(Received for publication, July 24, 1995; and in revised form, September 29, 1995)
From the
We have previously described a protocol for the simultaneous
isolation and reconstitution of a protein kinase A (PKA)-sensitive
outwardly rectified chloride channel (ORCC) and the cystic fibrosis
transmembrane conductance regulator (CFTR) from bovine tracheal
epithelium. Immunoprecipitation of CFTR from this preparation prevented
PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and
that this regulatory relationship was preserved throughout the
purification procedure. We now report the purification of CFTR from
bovine tracheal epithelia and the purification of a CFTR conduction
mutant (G551D CFTR) from retrovirally transduced mouse L cells using a
combination of alkali stripping, Triton-X extraction, and
immunoaffinity chromatography. Immunopurified CFTR proteins were
reconstituted in the absence and presence of ORCC. To test the
hypothesis that only functional CFTR can support activation of ORCC by
PKA and ATP, we used an inhibitory anti-CFTR peptide antibody or G551D CFTR. When anti-CFTR
peptide antibodies were present prior to the addition of PKA and
ATP, activation of both the ORCC and CFTR was prevented. If the
antibody was added after activation of the ORCC and CFTR Cl
channels by PKA and ATP, only the CFTR Cl
channel was inhibited. When ORCC and G551D CFTR were
co-incorporated into planar bilayers, only the ORCC was recorded and
this channel could not be further activated by the addition of PKA and
ATP. Thus, functional CFTR is required for activation of the ORCC by
PKA and ATP. We also tested the hypothesis that PKA activation of ORCC
was dependent on the extracellular presence of ATP. We added ATP on the
presumed extracellular side of the lipid bilayer under conditions where
it was not possible to activate the ORCC, i.e. in the presence
of inhibitory anti-CFTR
antibody or G551D CFTR.
In both cases the ORCC regained PKA sensitivity. Moreover, the addition
of hexokinase + glucose to the extracellular side prevented
activation of the ORCCs by PKA and ATP in the presence of CFTR. These
experiments confirm that both the presence of CFTR as well as the
presence of ATP on the extracellular side is required for activation of
the ORCC by PKA and ATP.
The cystic fibrosis transmembrane conductance regulator (CFTR) ()and outwardly rectified chloride channels (ORCC) both
contribute to cAMP-activated chloride conductance in normal, but not in
cystic fibrosis (CF), airway cells (1, 2) . ORCCs are
present in the apical membranes of CF airway epithelia, but they cannot
be activated by protein kinase A (PKA) and
ATP(3, 4, 5) . CFTR channels are regulated by
PKA and ATP and are inhibited by the channel blocker
diphenylamine-2-carboxylate (DPC) but not by
4,4`-diisothiocyanostilbene-2,2`-disulfonic acid
(DIDS)(6, 7) , whereas ORCCs are DIDS-sensitive.
Although CFTR and ORCC channels are distinct proteins, they are linked
through unknown regulatory pathways. Different mechanisms by which CFTR
might regulate the ORCC can be envisioned. For example, CFTR could
interact directly with the ORCC protein in the apical membrane,
regulation could be a consequence of the channel function of CFTR, or
both. It has been shown that CFTR can act as both an ATP and a
Cl
channel(6) . Recently Guggino's
group (7) demonstrated that ATP transported through CFTR acts
as an autocrine stimulator of the ORCC. The proposed mechanism of
regulation is via a P
receptor that, either through a
direct coupling to the ORCC or through a G protein-coupled signaling
pathway, stimulates the ORCC.
Increasing evidence suggests
heterogeneity in the molecular pathogenesis of CF. Some mutations, such
as the deletion of a phenylalanine at position 508 (Phe-508) of
CFTR, cause CFTR to be processed improperly within epithelial cells,
and disease may result because the mutant protein fails to traffic to
the apical cell membrane. Other mutations, such as the relatively
common glycine
aspartic acid replacement at CFTR position 551
(G551D, 3% of all mutations), appear to be processed and targeted
normally to the plasma membrane, but lack responsiveness to stimulation
by cAMP(8) . It is not known how different mutational forms of
CFTR affect regulation of the ORCC by PKA.
