(Received for publication, August 1, 1994; and in revised form, November 4, 1994)
From the
Our laboratory has developed a protocol for the isolation of a
140-kDa protein that forms an anion-selective channel when
reconstituted into planar lipid bilayers. Polyclonal antibodies have
been raised against the 38-kDa component of this purified protein. This
channel has a linear current-voltage relationship and is not activated
by protein kinase A (PKA) plus ATP. Using the same antibody and a
modified purification protocol (eliminating the ion exchange
chromatography steps), we isolated and reconstituted two other anion
channels from tracheal membrane vesicles. In vitro phosphorylation of these isolated proteins by PKA and ATP revealed
four bands migrating at 52, 85, 120, and 174 kDa. Immunoprecipitation
experiments with anti-CFTR antibodies indicate that the 174-kDa
phosphoprotein was CFTR. Upon incorporation of these isolated proteins
into planar bilayers, an anion channel that exhibited a marked outward
rectification in symmetrical Cl solutions with a
slope conductance of 82 pS at depolarizing voltages was observed. PKA
and ATP increased channel activity but only from one side of the
bilayer. However, channel activity was unaffected by addition of ATP
alone from either side of the membrane. DIDS (100 µM)
applied to the opposite side of the bilayer to which PKA and ATP act,
blocked channel activity. A linear anion-selective channel with a
conductance of 16 pS could be also resolved after inhibition of the
outwardly rectified anion channel by DIDS in the presence of PKA and
ATP. This small conductance channel was inhibited by 300 µM diphenylamine-2-carboxylic acid. Immunodepletion of the 174-kDa
phosphoprotein from the preparation prevented activation of the 82-pS
outwardly rectified anion channel by PKA and ATP. However, the
PKA-dependent in vitro phosphorylation of the 52-, 85-, and
120-kDa phosphoproteins was unaffected by the absence of CFTR. Our
results suggest a direct regulatory relationship between an outwardly
rectified anion channel and CFTR.
Maintenance of a fluid layer on the apical surface of alveolar
and tracheal epithelial cells primarily involves active transepithelial
Cl secretion. The overall rate of transepithelial
Cl
movement is determined by the activity of
Cl
channels located in the apical membrane of airway
epithelial cells. These channels are regulated by intracellular second
messengers such as cAMP and Ca
as well as voltage and
cell volume(1, 2) . Cytoplasmic addition of either
protein kinase A (PKA) (
)or protein kinase C plus ATP opens
secretory Cl
channels in patches excised from airway
epithelial cells(3, 4, 5) . Malfunction of
secretory regulation occurs in cystic fibrosis (CF) such that PKA does
not open anion channels from diseased cells, even though depolarization
of the patch demonstrates that functionally competent channels are
present(5, 6, 7) .
Two recent single
channel patch-clamp studies suggest that both outwardly rectified
chloride channels (ORCCs) and CFTR Cl channels
contribute to cAMP-activated Cl
current in normal
airway epithelial cells(8, 9) . Furthermore, defective
regulation of outwardly rectifying Cl
channels by
protein kinase A was corrected by insertion of normal copies of CFTR
cDNA(8) .
The biophysical properties of ORCC and CFTR
Cl channels are very distinct and well established.
ORCCs have a nonlinear current-voltage (I/V) relationship with a
20-40-pS single channel conductance at hyperpolarizing voltages
and a 60-80-pS conductance at depolarizing
voltages(1, 2, 3, 5, 10, 11) .
ORCCs are blocked by a wide variety of molecules including DIDS and the
calixarenes (12) and have a halide permeability sequence of
I
> Cl
>
Br
. PKA and protein kinase C can activate these ORCCs (13, 14, 15, 16) . Conversely, the
CFTR Cl
channel has a linear I/V relationship with an
8-16-pS single channel conductance. Channel activity can be
blocked by diphenylamine-2-carboxylic acid (DPC), but not by DIDS. The
halide permeability sequence for CFTR is Br
>
Cl
>
I
(27, 28) .
