 mg/ml, each in n-decane, to a hole of about 100-120 µm in diameter in a thin TeflonTM film separating two compartments that contained defined salt solutions (31). Channels were incorporated from a suspension of microsomes prepared from HEK293 cells expressing wild-type CFTR. The microsomes were added to the cis chamber in small aliquots, and incorporation occurred directly from the experimental solutions. Currents associated with CFTR channels were observed shortly after the bilayer system was exposed to protein kinase A (100 units/ml). The specific conditions include a KCl gradient (cis = 200 mM; trans = 50 mM), (1 mM) MgCl2, and ATP (2 mM) in cis, and 10 mM Tris/HEPES in cis and trans, adjusted to a final pH of 7.0. Single channel currents were recorded using a patch-clamp amplifier (AXOPATCH-1D equipped with a CV-4B 0.1-100 Bilayer headstage, Axon Instruments, Foster City, CA), and data were stored on magnetic tape using a pulse-code modulation/video cassette recorder digital system (Toshiba) with a frequency response in the range from direct current to 25,000 Hz.
Statistical Evaluation and Channel Current
Analysis--
Off-line analysis of the recorded CFTR channel activity
was carried out using the software package pClamp 5.51 and 6. (Axon Instruments, Foster City, CA). Data base files were obtained from playbacks of the experimental records digitized using a 12-bit analog
to digital converter (TL-1 DMA interface, Axon Instruments) using the
fetchex subroutine. The channel current signal from the PCM-VCR was fed
through a low pass filter (eight-pole Bessel 902 LPF; Frequency Devices
Inc., Haverhill, MA) in series with the ADC module. The filtering level
was set between 50 and 100 Hz. As a rule the data base used for open
time and close time distribution corresponded to filtered recordings
with a signal to noise ratio of 4:1.
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RESULTS |
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Single Channel Recordings of CFTR-- HEK293 cells expressing a permanently transfected CFTR gene were grown and microsomes were prepared by differential centrifugation (30). Microsomal membrane vesicles carrying the expressed protein were then incorporated into a planar lipid bilayer, and ionic currents measured in the presence of a chemical and potential gradient. Currents associated with CFTR channels were observed shortly after the bilayer system was exposed to protein kinase A (100 units/ml). The specific ionic conditions, as described under "Materials and Methods," were maintained throughout all the experiments described in this work. As previously reported, these channels are insensitive to DIDS (50 µM), blocked by diphenylamine-2-carboxylate (300 µM), and are selective for chloride (26, 27, 30).
Fig. 1A, illustrates typical current events observed upon application of electrical potentials (
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Influence of CPX on CFTR Channel Activity--
CFTR was
incorporated into the planar lipid bilayer, and the channel modestly
activated by addition of PKA and ATP (see "Materials and Methods"),
Fig. 2A. These data represent
the control condition, in which the dominant motif is relatively low
CFTR channel activity at the conductance level of 8.3 pS, and generated
by a 50 mV driving force potential. Fig. 2B shows
continuous recordings of CFTR channel activity, generated by the same
driving force 5 min after 500 nM CPX was added to the
cis side of the planar lipid bilayer system. The figure
represents 2 min of continuous recording. The overall general activity
is considerably increased, and the system exhibits multilevels of
current that were not observed under control conditions. Upon elevation
of the CPX concentration to 2 µM (see Fig.
2C), the general pattern of activity is reduced relative to
the maximum at 500 nM CPX. However, it remains higher than
control. This type of "bell-shaped response" is precisely what
would have been predicted from our previously published results with
the effect of CPX on chloride efflux from CF cells (19, 23). A kinetic
analysis of these data, shown below, indicates that the effect of CPX
is to increase the number and duration of open events of CFTR channels
without modifying their conductance and selectivity.
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I-V Curve of CFTR Channels in the Presence of CPX--
The I-V
relationship for CFTR channels under control and 500 nM CPX
conditions are shown in Fig.
3A. To prepare these data we
plotted the arithmetic mean current amplitude of unitary events recorded at different electrical potentials (59 events in control conditions and
257 events in the presence of CPX, for each
potential). Regression lines fit for the two conditions are
statistically identical, indicating that the slope conductances (8.2 pS) and equilibrium potentials (12 mV) for ion fluxes are the same for both conditions. This indicates that the ionic selectivity and the
unitary conductance of CFTR channels are not affected by the interaction with the drug. Rather, the increased current activity of
the system must be due to a direct effect of CPX on the kinetics of the
CFTR channel behavior.
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Influence of CPX on Amplitude of CFTR Current Levels--
The
action of CPX on the CFTR conductance can be most clearly visualized by
construction of amplitude histograms from current records. Fig.
3B, left panel, shows such an amplitude histogram from a
control record summarizing the distribution of amplitudes at 50 mV.
The Gaussian distribution of the mean amplitudes of these events gives
an average amplitude of 0.48 pA (59 unitary events). In the presence of
500 nM CPX (Fig. 3B, right panel) the amplitude
histogram of all events occurring during 2 min recording reveals that
the events are distributed in three levels. The principal Gaussian peak
(number 1)(749 events) is very similar to that of the control in the
absence of CPX. The additional peaks (number 2 (257 events) and number
3 (9 events)) are exact multiples of the principal peak. These data
indicate that the CPX-induced increase in CFTR activity are likely to
be additional CFTR channels.
