(Received for publication, October 24, 1995; and in revised form, December 19, 1995)
From the
Experiments were designed to test if immunopurified outwardly
rectified chloride channels (ORCCs) and the cystic fibrosis
transmembrane conductance regulator (CFTR) incorporated into planar
lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100
ng/ml) + NAD (1 mM) + ATP (1 mM) treatment
of ORCC and CFTR in bilayers resulted in a 2-fold increase in single
channel open probability (P) of ORCC but not of
CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical
properties of either channel. Further, PTX conferred a linearity to the
ORCC current-voltage curve, with a slope conductance of 80 ± 3
picosiemens (pS) in the ± 100 mV range of holding potentials.
PKA-mediated phosphorylation of these PTX + NAD-treated channels
further increased the P
of the linear 80-pS
channels from 0.66 ± 0.05 to >0.9, and revealed the presence
of a small (16 ± 2 pS) linear channel in the membrane. PTX
treatment of a CFTR-immunodepleted protein preparation incorporated
into bilayer membranes resulted in a similar increase in the P
of the larger conductance channel and restored
PKA-sensitivity that was lost after CFTR immunodepletion. The addition
of guanosine 5`-3-O-(thio)triphosphate (100 µM)
to the cytoplasmic bathing solutions decreased the activity of the ORCC
and increased its rectification at both negative and positive voltages.
ADP-ribosylation of immunopurified material revealed the presence of a
41-kDa protein. These results demonstrate copurification of a
channel-associated G-protein that is involved in the regulation of ORCC
function.
Cystic fibrosis (CF) ()is an autosomal recessive
disease that is common in North America. A characteristic of the
disease is impaired Cl
transport across several
tissues including those of the airway epithelia. The gene responsible
for impaired Cl
transport encodes the cystic fibrosis
transmembrane conductance regulator (CFTR), a protein that acts as a
small linear Cl
channel at the plasma membrane. A
second Cl
channel in the apical membrane of affected
tissue (the so-called outwardly rectified chloride channel or ORCC) is
also affected in CF such that it cannot be activated by PKA and ATP in
cells with the CF phenotype. The presence of a functional CFTR in the
membrane is required for the PKA/ATP activation of the
ORCC(1, 2, 3) . The biophysical properties of
ORCC and CFTR Cl
channels are 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(3, 4, 5, 6, 7, 8) .
ORCCs are blocked by a wide variety of molecules including DIDS and the
calixarenes (9) and have a halide permeability sequence of
I
> Cl
>
Br
. ORCCs can be activated by PKA and protein kinase
C (10, 11, 12, 13) . 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) and
glibenclamide(13) , but not by DIDS. The halide permeability
sequence for CFTR is Br
> Cl
> I
(14, 15) .
Heterotrimeric GTP-binding proteins (G-proteins) regulate ion
channels in a variety of tissues including respiratory
epithelia(16) . Regulation can be direct or indirect through a
cytoplasmic pathway involving second messengers and protein
kinases(16, 17) . Heterotrimeric G-proteins can also
regulate intracellular vesicle trafficking(18) . Both
regulatory mechanisms (direct regulation and regulation of endo- and
exocytosis) have been implicated in cAMP-dependent Cl secretion in normal and CF epithelia(17, 18) .
It was shown in whole-cell patch clamp studies (18) that
pertussis toxin (PTX), which uncouples heterotrimeric G
proteins from their receptors, increases Cl
transport and restores cAMP-activated Cl
currents in airway epithelial cells isolated from CF patients.
Additional studies suggested that the heterotrimeric G-protein
G
i-2 regulates CFTR Cl
conductance in human
airway epithelial cells by modulating vesicle trafficking and the
delivery of CFTR Cl
channels from an intracellular
vesicular pool to the plasma membrane(18) . The same authors
also demonstrated that the only pertussis toxin-sensitive G-protein
expressed in human airway cells was G
i-2(18) . As the
apical membrane of airway epithelial cells contains two cAMP-activated
Cl
channels (ORCC and CFTR), the possibility exists
that both of them are regulated by G-proteins. G
i-2 inhibits CFTR
function solely by preventing trafficking of the protein to the apical
membrane. However, G
i-2 may regulate ORCCs by a direct mechanism
independent of second messenger involvement(17, 18) .