Despite the fairly
detailed information concerning the biophysical characteristics of
these Cl channels, relatively few biochemical
investigations of individually isolated Cl
channels
have been undertaken. The protein that forms the ORCC is unknown, as
are the mechanisms by which CFTR regulates this protein. In this paper
we report the immunopurification from bovine tracheal epithelia and
functional reconstitution in planar lipid bilayers of an outwardly
rectified anion channel (ORAC) and the CFTR Cl
channel and demonstrate a direct regulatory relationship between
these channels.
Planar
lipid bilayers, composed of a mixture of 25 mg/ml
phosphatidylethanolamine, phosphatidylserine, and oxidized cholesterol
in a 2:1:2 (w/w/w) ratio, were painted with a fire-polished glass
capillary over a 200-µm hole drilled in a polystyrene chamber, as
described previously(24) . Bilayer formation was monitored by
the increase in membrane capacitance to a final value of 200-400
picofarads. Artificial 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. In some
experiments, KCl was substituted with 100 mM choline chloride
or N-methyl-D-glucamine chloride. The observation of
a stepwise increase in current was taken as an indication of channel
incorporation into the lipid bilayer. Current measurements were
performed with a high gain amplifier circuit based on a design
previously described(25) . Steady-state single channel
current-voltage (I/V) curves were measured after channel incorporation
by applying a known voltage and measuring individual channel current.
Single channel open probability (P) was calculated
from P
= I/n
i, where i is the unitary
current, I is the mean current, and n is the total
number of active channels. I, n, and i were
estimated from an all points current amplitude histogram and events
list produced by pCLAMP software (version 5.5; Axon Instruments). The dashed line in the figures represents the zero current level.
Figure 1:
Western blot analysis of purified anion
channel proteins following two different purification procedures.
Purified proteins were separated on a 10% SDS gel under nonreduced
conditions, transferred to nitrocellulose, and probed with either
preimmune IgG or immune (p38) IgG. Lane 1, solubilized
tracheal apical membrane vesicles were subjected to cation exchange
chromatography followed by immunopurification and then probed with
preimmune IgG; lane 2, proteins purified using the same
procedure as for lane 1 except immune
p38 antibodies were
used; lane 3, solubilized tracheal apical membrane vesicles
were subjected to immunopurification without prior cation exchange and
probed with immune IgG (
p38 antibodies); lane 4, proteins
purified using same procedure as for lane 3 except probed with
preimmune IgG.
Figure 2:
Autoradiograph of in vitro phosphorylated purified anion channel proteins from bovine
tracheal epithelia. The phosphorylation reaction mixture consisted of
100-200 ng of purified Cl channel protein, 30
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. Lane 1, all
reaction components except the catalytic subunit of the cAMP-dependent
protein kinase; lane 2, all reaction components except the
immunopurified channel protein; lane 3, all reaction
components. Phosphorylated products are present at 52, 85, 120, and 174
kDa. Lane 4, all reaction components plus 100 µg of
protein kinase inhibitor. Lane 5, all reaction components,
except that immunopurification was performed with nonimmune
IgG.
Figure 3:
Autoradiograph of CFTR immunoprecipitation
and in vitro phosphorylation of immunopurified anion channel
proteins after CFTR precipitation. Purified anion channel proteins
(100-200 ng) were incubated with monoclonal anti-CFTR antibody
(Genzyme) and then precipitated with poly(A)-Sepharose beads.
Precipitated protein was phosphorylated in the presence of 300 ng of
catalytic subunit of PKA and 2 nmol of
[-
P]ATP for 60 min at 30 °C. Proteins
that remained after the CFTR precipitation were phosphorylated in
vitro under conditions identical to those described in the legend
to Fig. 1. Lane 1, protein precipitated with monoclonal
anti-CFTR antibody (SDS-PAGE run under reducing conditions). A 174-kDa
phosphoprotein is present. Lane 2, phosphoprotein remaining
after CFTR immunodepletion. Phosphorylated products are present at 52,
85, and 120 kDa. The 174-kDa phosphoprotein was removed by the
anti-CFTR antibody.
