(Received for publication, September 26, 1995; and in revised form, December 20, 1995)
From the
The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes a linear conductance chloride channel, which is regulated by cAMP-dependent protein kinase phosphorylation at multiple sites located in the intracellular regulatory (R) domain. Studies in a lipid bilayer system, reported here, provide evidence for the control of CFTR chloride channel by its R domain. The exogenous R domain protein (encoded by exon 13 plus 85 base pairs of exon 14) interacted specifically with the CFTR molecule and inhibited the chloride conductance in a phosphorylation-dependent manner. Only the unphosphorylated R domain protein blocked the CFTR channel. Such functional interaction suggests that the putative gating particle of the CFTR chloride channel resides in the R domain.
CFTR ()belongs to a family of ABC
transporters(1) , each of which contains two transmembrane
domains and, usually, two nucleotide binding folds. CFTR contains, in
addition, a large intracellular regulatory (R) domain(2) . The
CFTR protein constitutes a linear conductance Cl
channel, the opening of which requires both phosphorylation of
the R domain by cAMP-dependent protein kinase (PKA) (3, 4) and ATP binding and hydrolysis by the
nucleotide binding folds(5, 6) . The working
hypothesis for CFTR activation by the R domain has been that
phosphorylation by PKA presumably induces a conformational
change(7) , which relieves the inhibitory action of the R
domain on the Cl
conductance, as CFTR with its R
domain removed generates a constitutively open Cl
channel (without requiring PKA
phosphorylation)(8, 9) .
Some voltage-dependent ion
channels, such as the Shaker K channel,
inactivate or close by the ``ball and chain''
mechanism(10) . The amino-terminal portion of the Shaker
K
channel serves as the ``ball'' that
occludes the conductance pathway of the open K
channel(11) . It is not known, however, whether the R
domain of CFTR functions as a gating particle for the Cl
channel. In favor of this possibility is the observation that
deletion of 130 amino acids from the R domain results in a
constitutively open channel(8) . On the other hand, the R
domain of CFTR is quite large (over 250 amino acids) compared with the
putative ``ball'' for the Shaker K
channel,
which is less than 25 amino acids in length.
We took a
reconstitutional approach in an attempt to search for the putative
gating particle of the CFTR Cl channel. First, the R
domain protein (RDP) was expressed as a peptide in vitro in a
rabbit reticulocyte lysate system. Second, the wild type CFTR cDNA was
subcloned into an eukaryotic expression vector pCEP4 and expressed in a
human embryonic kidney cell line (HEK 293), from which the microsomal
membrane vesicles were isolated and incorporated into lipid bilayers
where the CFTR channel activity can be directly assessed.
Alternatively, CFTR channels were incorporated into the planar lipid
bilayer from vesicles prepared from T84 cells, a human colon carcinoma
cell line that expresses abundant CFTR. The effect of RDP on the CFTR
channel was then tested in the lipid bilayer reconstitution system. We
reasoned that if the R domain, or portions of it, function as a
``ball'' or gating particle for CFTR, then exogenous R domain
protein might enter the CFTR pore or vestibule when the channel is open
and block it. Alternatively, if the open configuration of CFTR is
stabilized by intramolecular interactions of the R domain with other
portions of the molecule, then exogenous R domain might hold the
channel in the closed state by occupying this putative intramolecular
binding site and preventing the endogenous R domain from assuming the
open configuration. Based on the function of intact CFTR, in which
phosphorylation at a single site in the R domain is sufficient to
induce channel opening in the presence of ATP(9) , we
postulated that the open channel block model, in which R domain protein
blocks the conduction pore, would most likely require unphosphorylated
R domain to block, whereas block of the closed channel by the R domain
protein occupying an intramolecular stabilization site would probably
be favored by phosphorylation of the exogenous R domain.
Two
control translation mixes were prepared. For one, no exogenous DNA or
RNA was added to the reticulocyte lysate system, and when
[S]methionine was added to this mix and
incubated exactly as for the R domain translation, no translated
protein bands were detected on autoradiography (Fig. 1A, lane 1). This mix does, however,
contain all of the endogenous reticulocyte lysate proteins. The second
control translation mix was obtained by expressing the six proteins of
the brome mosaic virus, one of which migrates on electrophoresis at
about 35 kDa, where the R domain protein migrates. This control
provides not only the ribosomes and proteins of the reticulocyte but
also complete and possibly nascent proteins of about the same molecular
weight as the R domain translation product.