We recently reported the simultaneous isolation and functional
reconstitution of an ORCC and CFTR from apical membrane vesicles of
bovine tracheal epithelial cells(3) . Isolated channels were
functionally preserved and exhibited similar regulatory features as
native channels recorded in patch clamp studies from airway epithelia.
In light of these observations, the goals of this study were to
determine whether a PTX-sensitive G-protein copurified with these
Cl channels and to examine the regulatory
relationship between the isolated channels and the copurified
G-protein.
Planar lipid bilayers, composed of a mixture of 25 mg/ml diphytanoyl-phosphatidylethanolamine, diphytanoyl-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(26) . Bilayer formation was ascertained by an increase in membrane capacitance to a final value of 300-400 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. 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 records were analyzed using pCLAMP software (Axon Instruments, CA), as described previously(25, 26) . Single channel data were stored digitally and for analysis were filtered at 500 Hz with an 8-pole Bessel filter and acquired at 1 ms/point. The dashed line in the figures represents the zero current level.
Figure 1: Autoradiography of pertussis toxin-induced ADP-ribosylation of immunopurified anion channel proteins from bovine tracheal epithelia. Pertussis toxin induced ADP-ribosylation of solubilized protein from apical tracheal vesicles (lane 1) and proteins immunopurified with p38 antibodies before CFTR precipitation (lane 2) and after CFTR precipitation (lane 3). Specificity of ribosylation was confirmed by lack of 41-kDa ribosylated protein (lane 4) when immunopurified proteins were not added to the reaction mixture.
Figure 2:
Effect of GTPS and subsequent PKA
phosphorylation on immunopurified ORCC and CFTR incorporated into
planar lipid bilayers. Control channel recordings were performed at a
holding potential of +80 mV. The addition of 100 µM GTP
S from the cytoplasmic side decreased channel activity of
the incorporated channel (from 0.39 to 0.18; n = 9).
Consecutive addition of PKA (1.85 ng/ml) and ATP (100 µM)
increased channel activity and induced the appearance of a small CFTR
Cl
channel on top of the opening and closing of the
ORCC. The addition of 100 µM DIDS completely blocked ORCC,
and only CFTR was recorded in this condition. 300 µM DPC
blocked CFTR channel activity (bottom trace). The dashed
line represents zero current. Records were filtered at 100
Hz.
Figure 3: Effect of PTX and subsequent PKA phosphorylation on the immunopurified ORCC and CFTR incorporated into planar lipid bilayer. Control single channel recordings were performed at a holding potential of +80 mV. PTX (100 ng/ml), NAD (1 mM), and ATP (1 mM) were added to the presumptive cytoplasmic side of incorporated channels. Consecutive addition of PKA + ATP, DIDS, and DPC 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. The dashed line represents zero current. Records were filtered at 100 Hz.
Figure 4: Effect of PTX and subsequent PKA phosphorylation on the immunopurified ORCC (in the absence of CFTR) incorporated into planar lipid bilayer. Holding potential, doses of PTX, PKA + ATP, DIDS, and DPC were as described for Fig. 3. 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. The dashed line represents zero current level. Records were filtered at 100 Hz.
The addition of PTX (100
ng/ml), an agent known to inactivate G proteins, together
with NAD (1 mM) and ATP (1 mM), also significantly
increased the P
of the ORCC from 0.38 ±
0.03 to 0.66 ± 0.05 (n = 11, Fig. 3).
Furthermore, PTX conferred a linearity to the ORCC current-voltage
curve, which had a slope conductance of 80 ± 3 pS at a holding
potential of ±100 mV (see Fig. 5). As with GTP
S,
PKA-mediated phosphorylation of these PTX + NAD-treated channels
revealed the presence of a small (16 ± 2-pS), linear channel in
the bilayer membrane (Fig. 3). Inhibition of the ORCC by DIDS
afforded us the opportunity to compare the activity of phosphorylated
CFTR following PTX treatment. There was no significant difference in
the channel P
of CFTR for either the
GTP
S-treated channel (P
= 0.63
± 0.08 (n = 9); Fig. 2) or the
PTX-treated channel (P
= 0.58 ± 0.05 (n = 9); Fig. 4).