Figure 4: Immunopurified anion channels incorporated into planar lipid bilayers. Immunoaffinity purified anion channel proteins were reconstituted into artificial liposomes and then incorporated into planar lipid bilayers. Bilayers were composed of a mixture of phosphatidylethanolamine, phosphatidylserine, and oxidized cholesterol in a 2:1:2 ratio. The final lipid concentration was 25 mg/ml. The bilayer was bathed with symmetrical solutions of 100 mM KCl and 10 mM MOPS (pH 7.0). The dashed line represents the zero current level. The records were filtered at 100 Hz. A, simultaneous presence of a small (30 pS) and large (80 pS) conductance anion channel in the bilayer. B, small conductance anion channel. C, large conductance anion channel.
Fig. 5shows typical
current traces of channel activity recorded at ±100 mV following
incorporation of these stored proteoliposomes into planar bilayers. The
channels were anion-selective, the ratio of cation to anion
permeability being 0.11 ± 0.03 at a 10-fold KCl gradient (n = 3), and had a single channel conductance of 82 ± 9
pS at +100 mV and 35 ± 4 pS at -100 mV. Current through the
open channel was greater at positive versus negative applied
potentials, but single channel open probability (P) was independent of voltage. Under these
conditions, P
averaged 0.41 ± 0.07. Thus,
this anion channel had kinetic characteristics similar to those were
previously described in patch clamp studies of airway
epithelia(3, 5, 10, 26) , including
outward rectification (see Fig. 8).
Figure 5: Reconstituted immunopurified anion channel proteins: single channel characteristics, effect of phosphorylation by PKA + ATP, and effect of DIDS. Top traces, single channel recordings at holding potentials of ±100 mV (two channels were present in the bilayer). Middle traces, single channel recordings at holding potentials of ±100 mV after addition of ATP (100 µM) and the catalytic subunit of PKA (1.85 ng/ml). Note the appearance of small, well resolved current steps at both positive and negative voltages. Bottom traces, single channel recording at holding potentials of ±100 mV after addition of 100 µM DIDS added to the opposite side of bilayer from which PKA was active. DIDS completely blocked the outwardly rectified anion channel and made it possible to resolve a second small anion channel with a unitary conductance of 16 pS.
Figure 8: I/V plot of immunopurified and reconstituted anion channels before and after activation by PKA and ATP. A, I/V plots of mean current of purified anion channels reconstituted from two different preparations (with or without CFTR) under control conditions, after phosphorylation with PKA + ATP, and after phosphorylation and addition of DIDS. Conditions are indicated by the symbols defined on the figure. B, I/V plots of single channel current of purified anion channels recorded under conditions identical to those indicated in A. For both A and B, symbols indicate mean values and error bars indicate ± S.E. (n = 8). In B, errors are within the size of the symbol.
Inside-out patch clamp of apical membranes of tracheal epithelial cells showed that the native ORCC could be activated from the cytoplasmic side by PKA and ATP(13, 14, 15, 16) . Consequently, we tested whether this purified outwardly rectified anion channel could be activated by PKA plus ATP. As can be seen in Fig. 5, addition of 100 µM ATP and the catalytic subunit of PKA to the incorporated channel increased channel activity at the same holding potential. PKA plus ATP activation could be achieved from only one side of the bilayer. However, because the orientation of the incorporated channel was random, we generally added PKA and ATP to both sides of the bilayer. Addition of PKA or ATP alone to either or both sides of the bilayer produced no change in channel activity (data not shown). Inspection of the current records after PKA + ATP activation revealed the appearance of another small conductance channel. Because of the observation of the presence of a phosphoprotein at 174 kDa in our preparation ( Fig. 2and 3), we suspected that this small conductance channel might be CFTR. To test this hypothesis, we applied the inhibitor DIDS (100 µM) to the bilayer solution with the expectation that DIDS would inhibit the large conductance outwardly rectified channel but leave the smaller one unaffected. This prediction was borne out by experiment (Fig. 5). During the first 2 min after the addition of DIDS, bursts of outwardly rectified anion channel activity were sometimes observed as shown in Fig. 4, but after this time they were never again seen. The small anion channel had a unitary conductance of 16 pS and a linear current/voltage relationship (see Fig. 8). This small, linear channel was found to be sensitive to DPC, a known blocker of CFTR (Fig. 6). Increasing concentrations of DPC were added to the presumptive extracellular side of the incorporated channel. Doses of 200 µM of DPC inhibited approximately 50% of channel activity. When 300 µM DPC was added, channel activity was completely blocked.
