(Received for publication, December 15, 1994; and in revised form, June 14, 1995)
From the
A unique feature of the cystic fibrosis transmembrane
conductance regulator (CFTR) Cl channel is regulation
by ATP through the two cytoplasmic nucleotide-binding domains (NBDs).
To better understand this process, we asked how channel activity is
affected by inorganic pyrophosphate (PP
), a compound that
binds to NBDs in other proteins. PP
and three
nonhydrolyzable PP
analogs reversibly stimulated the
activity of phosphorylated channels. Kinetic modeling of single channel
data demonstrated that PP
affected two distinct steps in
channel regulation. First, PP
increased the rate at which
channels opened. Second, once channels were open, PP
delayed their closure. PP
could only stimulate
channels when it was applied in the presence of ATP. PP
also increased the photolabeling of CFTR by an ATP analog. These
two findings suggest that PP
modifies the activity of
ATP-dependent CFTR channel gating. Based on these and previous data, we
speculate that the effects of PP
are mediated by binding of
PP
to NBD2 where it regulates channel opening by NBD1, and
then, because it is not hydrolyzed, it slows the rate of NBD2-mediated
channel closing. Because PP
stimulated wild-type channels,
we tested its effect on CFTR containing the cystic fibrosis mutations:
F508, R117H, and G551S. PP
stimulated all three.
PP
also stimulated endogenous CFTR in the apical membrane
of permeabilized T-84 epithelia. These results suggest that PP
or an analog might be of value in the development of new
approaches to the treatment of cystic fibrosis.
The cystic fibrosis transmembrane conductance regulator (CFTR) ()is an epithelial Cl
channel with novel
structure and regulation (for reviews see Refs. 1 and 2). CFTR is
composed of two membrane-spanning domains that contribute to formation
of the ion conducting pore and three cytoplasmic domains that regulate
channel activity: two nucleotide-binding domains (NBDs) and the R
domain. Dysfunction of the CFTR Cl
channel causes
cystic fibrosis (CF), a common lethal genetic
disease(3, 4) . An important goal of CF research is to
understand the function of CFTR and to use that knowledge to develop
better treatments for the disease.
The presence of two NBDs confers a complex mechanism of regulation on channel activity. Phosphorylation of the R domain by cAMP-dependent protein kinase is necessary but not sufficient for channel activity. Once the R domain has been phosphorylated, ATP is required to open the channel(5) . Functional studies of ATP regulation of variant channels and studies of nucleotide binding to full-length CFTR and to NBD peptides suggested that ATP may interact with both NBDs(6, 7, 8, 9, 10, 11, 12) . The inability of nonhydrolyzable analogs to open the channel suggested that ATP hydrolysis is required for activity(5, 8, 13, 14) . Although NBD1 and NBD2 have some sequence similarity, functional studies of CFTR containing site-directed mutations in the NBDs provided evidence that the two NBDs have distinct functions in controlling the channel(6, 7, 8) . Through work with nucleotide analogs, vanadate, and beryllium, Gadsby and co-workers (13, 15) suggested that hydrolysis of ATP is not only required for channel opening but is also involved in channel closure from the bursting state. This conclusion was supported and expanded upon by our work in which mutations expected to decrease the rate of hydrolysis by NBD2 but not by NBD1 increased the duration of bursts(8) . Thus ATP binding and hydrolysis at the two NBDs appear to regulate both channel opening and closing, perhaps with the rate of ATP hydrolysis at NBD1 regulating opening and the rate of ATP hydrolysis at NBD2 regulating closure.
To further understand how the
two NBDs function to regulate channel activity, we examined the effect
of inorganic pyrophosphate (PP) on CFTR channel gating.
Gunderson and Kopito (14) recently showed that PP
could prolong bursts of activity, an effect similar to that
observed with the nonhydrolyzable ATP analog
AMP-PNP(8, 15) . However, the mechanism of this effect
was not examined. We hypothesized that PP
might interact
with the NBDs of CFTR to alter ATP-dependent channel activity. Previous
studies have demonstrated that PP
binds with high affinity
to the NBDs of other ATP-binding proteins. For example, PP
interacts with both intact mitochondrial F
-ATPase (16, 17, 18) and a 50-amino-acid
F
-ATPase peptide(19) . PP
also binds to
myosin, producing a charge change effect similar to that produced by
ATP binding(20) , and produces dissociation of S-1 myosin from
actin (21) without being hydrolyzed(22) .