Effect of CPX on the Activity of the 2.5 pS CFTR
Conductance--
As mentioned above, the smaller 2.5 pS conductance
associated with CFTR expression can, on occasion, be incorporated as a single channel in isolation from the larger 7-10 pS CFTR conductance. We therefore took advantage of several such instances to study the
influence of CPX on the smaller conductance. Addition of CPX also
increased the overall activity of the 2.5 pS conductance. For a better
signal to noise ratio, we analyzed this increased activity at a large
potential. As shown in Fig.
4A, control conditions at a
membrane potential of 100 mV (upper panel) are
characterized predominantly by brief spikes, which under the 50 Hz
filtering conditions give an apparent average open time of 8 ms and an
apparent arithmetic mean amplitude of 0.08 pA. After the addition of
500 nM CPX the activity of the channel increases
profoundly, with the apparent arithmetic mean amplitude increasing to
0.14 pA (Fig. 4A, lower panel). The effect of CPX is to
increase the duration of single events. Quantitatively, the open time
probability increases from about 8% in control conditions to 35-38%
in the presence of CPX. Since the control amplitude of 0.08 pA is the
average spike amplitude observed under high filtering conditions (50 Hz), the apparent increase in current amplitude mediated by CPX may not
be the real effect of the drug. This is because the amplitudes of
longer events are less attenuated by the filtering condition. Thus,
alternatively, the apparent increase in current amplitude could also be
a consequence of the ability of CPX to prolong the open time of
individual events.
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Influence of CPX on Open Time Distributions of the 2.5 pS CFTR Conductance-- To test the latter hypothesis we plotted open time histograms of the 2.5 pS CFTR conductance for both control and 500 nM CPX conditions. Channel activity of the 2.5 pS conductance under control conditions is characterized by predominantly brief spikes with an average duration of 8 ms (see upper panel of Fig. 4B). A minor peak is observed at approximately 24 ms. In the presence of CPX (see lower panel, Fig. 4B) the increase in open time duration is not monotonic but is distributed into three main Gaussian populations, with peak durations of 9, 27, and 44 ms, respectively. However, we observe that the 27 ms population in the CPX treated condition is actually present to a very minor extent in the drug-free condition (viz. the 24 ms population in the control histogram in the upper panel of Fig. 4B). These data therefore suggest that the CPX may activate or modify existing 2.5 pS CFTR channels, rather than merely recruit cryptic or new CFTR channels. We conclude that these data support the concept that addition of CPX not only increases the number of events, but also their duration.
Influence of DAX on CFTR Channel Activity--
DAX is also a
potent activator of chloride conductance in CF cells, much like CPX,
which we hypothesized might also activate CFTR channels. To study this
possibility we followed the same protocol used to study CPX. Therefore,
prior to the addition of DAX we established an active CFTR channel
using PKA/ATP (see "Materials and Methods"). We then added DAX at
various concentrations and studied the channel activity at various
membrane potentials. In all conditions, addition of DAX produced a
profound increase in basal channel activity. Fig.
5A summarizes one of those
results for a driving force potential of 50 mV. The data representing the control condition show a dominant motif of relatively low activity
at the level of 7-10 pS. The 2.5 pS conductance is absent from this
example. DAX (500 nM) is then added to the cis
side of the planar lipid bilayer system. As shown in Fig.
5B, the number of channel events is considerably increased.
By inspection, at least four different levels can be observed.
Furthermore, the apparent duration of channel events appears elevated.
Then, when the DAX concentration is increased to 1.5 µM
(Fig. 5C), the general pattern of activity is only slightly
reduced compared with the more profound suppressive effect of a similar
concentration of CPX (compare with Fig. 2C).
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Comparison of CPX and DAX Effects on CFTR Channel Activity-- The data above suggest that CPX and DAX are similar in their ability to activate the CFTR channel, but that they differ in terms of potency and kinetics. To further examine this possibility we titrated CPX and DAX in a stepwise, dose-escalating fashion on CFTR channels in the large 7-10 pS range. Fig. 6 shows drug titrations on two different channel incorporations in which it was possible to follow the activity of each of the channels systematically, up to 2000 nM for CPX or 1500 nM for DAX. Although the drugs were tested on different channels, the control open probabilities for the two channels were very similar before the addition of the drug (2 and 5% for CPX and DAX, respectively). Therefore, in Fig. 6 we superimposed all the resulting data in the same plot in an attempt to compare the effects of the two drugs.
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Influence of PKA and CPX on CFTR Channel Kinetics-- To further evaluate the possibility of direct activity of CPX on the CFTR channels, we activated the channels in the usual way, and then removed PKA and ATP from the chamber. As shown in Fig. 7A (see control currents), basal activity was sustained. Indeed, we saw no substantive change in the open probability of CFTR channels. This observation is not surprising since it is widely appreciated that once the channel is phosphorylated removal of PKA has no effect on sustained activity (38). Dephosphorylation is a counteracting process which is known to reduce CFTR activity (38, 39). Thus the sustained activation of CFTR after removal of PKA in our system indicates that the functional phosphatase activity incorporated with these channels is minimal.