Figure 6: Effect of DPC on small anion channel remains after inhibition of the outwardly recitified anion channel by DIDS. Conditions were the same as for Fig. 5. Dashed lines represent the zero current level. The record was filtered at 100 Hz, and only the current traces at +100 mV are shown. Top trace, single channel recording of small anion channel resolved after inhibition of outwardly rectified anion channel by DIDS. Two current levels were observed, indicating the presence of two identical anion channels in the membrane (Control). Middle trace, single channel recording after addition of 200 µM DPC on the presumptive extracellular side of the incorporated channel. Bottom trace, single channel recording after addition of 300 µM of DPC to the presumptive extracellular side of the incorporated channel.
Figure 7: Reconstituted immunopurified anion channel proteins after CFTR precipitation: single channel characteristic and effect of PKA and DIDS. Immunopurification and reconstitution of purified protein was the same as described in the legend of Fig. 3except that immunopurified protein was immunodepleted of CFTR with anti-CFTR antibodies. Incorporated channels were recorded following a procedure identical to that described for Fig. 5. Dashed lines represent the zero current level. The record was filtered at 100 Hz. Top traces, single channel recording at holding potentials of ±100 mV (two channels were present in the bilayer). Middle traces, single channel recording at holding potentials of 100 mV after addition of ATP (100 µM) and the catalytic subunit of PKA (1.85 ng/ml) to both sides. Note that PKA + ATP did not alter channel activity. Bottom traces, single channel recording at holding potentials of ±100 mV after addition of 100 µM DIDS. Channel activity was completely blocked by DIDS. The small anion channel was not present in this preparation.
Fig. 8presents summary I/V curves for the immunopurified anion channels recorded under control conditions with (open circles) and without (open triangles and diamonds) CFTR and after phosphorylation with PKA plus ATP (solid circles and squares). Fig. 8A shows the mean current versus voltage curves, whereas Fig. 8B displays the single open channel I/V relations. It is apparent that PKA-induced phosphorylation activates an outwardly rectified anion channel (with no change in single channel current), but only when CFTR is present in the bilayer. After phosphorylation but in the presence of DIDS, only a small, linear conductance remained in the bilayer (solid squares).
Our results demonstrate that we have immunopurified from
bovine tracheal epithelia and functionally reconstituted into planar
lipid bilayers an outwardly rectified anion channel and the CFTR
Cl channel. The purified material contained four
phosphoproteins with apparent molecular masses of 52, 85, 120, and 174
kDa. We demonstrated that the 174-kDa protein is CFTR by precipitation
of this protein with anti-CFTR antibodies. In bilayers, the CFTR
channel was activated by PKA + ATP, had a linear I/V curve, was
inhibited by DPC, and was insensitive to DIDS, properties similar to
those reported for CFTR(27, 28) . Western blot
analysis of the immunopurified material revealed that the 52-kDa
protein was recognized by
p38 antibodies. The biophysical
characteristics of the isolated and reconstituted anion channels were
very similar if not identical to the characteristics of similar
channels recorded in patch clamp studies from airway epithelia (i.e. activation by PKA + ATP, conductance, I/V
properties, inhibitor sensitivities, etc.), suggesting that the
isolated channels were well preserved. Because an antibody (
p38)
that was raised against another anion channel, namely a 30-40-pS,
linear, Ca
-activated anion
channel(19, 29) , was used for immunopurification, we
also observed this channel in bilayers. However, because this channel
is extremely labile, its appearance in the bilayer could be controlled
by preparing proteoliposomes and storing them at 4 °C overnight or
longer. Under these conditions, the Ca
-activated
anion channel was never observed (in over 50 experiments) after
incorporation of the stored immunopurified material into the bilayer.