Figure 5: Single channel current-voltage relationship of immunopurified and reconstituted ORCC in the presence and absence of CFTR before and after the addition of PTX. Conditions are defined in the symbol legends on the graph. Symbols indicate mean value, and error bars indicate ± S.D. for at least five separate experiments for each condition.
To further explore the
regulatory relationship between G-proteins and the ORCC, we
immunodepleted CFTR from the immunopurified tracheal preparation prior
to reconstitution of this material into the lipid bilayer. As
previously shown, immunodepletion of CFTR did not affect the
biophysical properties of the ORCC, although under these conditions the
ORCC could not be activated by PKA + ATP(3) . The addition
of PTX to the presumed cytoplasmic face of the incorporated channel in
the absence of CFTR resulted in a similar increase in P of the ORCC as had been observed in the presence of CFTR (Fig. 4). Moreover, PTX restored sensitivity to PKA + ATP
to the ORCC, as P
was increased from 0.63 ±
0.06 to 0.91 ± 0.08 (n = 12) under these
conditions, even in the absence of CFTR (Fig. 4). Furthermore,
PTX treatment also conferred a linearity to ORCC, as was the case in
the presence of CFTR. I/V curves, derived from these
experiments are shown in Fig. 5. It is clear from these plots
that PKA-mediated phosphorylation of ORCC partially restored
rectification properties after the addition of PTX. This effect of PTX
was independent of the presence or absence of CFTR. GTP
S-treated
ORCC, in contrast, was significantly more rectified at both negative
and positive holding potentials (100 pS at +40 to +100 mV and
16 pS at -40 to -100 mV). Interestingly, the negative
branch of the I/V curve of ORCC in the absence of CFTR was
almost identical to the I/V curve of ORCC treated with
GTP
S in the presence of CFTR.
The results presented in this study show that a pertussis
toxin-sensitive G-protein copurifies with bovine tracheal ORCC and CFTR
channels and is involved in the direct regulation of the ORCC. We have
previously demonstrated that solubilized apical membrane vesicles from
bovine trachea, immunopurified with an antibody raised against an anion
channel protein, contain both an ORCC and CFTR(3) . The
addition of PKA + ATP to the cis (or cytoplasmic) bathing
solution of a bilayer containing this material activated a large (80 pS
at +80 mV) conductance, outwardly rectified anion channel that was
sensitive to DIDS. Inhibition of this 80-pS channel with 100 µM DIDS in the presence of PKA and ATP revealed a second, low
conductance anion channel (16 pS) that exhibited a linear I/V
relationship and that was sensitive to DPC. These data suggested that
the immunopurified tracheal material contained both a functional ORCC
and a CFTR. Moreover, these channels maintained a regulatory
relationship even after this harsh purification procedure, e.g. the presence of CFTR was required for PKA activation of
ORCC(3) . In contrast, the recent model proposed to explain
regulation of Cl transport via ORCC and CFTR in
epithelial cells (31, 32) includes an amplification
cascade initiated by an initial interaction of extracellular ATP with a
G-protein-coupled P
receptor. Our studies with
immunopurified proteins reconstituted into planar lipid bilayers
demonstrated that the ORCC could be activated by PKA in the presence of
G551D mutant CFTR, but only when ATP was added to both sides of the
channel-containing bilayer, consistent with the external ATP
stimulation part of this model(19) .
Several other studies
report copurification of channels or receptors with associated
G-proteins(24, 27, 28, 29) . In our
preparation, a PTX-sensitive, ADP-ribosylated G-protein remained with
the ORCC complex after CFTR precipitation. A direct regulatory
relationship between the ORCC and a G-protein was confirmed by the
addition of GTPS or PTX to the ORCC either by itself or
incorporated together with CFTR. The addition of pertussis toxin, which
prevents the dissociation of the heterotrimeric G-protein complex,
activated the ORCC. The addition of GTP
S decreased the activity of
the ORCC in the planar lipid bilayer. Uncoupling of the ORCC from the
G-protein also altered the rectification properties of the channel,
suggesting that channel rectification is influenced at least in part by
its interaction with a G-protein. Interestingly, the ORCC regained PKA
sensitivity after the addition of PTX, even in the absence of CFTR.