We
examined the effect of PP on CFTR channel activity using
the excised inside-out configuration of the patch-clamp technique. We
found that PP
strongly potentiated the activity of
phosphorylated channels in the presence but not the absence of ATP. We
also assessed the effect of PP
on photolabeling of CFTR
using the photolabile ATP analog 8-N
ATP. We then used
kinetic modeling of single channel data to investigate the mechanism of
how PP
stimulated channel activity. Our results led us to
test the effect of PP
on three CF-associated mutant CFTRs
and in a permeabilized epithelial preparation to explore the
possibility that PP
or an analog might be utilized in the
development of new approaches to therapy.
For experiments with excised inside-out membrane patches, the
pipette (extracellular) solution contained (in mM): 140 N-methyl-D-glucamine, 100 aspartic acid, 35.5 HCl, 5
CaCl, 2 MgCl
, 10 HEPES, pH 7.3, with 1 N NaOH. The bath (intracellular) solution contained (in
mM): 140 N-methyl-D-glucamine, 135.5 HCl, 3
MgCl
, 10 HEPES, 4 cesium, and 1 EGTA, pH 7.3, with 1 N HCl ([Ca
]
<
10
M). PP
and PP
analog stock solutions were 200 mM in cesium EGTA-free
bath solution, pH 7.3, and were diluted to desired the final
concentration in bath solution, except for etidronate disodium, which
was diluted from commercial preparation. For Ussing chamber
experiments, the mucosal (apical) solution contained (in mM):
135 NaCl, 1.2 MgCl
, 1.2 CaCl
, 2.4
K
HPO
, 0.6 KH
PO
, and 10
HEPES, pH 7.3. The submucosal (basolateral) solution contained (in
mM): 135 sodium gluconate, 7 mM MgSO
, 2.4
K
HPO
, 0.6 KH
PO
, 10
HEPES, 10 dextrose, no added calcium, and 1 magnesium ATP, pH 7.3.
For excised macro-patch data, replayed records were filtered at 1 kHz using a variable 8-pole Bessel filter (Frequency Devices Inc., Haverhill, MA) and digitized at 2 kHz. Each time course data point represents the average current from 1 s with one data point collected every 5 s. Average currents for an intervention were determined as the average of the last 12 data points (the last minute) during the intervention. To compensate for any channel run-down during an experiment, specific interventions were bracketed when possible with current measurements made with similar concentrations of ATP but without the test compound; the intervention current was then compared with the average of pre- and post-intervention currents. For single channel analysis, replayed data were filtered at 1 kHz using a variable 8-pole Bessel filter, digitized at 5 kHz, and digitally filtered at 500 Hz. Idealized records were created using a half-height transition protocol; transitions less than 1 ms in duration were not included in the analysis. For the purpose of illustration, time course figures are inverted so that an upward deflection represents an inward current, data points during solution perfusion were not included in some figures, and single channel traces were digitally filtered at 200 Hz.
Burst analysis was performed as
described previously(8, 27) , using a t (the time which separates interburst closures from intraburst
closures) of 20 ms. This value was derived from analysis of wild-type
CFTR closed-time histograms derived from excised inside-out membrane
patches containing a single channel studied in the presence of 1 mM ATP plus cAMP-dependent protein kinase and by the method of
Sigurdson et al.(28) . Closures longer than 20 ms were
considered to define interburst closures, whereas closures shorter than
this time were considered gaps within bursts. We used a 20-ms
interburst discriminator for studies done in both the absence and
presence of PP
and analogs; this is justified by the facts
that closed-time histograms showed a similar minimum at approximately
20 ms in the presence of PP
and that kinetic analyses
showed little change in rates within bursts (note that in Fig. 5the
is 20 times slower than
). Burst data for PP
, PNP, and PCP were
derived from experiments in which the membrane patch contained one
active channel. For experiments with etidronate and
F508 CFTR,
burst data were from patches containing four or fewer active channels;
bursts in which there were no superimposed openings and that were
separated from other bursts by greater than 20 ms were included in the
analysis. We have previously shown that no discernible bias is observed
by including burst data from patches with more than one
channel(8, 27) .
Figure 5:
Effect of PP on kinetically
modeled rate constants. A, a linear three state model of CFTR
channel activity composed of two closed states (C
and
C
), one open state (O), and four rate constants
(
,
,
, and
). B-E show values of rate constants
before and after the addition of 5 mM PP
. Rate
constants were derived as described under ``Experimental
Procedures'' from four experiments in which the membrane patch
contained only one active channel, studied in the presence of 0.3
mM ATP and 75 nM cAMP-dependent protein kinase. The asterisks indicate p <
0.05.