Amino acid sequence
analysis suggests that CFTR is composed of two motifs, each containing
a membrane-spanning domain (MSD) and a nucleotide-binding domain (NBD)
linked by a regulatory R domain(9) . Recently, Welsh and
colleagues (10) demonstrated that the amino-terminal portion of
CFTR, which includes MSD1, NBD1, and the R domain, forms a regulated
Cl channel. It has been shown previously that an even
smaller part of CFTR, namely NBD1 (amino acids 426-588), can form
an anion channel when reconstituted in planar lipid
bilayers(11) . It is not known if these portions of the CFTR
molecule can also transport ATP or if they are sufficient to replace
intact CFTR in the regulation of the ORCC.
We recently reported the simultaneous isolation and functional reconstitution of an ORCC and CFTR from bovine tracheal epithelia(12) . The regulatory relationship between these channel proteins was preserved throughout the purification procedure, as demonstrated by the activation of the ORCC by PKA and ATP. Immunoprecipitation of CFTR from this preparation prevented the activation of the ORCC by PKA and ATP, suggesting that the presence of CFTR is required for PKA-dependent activation of the ORCC. However, these experiments did not distinguish if functional CFTR was essential for conferring PKA sensitivity to the ORCC, if only the physical interaction of CFTR was required, or both.
We thus tested
the hypothesis that the functional form of CFTR is required for PKA
activation of the ORCC. In order to achieve this goal, we used an
inhibitory anti-CFTR peptide antibody (13, 14, 15) to block CFTR transport function
or replaced functional CFTR with a nonfunctional mutant form of CFTR,
namely G551D CFTR(8) . Our results indicate that only when CFTR
is functional can the ORCC be activated by PKA and ATP. Our results
also are consistent with the hypothesis of Schwiebert et al. (7) that the transport of ATP by CFTR to the extracellular side
of the cells is necessary for PKA activation of the ORCC.
Planar lipid bilayers, composed of a
mixture of diphytanoyl-phosphatidylethanolamine,
diphytanoyl-phosphatidylserine, and oxidized cholesterol in a 2:1:2
(w/w/w) ratio (final concentration = 25 mg/ml in n-octane), were painted with a fire-polished glass capillary
over a 200-µm hole drilled in a polystyrene chamber, as described
previously(22) . Bilayer formation was monitored by the
increase in membrane capacitance to a final value of 300-400
picofarads. Liposomes containing anion channel proteins were
incorporated into bilayers bathed with symmetrical solutions of 100
mM KCl and 10 mM MOPS adjusted to pH 7.4. Channel
incorporation was indicated by the appearance of quantal step increases
in current with applied voltage. Current measurements were performed
with a high gain amplifier circuit based on a design described
previously(23) . Single channel records were analyzed with
pCLAMP, version 5.5. Steady-state single channel current-voltage (I/V) curves were measured after channel
incorporation by applying a known voltage and measuring individual
channel current (i). Single channel open probability (P) was calculated from P
= I /ni, where i is the unitary current, I is the mean current, and n is the total number of
active channels. Both n and i were estimated from an
all points current amplitude histograms produced by pCLAMP software.
The dashed line in the figures represents the zero current
level (or close state). All bilayer experiments were repeated a minimum
of four times under a specified set of experimental conditions.
Figure 1:
Autoradiograph of in vitro phosphorylated, immunopurified bovine CFTR and G551D CFTR. The
phosphorylation reaction mixture consisted of the protein sample
dissolved in 40 µl of buffer (50 mM Tris, pH 7.5, and 10
mM MgCl), 300 ng of the catalytic subunit of
protein kinase A, and 2 nmol of [
-
P]ATP.