Hence, only the outwardly rectified and CFTR anion channels could be
seen in the bilayer.
As both channels were present in the same
preparation, it was possible to explore the proposed regulatory
relationship between the two channels. The outwardly rectified anion
channel was not affected by voltage or [Ca]
or by ATP from either side of the membrane. This finding is in contrast
to previous reports that ORCCs are activated by ATP from the
extracellular side of the apical membrane (30, 31, 32, 33) .
We demonstrate
that the reconstituted outwardly rectified anion channel was activated
by PKA and ATP only when CFTR was present in immunopurified material.
This finding is in agreement with three recent patch clamp
studies(8, 9, 12) . Egan et al.(8) showed that transfection of CF airway epithelial cells
with the wild-type CF gene corrected cAMP regulation of Cl secretion, induced the appearance of low conductance
Cl
channels attributable to CFTR, and permitted PKA
to activate ORCC. Gabriel et al.(9) demonstrated that
PKA regulation of ORCC was defective in nasal epithelial cells isolated
from a transgenic CF (-/-) mouse in which both CFTR mRNA
and protein were absent. Third, in whole-cell studies of normal human
tracheal epithelial cells and cell lines of CF tracheal cells
complemented with the normal CF gene, Schwiebert et al.(12) concluded that both CFTR and ORCC contribute to
whole-cell anion channel current and that CFTR is necessary and
required for cAMP regulation of ORCC. At least two possibilities exist
to account for these observations. First, because mutations in CFTR
lead to diminished CFTR trafficking to the apical plasma
membrane(34, 35) , the absence of membrane CFTR would
prohibit cAMP activation of ORCC. This hypothesis is consistent with
the results presented in this paper. Second, assuming some mutated CFTR
does get to the membrane, a mutation in the CF gene may alter the
activation process of the ORCC, interfering with the ability of kinase
A to phosphorylate the channel. Alternatively, defective CFTR may
interfere with the channel gating mechanism that controls PKA-induced
phosphorylation-mediated entry of the channel into its open state.
Our study on purified and reconstituted anion channels resolves several questions concerning the relationship between CFTR and outwardly rectified anion channels. We demonstrated that in vitro phosphorylation of the isolated proteins was not affected by CFTR precipitation but that the outwardly rectified anion channel could not be activated by PKA and ATP in the absence of CFTR. The biophysical properties of the outwardly rectified anion channel were independent of the presence of CFTR in the absence of PKA and ATP, indicating that CFTR itself does not affect channel conduction or gating. This observation is consistent with earlier patch clamp studies where it was possible to record ORCCs in CF (-/-) mouse cells and CF human trachea in cells activated by sustained strong depolarization(12) . Our study suggests that a direct interaction between CFTR and outwardly rectified anion channels is required for PKA activation, although it does not exclude the possibility that intermediate steps may still be involved.
In
summary, we have isolated and reconstituted an outwardly rectified
anion channel and the CFTR Cl channel from bovine
tracheal epithelia. These isolated channels were functionally very well
preserved as demonstrated by the similar biophysical characteristics of
reconstituted channels with the corresponding channels recorded in
patch clamp studies of native airway epithelia. We also demonstrated
that the outwardly rectified anion channel can be activated by PKA and
ATP. Functional channel activation by the protein kinase A was possible
only when CFTR was contained in the immunopurified material in spite of
the fact that there was no difference in phosphoprotein distribution.
After precipitation of CFTR, PKA and ATP did not activate these
outwardly rectified anion channels in bilayers. This result suggests
that the mechanism of regulation of these outwardly rectified anion
channels by CFTR involves an interaction between the two channels. In vitro phosphorylation of purified proteins by PKA and ATP
was unaffected by CFTR precipitation, suggesting that CFTR does not
play a role in phosphorylation of protein(s) that forms these
conductive pathways. The purification of an outwardly rectified anion
channel now raises the possibility of direct biochemical and molecular
studies on this important family of anion channels, particularly
important in light of our finding that CFTR can modulate PKA-mediated
regulation of this channel.