Photolabeling was performed by preincubating membranes (50 µg of
membrane protein/sample) on ice with
[-
P]8-N
ATP (30 µM,
6-12 Ci/mmol) and PP
(in mM) as indicated in
the figures. After 60 s of UV irradiation, CFTR was solubilized and
immunoprecipitated as described (12) using antibodies raised
against the R domain (M13-1, 0.3 µg/sample) and against the C
terminus (M1-4, 10 µg/sample). Immunocomplexes were analyzed
by SDS-polyacrylamide gel electrophoresis, and incorporation of
[
-
P]8-N
ATP was quantitated with
an AMBIS radioanalytic imaging system (AMBIS Systems, Inc., San Diego,
CA). Data are expressed as the percentage of radiolabel incorporation
relative to control that had no added PP
. We have
previously shown the specificity of the labeling(12) .
Results are the means ± S.E. of n observations. Statistical significance was assessed using a paired, unpaired, or one-population Student's t test as appropriate.
Figure 1:
A, effect of PP on CFTR
Cl
current. Data show the time course of current in
an excised membrane patch from a HeLa cell transiently expressing
wild-type CFTR. Prior to starting the time course, CFTR Cl
channels had been phosphorylated with 75 nM cAMP-dependent protein kinase and 0.3 mM ATP (not shown).
ATP (0.3 mM) and PP
(5 mM) were present
during times indicated by bars. Data were not collected while
solutions were changed. B, effect of PP
concentration on CFTR Cl
currents. All values
were determined in the presence of 0.3 mM ATP and are
expressed as the percentages of current supported by 0.3 mM ATP. Data points are mean ± S.E. of 3-15 observations
at each point; some errorbars are smaller than data
symbols.
To determine
how PP stimulated CFTR currents, we studied membrane
patches that contained only a single active channel. Examination of the
traces in Fig. 2A suggests four things. First, PP
did not increase Cl
current by changing the
amplitude of current flowing through a single channel. In eight
experiments, current amplitude was 0.90 ± 0.05 pA before and
0.91 ± 0.02 pA after addition of 5 mM PP
(p = 0.812). Second, it is apparent that the
increase in total current is due to an increase in the probability that
single channels are in the open state (P
). Third, it
appears that the increased P
is at least in part caused by
an increase in the duration of bursts of activity. (Note that a burst
is defined as the time in which the channel is open with only brief
flickers to the closed state. A closure of greater than 20 ms separates
bursts). Fourth, PP
appeared to decrease the duration of
the long closed states between bursts of activity.
Figure 2:
Effect of PP on the single
channel characteristics of wild-type CFTR. A, traces from an
excised inside-out membrane patch from a C127 cell containing a single,
active channel. Dotted lines show closed state, and downward deflections correspond to openings. ATP and PP
concentrations (in mM) are indicated; cAMP-dependent
protein kinase concentration is 75 nM. B and C, effect of 5 mM PP
on channel open
probability (P
) and burst duration. Asterisks indicate p < 0.001; each of eight excised patches was
studied an average of 3.0 ± 0.5 and 2.7 ± 0.8 min for
control and PP
, respectively.
Some of the
changes that are apparent from visual inspection of tracings of single
channels are quantitated in Fig. 2, B and C.
PP increased P
from 0.39 ± 0.02 to 0.81
± 0.03 (n = 8, p < 0.001) and
increased mean burst duration from 175 ± 6 ms to 1568 ±
219 ms (n = 8, p < 0.001). We considered
that PP
might increase P
through a ``foot
in the door'' blocking mechanism, in which blockers can actually
increase channel opening duration (and sometimes opening rate) due to
increased residence times within the pore(32, 33) .
However, such a mechanism cannot explain the kinetic effects of
PP
because it did not decrease macroscopic or single
channel current, whereas a decrease would have been expected if
PP
were binding to a site within the pore of CFTR.