The reaction was carried out at 30 °C for 60 min. A,
bovine CFTR. Lane 1, total in vitro phosphorylated
proteins from solubilized tracheal apical membrane vesicles. Lane
2, phosphoprotein eluted from immune Acti-Disk (polyclonal
anti-CFTR
antibody). A 170-kDa phosphoprotein is
present. Lane 3, phosphoproteins eluted from a non-immune IgG
bound Acti-Disk. B, G551D CFTR from mouse L cells. Lane
1, total in vitro phosphorylated proteins from L cell
lysate. Lane 2, phosphoprotein eluted from immune IgG-bound
Acti-Disk (polyclonal anti-CFTR
antibody). A 170-kDa
phosphoprotein is present. Lane 3, phosphoproteins eluted from
non-immune IgG-bound Acti-Disk.
Figure 2:
Single channel activity of immunopurified
bovine CFTR reconstituted into planar lipid bilayer. Control single
channel recordings were performed at holding potentials of +80 mV
in the presence of 100 µM ATP and the catalytic subunit of
PKA (1.85 ng/ml). Anti-CFTR antibodies 200 µg/ml
were added to both sides of the bilayer, followed by addition of 100
µM DIDS to both compartments of the bilayer chamber.
Addition of 100 µM DIDS in a separate experiment (n = 13) to either side (or both) of the channel-containing
bilayer prior to addition of anti-CFTR
antibodies had no
effect on channel activity. In the experiment shown (representative of
18 out of a total 33), 300 µM DPC completely blocked
channel activity when added to one side of the bilayer (usually trans). In the other 15 experiments, DPC was ineffective when
added to this one side of the bilayer. Thus, addition of 300 µM DPC was made to the opposite side; this resulted in abolished
channel activity. In the experiment shown, anti-CFTR
antibodies (50 ng/ml) were added to one side of the bilayer
(usually cis). Likewise in the DPC inhibition experiments, if
channel activity remained unaffected (11 experiments from total 28),
antibodies were added to the opposite side with subsequent
inhibition.
Figure 5: Single channel current-voltage relationships of immunopurified and reconstituted ORCC in the presence or absence of wild type CFTR or G551D CFTR before and after activation by PKA and ATP in planar lipid bilayer. Conditions are defined in the symbol legends on the graph. Symbols indicate mean values, and error bars indicate ± S.D. for at least four separate experiments for each condition.
We found that anti-CFTR antibodies
had no effect on base-line ORCC activity, confirming their specificity
toward CFTR. If inhibitory anti-CFTR
antibodies
were added to the presumptive cytoplasmic bathing solutions after PKA
and ATP activation of the incorporated channels, only CFTR
Cl
channel activity was inhibited, while ORCC
remained activated with a P
0.78 ± 0.06 (n = 21; Fig. 3A). Amplitude histograms
of channel recordings made in the presence of PKA and ATP (Fig. 3B) showed that at least three CFTR
Cl
channels accompanied one ORCC channel (only one
ORCC was active in the bilayer after inhibition of CFTR using
anti-CFTR
antibodies). However, it would be
premature to draw any conclusions about the exact molar ratio of ORCC
and CFTR necessary for interaction from these bilayer experiments,
because in other comparable experiments two to five CFTR channels per
single ORCC have been observed. In contrast to these results obtained
with anti-CFTR
antibodies added after PKA
+ ATP activation of incorporated channels, addition of inhibitory
anti-CFTR
antibodies prior to addition of PKA
and ATP prevented activation of both CFTR and ORCC by PKA + ATP (n = 11; Fig. 4, A and B).
Figure 3:
Effect of anti-CFTR antibody on PKA- and ATP-activated ORCC and CFTR in planar lipid
bilayer. A, traces shown are representative of 21 experiments.