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DISCUSSION |
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The experimental results presented here provide evidence that CFTR
chloride channels, incorporated in planar lipid bilayers and activated
by pKA and ATP, can be further activated by the addition of the
xanthines CPX or DAX. This enhanced activation appears to be the effect
of direct interaction of these drugs with the CFTR channel molecules.
Earlier data leading to these studies with intact cells had shown that
CPX could activate chloride efflux (17-19) or chloride current (20)
from cells expressing either wild-type or mutant (F508) CFTR,
respectively. In addition, these drugs have been shown to bind
selectively to a specific domain (NBF-1) of the CFTR molecule (24, 25).
However, until the present study a direct connection between CPX or DAX
binding and CFTR channel activation, per se, has been
missing. The data presented in this work therefore provide strong
evidence that the mechanism of activation is indeed very likely to be
by direct interaction between CPX or DAX and the CFTR molecule.
CFTR channel activation, measured as a pronounced increase in the total open time probability or total conductive time, is a combined result of an increase in the frequency and in the duration of channel opening events. The observed increased frequency in the number of events could have several origins, since the source of CFTR channels are microsomes from transfected cell membranes which fuse to the lipid bilayer. We envision at least three possible activation mechanisms to explain the observed overall channel activation. First, the drugs may further activate an already activated channel. For example, we do observe an increase in the frequency and duration of unitary CFTR channel events after drug addition. Second, the drugs might activate otherwise inactive but incorporated channels. The evidence is that multiple channels become evident only after the drugs are added. However, all channels in the experimental chamber are potentially activated since they are permanently bathed in PKA and ATP, and we do not see activation by the drugs if channels have not been previously activated by PKA. Finally, it is possible that the drugs might facilitate the fusion to the bilayer of new microsomes carrying active channels. For example, CFTR can facilitate endosome-endosome fusion (32), and recombinant NBF-1 promotes phosphatidylserine liposome interaction (25, 33). However, we do not detect new conductances after drug addition, which might have supported this third possibility. It therefore follows that the available data do not rule out any of these mechanisms. However, the data presented here do tend to favor the possibility that CPX and DAX further activate otherwise active CFTR channels by direct interaction with the protein.
From a different perspective, recently published radioligand binding
data have indicated that both CPX and DAX bind with high affinity to
the recombinant first nucleotide-binding fold domain (NBF-1) of CFTR
(24). In addition, the F508-NBF-1 binds both ligands with
substantially greater affinity than wild-type NBF-1 (24). Thus, taken
together both the binding data and the channel data presented here
provide strong arguments for the ability of CPX and DAX to activate
CFTR by direct interaction with the CFTR protein. These results also
further exclude the possibility that simultaneous actions of CPX on
adenosine A1 or adenosine A2 receptors might be
responsible for the unusual kinetic activity on CFTR function. An
important pharmacologic difference between CPX and DAX is that while
CPX is a quite potent adenosine A1 antagonist, DAX is
relatively poor. Yet they both activate CFTR in cells and bilayers with
intrinsic but proportional differences in both systems.
Finally, we can speculate about the possible mechanisms by which these xanthine drugs might activate CFTR channel activity. The data in Fig. 7 indicate that to observe the channel, continued presence of PKA in the chamber is not needed once the channel is phosphorylated. Furthermore, the continued presence of PKA is not needed to observe channel activation by CPX. The sustained activity after removal of PKA indicates that endogenous phosphatase activity is minimal, and that the CPX activation is probably not through inhibition of this otherwise minimal activity. Thus CPX activation does not appear to be mediated by action on ancillary activation or inhibition systems, leaving only direct action on the CFTR protein as the likely mechanism. In searching for the most likely site of CPX interaction on CFTR, attention could immediately turn to the NBF-1 domain of CFTR for a variety of circumstantial but relevant reasons. First, both CPX and DAX also bind with high affinity to the recombinant first nucleotide-binding fold domain (NBF-1) of CFTR, in the vicinity of F508 (24). Second, the NBF-1 domain is immediately contiguous with the cytosolic aspect of the sixth transmembrane domain of CFTR (TMD6), and Akabas and colleagues (34, 35) have shown that this domain is important in defining the selectivity filter of CFTR. This sequential contiguity provides a structural basis for understanding how ATP might affect channel function, and might also provide a basis for direct CPX or DAX actions on the channel. Finally, the fact that NBF-1 itself interacts intimately with artificial (31) and natural membranes (36, 37) could provide the energetic basis for hypothetical close interactions between TMD6 and NBF-1 within or near the plasma membrane.
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FOOTNOTES |
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* 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.
1 The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CPX, 8-cyclopentyl-1,3-dipropylxanthine; DAX, 1,3-diallyl-8-cyclohexylxanthine; PKA, protein kinase A; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; NBF, nucleotide-binding fold.
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REFERENCES |
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