Fig. 3shows
that PP produced a concentration-dependent increase in
8-N
ATP photolabeling of CFTR, suggesting that PP
alters the interaction of nucleotide with the protein. If
PP
and ATP both interact (in fact compete) at an NBD, why
would PP
increase photolabeling by 8-N
ATP? At
first inspection, the opposite effect would be predicted. However, the
observation can be explained if PP
binds to one active site
(one NBD) and thereby affects the properties of another active site in
the molecule (the other NBD). Note that 8-N
ATP
photolabeling does not measure equilibrium binding because the
photolabeling reaction was performed over a 60-s period and is
irreversible. Increased labeling of CFTR by PP
could be due
to an increase in the rate of nucleotide binding to an NBD, a decrease
in the rate of nucleotide release from an NBD, or exposure of more
ATP-binding sites. With which NBD does PP
interact? Neither
our present labeling data nor our previous study of 8-N
ATP
photolabeling of CFTR variants allow us to answer this question. We
previously showed that mutations predicted to disrupt hydrolysis at the
NBDs did not alter 8-N
ATP photolabeling(8) .
However, the data suggested that those mutations may have disrupted
interactions between the two NBDs.
Figure 3:
Effect of PP on
8-N
ATP photolabeling of membrane-associated CFTR. Membranes
of Sf9 cells infected with CFTR baculovirus were photolabeled with
[
P]8-N
ATP in the presence of
the indicated amount of PP
. Incorporation is expressed as
the percentage of photolabeling observed without added PP
.
Each value is average ± S.E. of 10-14 samples from four
separate experiments. The asterisk indicates p <
0.05.
Figure 4:
Effect
of nonhydrolyzable PP analogs on CFTR single channel
characteristics. A, traces are from two separate excised
membrane patch experiments from C127 cells that contained only a single
active channel. To the right of the traces are the chemical structures
of the PP
analogs. Traces shown were chosen to illustrate
the effects of PCP, PNP, and etidronate and may not be representative
of average P
or burst duration. B and C,
effect of PCP (n = 6 and 5), PNP (n = 7
and 6), and etidronate (n = 5 and 3) on P
and burst duration, respectively. The asterisks indicate p < 0.05; P
and burst duration were calculated
from greater than 3-min recording in each
experiment.
The effects of these PP analogs on P
and
average burst duration are shown in Fig. 4, B and C. Although all three nonhydrolyzable analogs altered channel
activity, the effects of PCP did not achieve statistical significance,
suggesting that small differences between compounds, such as the
electronegativity and/or the angle of the bridging group, can produce a
large difference in the ability to alter channel gating. This is
similar to the finding that AMP-PNP, a nonhydrolyzable ATP analog with
a structure very similar to ATP(38) , competes for
photolabeling at one-twentieth the potency of ATP(12) .
Although these data do not rule out the possibility that hydrolysis of
PP
may occur, they suggest that binding of PP
is sufficient for stimulation of the channel activity and
prolongation of burst duration.
Fig. 5(B-E) describes the average values of
the rate constants in the presence and the absence of 5 mM
PP. PP
produced large changes in both
and
and a smaller decrease in
and did not alter
. These results
suggest that PP
affects more than one step in channel
gating. The increase in burst duration shown in Fig. 2C is caused principally by a 6-fold decrease in
.
is a major determinant of burst duration because it
defines the rate at which the channel leaves the bursting mode (i.e. leaves C
, the closed state within a burst).
The duration of bursts can also be affected by
and
, the transition rates within a burst, but PP
did not alter
and decreased
by 37%. However, because
is one-tenth the
magnitude of
, the decrease in
produced an overall change in P
within a burst of
less than 5% (n = 4, not significantly different). Thus
the decrease in
had a minimal effect on net channel
activity. In addition to increasing burst duration, PP
also
increased
, the rate of transition from the long
closed state (C
) to the bursting state (C
O), suggesting that PP
facilitated opening by
ATP. The decrease in the duration of long closed times between bursts
can also be appreciated by examining the traces shown in Fig. 2and Fig. 4.
How
does PP increase the rate of channel opening (increase
)? There are four pertinent considerations. First,
PP
binding alone cannot open the channel. The stimulatory
effect of PP
required the presence of ATP, consistent with
previous data suggesting that ATP binding and hydrolysis are required
to open the channel(5, 8, 13, 15) .
Second, hydrolysis of PP
was not required for stimulation.
Third, PP
stimulated the only transition that is regulated
by ATP concentration (i.e.
). Fourth,
PP
increased photolabeling with 8-N
ATP. We
conclude that PP
must bind to a site in CFTR other than the
site at which ATP directly opens the channel. When PP
binds
it facilitates binding (and probably hydrolysis) of ATP at a separate
site, increasing the rate at which the channel opens. Given the
precedent for PP
interaction at the NBDs of other proteins,
the presence of two NBDs in CFTR, and the previous suggestion that
PP
competes with ATP(14) , we propose that PP
regulates channel activity by interacting with an NBD.