Holding potential and doses of PKA and ATP and
anti-CFTR
antibodies were as described for Fig. 2. Control refers to channel activity in symmetrical
bathing solutions containing 100 mM KCl, 10 mM MOPS,
pH 7.5. Additions were made sequentially as shown in the figure. B, all point amplitude histograms of traces shown in A. Histograms were generated by pCLAMP software from record of
5 min in length.
Figure 4:
Effect of anti-CFTR antibody on activation of ORCC and CFTR by PKA and ATP in planar
lipid bilayer. A, traces shown are representative of 11
experiments. Holding potential, doses of PKA + ATP, and
anti-CFTR
antibodies were as described for Fig. 2. Control refers to channel activity in symmetrical
bathing solutions containing 100 mM KCl, 10 mM MOPS,
pH 7.5. Additions were made sequentially as shown in the figure. B, all point amplitude histogram of traces shown in A. Histograms were generated by pCLAMP software from
recordings of 5 min in length.
Anti-CFTR antibodies did not affect the
rectification properties of ORCC, although by using these antibodies to
block CFTR channel activity we were able to determine more precisely
the I/V curves of phosphorylated ORCC. Notably,
rectification of ORCC upon PKA-induced phosphorylation did not change.
The slope conductance of ORCC at positive voltages was not altered
under any conditions. These results support the hypothesis that the
active form of CFTR is required for activation of ORCC by PKA and ATP.
The aforementioned results suggested that ATP secretion through CFTR may be required for the PKA-induced activation of the ORCC. Addition of ATP by itself from the presumptive extracellular side did not have any effect on the channel activity of the incorporated ORCC + CFTR. If transport of ATP through CFTR was the only requirement for PKA-mediated activation of the ORCC, it should be possible to activate ORCC by the addition of ATP to the extracellular solution and PKA + ATP to the cytoplasmic side of the system. However, when ORCC alone was incorporated into a bilayer, this channel could not be activated by addition of 100 µM ATP to the ``outside'' and PKA + ATP to the ``inside'' or by PKA + ATP to both bathing solutions (see Fig. 7of (12) ). These results suggest that transport of ATP through CFTR is not the only role that CFTR plays in the regulation of the ORCC.
Figure 7:
Channel activity of ORCC and G551D CFTR in
planar lipid bilayers and the effects of extracellular ATP and
intracellular PKA + ATP. Traces shown are representative of 11
experiments. Holding potential, doses of PKA + ATP, and
anti-CFTR antibodies were as described for Fig. 2. Control refers to channel activity in symmetrical
bathing solutions containing 100 mM KCl, 10 mM MOPS,
pH 7.5. Additions were made sequentially as shown in the
figure.
We therefore examined if the presence of ATP on the presumptive extracellular side of a bilayer containing ORCC and nonfunctional CFTR (i.e. either blocked with the inhibitory antibody or nonfunctional G551D CFTR) could restore PKA sensitivity to the ORCC. Under both conditions, namely, in the presence of inhibitory antibodies (Fig. 6) or G551D CFTR (Fig. 7), the ORCC regained PKA sensitivity when 100 µM ATP was added to the extracellular bathing medium. These findings also confirmed the hypothesis that transport of ATP is not the only role that CFTR plays in the regulation of ORCC. These results also indicate that G551D CFTR, like wild-type CFTR, incorporated into the membrane with a specific orientation in the presence of ORCC.
Figure 6:
Channel activity of ORCC and CFTR in
planar lipid bilayers in the presence of anti-CFTR antibody and the effects of extracellular ATP and intracellular
PKA + ATP. Traces shown are representative of 14 experiments.
Holding potential, doses of PKA + ATP, and
anti-CFTR
antibodies were as described for Fig. 2. Control refers to channel activity in symmetrical
bathing solutions containing 100 mM KCl, 10 mM MOPS,
pH (7.5). Additions were made sequentially as shown in the
figure.