These results are consistent with the findings of Schwiebert et al.(18) that G-proteins are involved in the regulation of
Cl
transport in airway epithelia. It would appear
that uncoupling of the effector (in our case ORCC) from its associated
G-protein restores PKA sensitivity to the channel. It is known that
phosphorylation of the
-receptor plays a role in desensitization
of this receptor(30) . Phosphorylation in this case prevents
interaction between a G-protein and the phosphorylated
-adrenergic
receptor and stops further mediation of signals from the stimulated
-receptor(30) . A similar mechanism between the ORCC and a
G-protein may also exist. Phosphorylation of the channel may uncouple a
G inhibitory (G
) protein from the channel and thus activate
it. Dephosphorylation of the channel would be predicted to permit the
G
protein to interact with the channel again and to inhibit
it. However, after activation with PTX (uncoupling of the channel from
the G-protein; (31) ), additional stimulation with PKA and ATP
was possible, suggesting that complete uncoupling of the channel from a
G-protein may uncover additional phosphorylation sites on the channel
protein. Moreover, these findings indicate that there are at least two
major independent regulatory inputs that converge on the ORCC.
Recently, Schwiebert et al. suggested that ATP transported
through CFTR acts as an autocrine stimulator of the ORCC(32) .
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. We have
previously shown that the active form of CFTR is required for
PKA-mediated activation of ORCC(19) , while in the present
study we have demonstrated the presence of a G-protein in our
preparation. However, in contrast to the findings of Guggino and
co-workers(32) , namely that extracellular ATP at nanomolar
concentrations stimulates the ORCC in normal or CF cells, we found that
under our experimental conditions ATP had no effect on channel activity
if CFTR was not present in the membrane. Moreover, ATP had no effect on
the ORCC if PKA + ATP was not present on the cytoplasmic side of
incorporated channels. There are several possible explanations for our
results. One possibility is that ATP (on the cis side of the
bilayer) may bind to the ORCC, which subsequently interacts with CFTR
and is thereby activated. Interaction of the ORCC with CFTR may
allosterically diminish interaction of the ORCC with its G-protein, as
both the G-protein (inhibition) and CFTR (activation) regulate the ORCC
and have opposite effects on the channel. Rectification of the ORCC may
be in part due to G-protein coupling, because treatment with PTX
confers a linearity to the otherwise outwardly rectified behavior of
the channel. CFTR also affects rectification, because when CFTR was
present, the ORCC exhibited less rectification in the negative voltage
quadrant. This finding supports the hypothesis that both molecules
(CFTR and a G-protein) are coupled to the ORCC and have opposite
effects on rectification (i.e. G-protein coupling induces
rectification, whereas CFTR decreases rectification).
A second possibility to consider is that ATP may bind to CFTR directly. Alternatively, a receptor for ATP (purinergic receptor) could be strongly associated with CFTR. If this hypothesis is correct, we may have copurified a purinergic receptor together with CFTR. However, it would seem unlikely that the appropriate signaling pathway would be maintained under our conditions. If, on the other hand, CFTR is both coupled to ATP and bound to a G-protein, ATP binding could uncouple the G-protein from its effector (in this case ORCC). This would allow the involvement of CFTR in the regulation of different processes through G-proteins. In either case, we have demonstrated that both a G-protein and CFTR are involved in the regulation of ORCC.
In summary, we have
shown that a pertussis toxin-sensitive G-protein copurifies with ORCC
and CFTR and that, after precipitation of CFTR, the G-protein remains
in the material that contains the ORCC. The effects of PTX and
GTPS on the ORCC were independent of the presence or absence of
CFTR. PTX and GTP
S did not have any effect on the CFTR
Cl
channel activity under our experimental conditions
(in presence of PKA and ATP from cytoplasmic side). These observations
are consistent with the hypothesis that G-proteins directly regulate
the ORCC, but not CFTR. Additionally, whereas we have previously
demonstrated that precipitation of CFTR prevents the PKA +
ATP-dependent activation of the ORCC(3) , the data presented in
this study have shown that even in the absence of CFTR, the ORCC can be
activated by PKA + ATP as long as PTX is present on the cis side. This observation suggests a possible regulatory role of CFTR
on the ORCC through a G-protein interaction.