How
does PP prolong burst duration (decrease
)? Our previous work indicated that mutations expected
to reduce the rate of hydrolysis at NBD2 increased the duration of
bursts(8) . Nonhydrolyzable analogs of ATP also prolonged the
duration of bursts(8, 13, 14) . These data
suggest that the rate of hydrolysis of bound nucleotide at NBD2 is the
primary determinant of burst duration. While ATP is bound, the burst
continues; upon hydrolysis, the burst is terminated. The most
straightforward interpretation of the data is that PP
prolongs the duration of bursts by binding to NBD2. Because
PP
is not hydrolyzed, the open state is not terminated by
hydrolysis and the burst duration is prolonged, and this is reflected
in the model as a decrease in
.
Obviously, the linear three state model shown in Fig. 5A is a minimal model, and each state or transition in the model may represent more than one physical event or conformational change. We previously proposed a more complex mechanistic model of how events at NBD1 and NBD2 give rise to the opening and closing of phosphorylated channels (see Fig. 8 and (8) ) and proposed that there are interactions between the two NBDs such that binding of ATP to NBD2 may regulate events at NBD1, including a step leading to channel opening.
In
keeping with that model, we speculate that PP interacts
primarily with NBD2, and in so doing it alters two functions of NBD2.
First, binding of PP
to NBD2 could mimic the effect of ATP
binding. It would effect NBD1, enhancing binding and/or hydrolysis of
ATP. Hydrolysis of ATP at NBD1 may then open the channel to the
bursting state. Second, because PP
is not hydrolyzed, it
prolongs the duration of the bursting state preventing channel closure.
Although this seems to be the most straightforward explanation and is
consistent with previous observations, we acknowledge the possibility
that PP
may interact at a site other than NBD2 or that
PP
interaction at more than one site may produce the
different effects observed. Consistent with the effects reported here,
we previously speculated that AMP-PNP, like PP
, interacted
with NBD2 to increase burst duration(8) . However, AMP-PNP was
much less potent (see Fig. 7and Ref 8). Based on our data with
PP
, we predict that AMP-PNP would also increase channel
opening (i.e. increase
), but because of its
low potency it was not possible to test this prediction.
Figure 7:
Effect of PP on apical
membrane Cl
currents in permeabilized T-84 epithelia. A, apical membrane Cl
current was recorded
after basolateral membrane was permeabilized with S. aureus
-toxin (100 µg/ml). Basolateral solutions contained 1
mM ATP. The bars indicate the presence of cAMP (10
µM) and PP
(in mM). The breaks in the traces omit recordings during change in basolateral
solutions because electrical contact was disrupted. B, effect
of PP
on apical membrane Cl
current in
the presence (solid bars; n = 10) or the
absence (hatched bars; n = 4) of cAMP (10
µM). The asterisk indicates p <
0.05.
Why would
PP show preferential binding to NBD2? Perhaps the binding
affinity or steric constraints of NBD2 are more favorable for PP
binding. The two NBDs do have significantly different amino acid
sequences, and there is substantial precedent for nucleotide-binding
sites that show preference between different nucleotide analogs and
PP
(17) .
We examined the
effect of PP on CFTR containing the CF-associated mutations
F508, R117H, and G551S. We studied these mutations because they
occur in different regions of the protein and have different mechanisms
of dysfunction(39) . CFTR-
F508, the most common CF-causing
mutation(40, 41) , is defectively processed and fails
to traffic to the plasma membrane. In addition to the processing
defect, the function of CFTR-
F508 is decreased as indicated by a
reduced P
(25, 42) . G551S, a mutation in
NBD1, is correctly processed but has altered ATP-dependent channel
regulation resulting in a reduced P
(6) . R117H,
which contains a mutation in the membrane-spanning domain, is also
correctly processed but has altered ion-conducting properties producing
an overall decrease in function(43) .
Fig. 6shows
current tracings from a membrane patch containing two active
CFTR-F508 channels. In the presence of 75 nM cAMP-dependent protein kinase and 0.3 mM ATP, channel
openings appear qualitatively similar to those of wild-type CFTR,
except that both P
and burst duration are less than those
of wild type. The addition of 5 mM PP
stimulated
F508 activity 3-fold (n = 5, Fig. 6, A and D). P
increased 3-fold (from 0.07
± 0.01 to 0.21 ± 0.022), and average burst duration
increased 8-fold (from 125 ± 19 ms to 1023 ± 330 ms, n = 3). A similar stimulation of channel activity was
observed when 1 mM PP
was added to CFTR-R117H and
CFTR-G551S (Fig. 6, B, C, and D).