We also tested whether a portion of CFTR, namely the NBD1 domain, could interact with the ORCC in the presence of extracellular ATP and be sufficient for PKA-mediated activation of ORCC. However, in four separate experiments, we found that NBD1 could not substitute for full-sized CFTR in the regulation of ORCC by PKA and ATP added from either or both sides of the membrane.
Our results demonstrate that we have immunopurified and functionally reconstituted into planar lipid bilayers bovine CFTR and nonconductive G551D CFTR from transduced L cells. A similar immunopurification procedure for the isolation and reconstitution of recombinant, and functionally competent, CFTR from Chinese hamster ovary and Sf9 cells was reported recently(24) . Our reconstituted proteins were used with purified and functionally reconstituted ORCC (as described previously) for further delineation of the mechanism of interaction between CFTR and the ORCC.
Immunopurified and reconstituted bovine CFTR Cl
channel had the same ion selectivity, inhibitor specificity, and
inhibition by anti-CFTR
antibodies as reported
previously in patch clamp studies or for reconstituted recombinant
CFTR(7, 13, 25) . The main difference between
the previously reported characteristics of the CFTR Cl
channel and the bovine CFTR Cl
channel was the
single channel conductance. In our experiments the conductance of
bovine CFTR was 16 picosiemens, somewhat larger than that previously
reported (8-13 picosiemens). The conductance of the CFTR channel
in our hands was the same regardless of the antibody that was used for
purification or the presence or absence of the ORCC in the
reconstituted material. The reason for this discrepancy is not
apparent.
Our study demonstrated that only when the transport function of CFTR was intact could the activation of the ORCC by PKA and ATP occur. Our results also support Guggino's (7) hypothesis that one function of CFTR in the activation of the ORCC is to transport ATP to the extracellular side of the cell membrane. This hypothesis is consistent with the findings of Reisin et al.(6) who have demonstrated directly that ATP can traverse the CFTR channel. We cannot at present exclude the co-purification of a purinergic receptor by our procedures. However, it is unlikely that a receptor-coupled mechanism would be active in the bilayer system because of the absence of necessary signaling molecules. However, the failure of ORCC to be activated by PKA + ATP in the presence of external ATP supports the idea that a purinergic receptor mediated pathway does not co-purify in our preparation. The second finding revealed by our study is that a nonconductive form of CFTR (G551D) was able to support activation of the ORCC by PKA when ATP was present on the extracellular side of the incorporated channels.
It
has been shown previously that the portion of CFTR that includes MSD1,
NBD1, and the R domain, or even smaller parts of CFTR (NBD1 amino acids
426-588), can form Cl channels(10, 11) . We tested whether NBD1 could
substitute for CFTR in the regulation of the ORCC by PKA by ATP. Under
the conditions used in these studies, NBD1 did not form a channel in
the bilayer and therefore was not able to substitute for CFTR.
Furthermore, a recombinant NBD1 purified from prokaryotic cells was
unable to support activation by PKA of the ORCC in the presence of
exogenously added extracellular ATP. Taken together these results
suggest that there is a minimal size requirement of CFTR necessary to
subserve its regulatory role in the PKA-related activation of the ORCC.
In summary, we have immunopurified and functionally reconstituted
CFTR from bovine trachea and nonconductive G551D CFTR from transduced L
cells. In combination with previously co-purified ORCC and CFTR from
bovine tracheal epithelia, and inhibitory anti-CFTR antibodies, we were able to explore the regulatory relationship
between the ORCC and CFTR. We have shown that functional CFTR is
required for activation of the ORCC by PKA and ATP. CFTR is apparently
required to transport ATP from the cytoplasm to the extracellular
bathing solution, where it can then interact with some external domain
of the ORCC. The observation that the ORCC cannot be activated by
extracellular ATP and PKA + ATP added to the cytoplasmic side in
the absence of the CFTR molecule also suggests that, in addition to the
transport (ATP) function of CFTR, a direct interaction between CFTR and
ORCC is essential to confer PKA sensitivity to the ORCC.