This amount of stimulation is similar to that observed with wild-type
CFTR (see Fig. 1B).
Figure 6:
Effect
of PP on CFTR Cl
channels containing
CF-associated mutations. A, traces from an excised membrane
patch from a C127 cell expressing two
F508 CFTR Cl
channels. The cell had been incubated at 25 °C to increase
protein levels at the plasma membrane(25) . Traces are plotted
as described in the legend to Fig. 2. B and C,
time course experiments from membrane patches containing multiple
channels from transiently transfected HeLa cells expressing R117H (B) or G551S (C) CFTR. Note that PP
concentrations for these experiments are 1 mM. Data are
plotted as described in the legend to Fig. 1A. D, effect of PP
on
F508 CFTR (5 mM
PP
) (n = 5), R117H (1 mM
PP
) (n = 9), and G551S (1 mM PP
) (n = 6). Data are expressed as the
percentages of current relative to current in presence of 0.3 mM ATP and 75 nM cAMP-dependent protein kinase. The asterisks indicate p < 0.05 compared with value in
the absence of PP
.
Fig. 7A shows examples of the results. After permeabilization, the
baseline Cl current was small, suggesting that in the
absence of cAMP, the apical membrane was relatively impermeable to
Cl
. As we previously reported, addition of cAMP to
the basolateral bathing solution stimulated Cl
current(30) . Subsequent addition of either 1 or 5 mM PP
to the basolateral bathing solution produced a
further increase in current. Fig. 7B (solid
bars) shows the average values of current 5 min after the addition
of the bathing solution with or without PP
.
Although
PP can function as a nonspecific phosphatase inhibitor, and
CFTR channel activity is regulated in part by
phosphorylation(46) , we think it unlikely that PP
stimulated the apical membrane Cl
current by
acting as a nonspecific phosphatase inhibitor because upon removal of
cAMP in the continued presence of PP
, current decreased to
baseline levels within 3 min. This rate is similar to that observed in
the absence of PP
(Fig. 7A). In other
experiments, the addition of 1 or 5 mM PP
to
permeabilized monolayers in the absence of cAMP did not increase
current above baseline levels (Fig. 7B, hatched
bars; n = 4). These results suggest that PP
only stimulates apical Cl
current in the
presence of cAMP, suggesting that PP
has a direct effect on
active channels and not an indirect effect via alteration of the
phosphorylation state of CFTR. The conclusion that PP
is
not acting primarily as a phosphatase inhibitor is consistent with the
results shown in Fig. 1and is also supported by the finding
that PP
stimulated CFTR
R-S660A in excised membrane
patch experiments (n = 6, p = 0.004,
data not shown). CFTR-S660A is a mutant in which most of the R domain
has been deleted and as a result it does not require and is not
stimulated by cAMP-dependent protein kinase(47) .
Could
PP or a related compound be of value to treat CF? That
possibility is suggested by the ability of PP
to stimulate
CFTR containing several different CF-associated mutations and the
ability of PP
to potentiate the activity of endogenous CFTR
located in the apical membrane of an epithelium. The compounds studied,
however, are hydrophilic and are not expected to partition through the
lipid membrane to gain access to the cytoplasmically located NBDs when
applied extracellularly. Thus delivery of PP
or a stable
analog may depend upon modification of the compound to allow
partitioning through the lipid membrane or use of a delivery vehicle
such as liposomes. Certainly, intracellular pyrophosphates or analogs
that lack specificity for CFTR might have many undesirable
consequences. However, a lipophilic bisphosphonate has been
administered systemically to alter cholesterol
biosynthesis(48) .
In addition, such a strategy must take
into account the specific type of CF mutation, because different
mutations have different molecular mechanisms of
dysfunction(39) . Poorly functional channels appropriately
localized to the plasma membrane, such as R117H and G551S, might be
most amenable to regulation by a PP analog. Conversely,
because little
F508 mutant is present at the apical membrane in
cells grown at 37 °C, an approach based on simulating channel
activity might also require a second strategy to increase the amount of
F508 CFTR at the plasma membrane. In summary, although the idea of
using PP
and PP
-like compounds to stimulate
defective CFTR remains preliminary, these findings suggest that further
investigation of PP
and PP
analogs may be
useful not only to understand the regulation of CFTR but also in the
evaluation of future therapeutic applications.