Figure 1:
Expression of CFTR and R
domain proteins. A, in vitro expression of R domain
protein. To visualize the newly made protein,
[S]methionine was added to the
transcription/translation reaction (lane 2). The doublet in lane 2 corresponds to an alternative starting site of
translation located 9 amino acids into the R domain of CFTR. Both bands
were recognized by the antibody against the R domain of CFTR (lane
3). As a control, mock transcription/translation was carried out
with all the reagents except a DNA template (lane 1).
Non-radioactive RDP was immunoprecipitated with R domain antibody and
protein G-agarose. Phosphorylation of non-labeled RDP was performed
with the immunoprecipitated RDP-antibody complex, in the presence of 10
µCi of [
-
P]ATP and 2 units of catalytic
subunits of PKA (lane 4). B, expression of CFTR in
HEK 293 cells. The wild type CFTR cDNA was subcloned into the
eukaryotic expression vector pCEP4 and expressed in HEK 293 cells.
These cells express abundant quantities of CFTR protein, as indicated
by the Western blot using antibody against the R domain of CFTR. Data
presented in this paper were obtained with eight separate preparations
of RDP and 11 separate preparations of CFTR transfected in HEK 293
cells.
To maintain stability of the bilayer membrane and the CFTR channel activities, designed pulse protocols were used to measure currents through the single CFTR channels. The bilayer membrane was kept at a holding potential of 0 mV and pulsed to different test potentials of either 2- or 5-s durations. The interval between consecutive episodes was 10 s. Single channel currents were recorded with an Axopatch 200A patch clamp unit (Axon Instrument). Data acquisition and pulse generation are performed with a 486 computer and 1200 Digidata A/D-D/A convertor (Axon Instrument). The currents were sampled at 1-2.5 ms/point and filtered at 60 Hz through an 8-pole Bessel filter. Single channel data analyses were performed with pClamp, TIPS, and custom softwares.
In a KCl concentration gradient of 200 mM (cis)/50 mM (trans), a single CFTR
channel had a linear conductance of 8.2 ± 0.6 pS, with a
reversal potential of +22 ± 1.4 mV. At -80 mV, the
channel had an average open probability of 0.318 ± 0.028, with
mean open lifetimes of o1 = 23.6 ms and
o2 =
110 ms (n = 76)(13) . Values were the same for
channels from T84 vesicles, except for a small difference in the
relative occurrence of
o1 and
o2(15) . An inhibitor
of the CFTR channel, diphenylcarboxylate (3 mM), when added to
the trans solution, completely blocked channel activity.
4,4`-Diisothiocyanate 2,2`-stilbene disulfonate (0.5 mM) added
to both solutions had no effect on the channel. These properties are
those reported for and expected for CFTR(16) . Vesicles
prepared from untransfected HEK 293 cells never displayed chloride
conductances with these properties.
Figure 2:
Transient inhibition of CFTR chloride
conductance by R domain protein. A single CFTR Cl channel was incorporated into the bilayer membrane from
microsomal membrane vesicles isolated from HEK 293 cells. Channel open
probabilities (Po) were calculated as the fractional time
occupied by the open state during the 5-s test pulse to -80 mV.
The calculated Po during consecutive test pulses was plotted
as a function of time (``diary plot''), as shown in the upper panel. RDP exhibited a biphasic effect on the CFTR
channel, promoting channel closure for
12 min, followed by
spontaneous recovery. Representative single channel traces were shown
for the control channel (A), 3 min after the addition of RDP
(
15 pM final concentration) (B), and 16 min
after the addition of RDP (C). Marks to the left of the single
channel traces show base-line (zero current, closed channel) levels.
Data are representative of seven separate
experiments.
Exogenous RDP blocked the CFTR Cl channel when added to the intracellular side (that is, the same
side as contains PKA and ATP) but not to the extracellular side (Fig. 3, n = 3). RDP had no effect on a small
conductance 4,4`-diisothiocyanate 2,2`-stilbene disulfonate-sensitive
Cl
channel detected in the T84 cells (3.2 pS,
distinct from the CFTR channel)(15, 17) (Fig. 4A, n = 6) nor on a
98-pS cation channel endogenous to the T84 cells (Fig. 4B, n = 5). The results indicate
that RDP binds specifically to the intracellular portion of the CFTR
protein, and the results are the same whether native CFTR or
heterologously expressed CFTR is studied.
Figure 3: Inhibition of the endogenous CFTR channel in T84 cells by R domain protein. Isolated microsomal vesicles from T84 cells were fused with the lipid bilayer membrane. Currents through a single CFTR channel was measured with a test potential of -80 mV (Control, A). The traces shown are consecutive episodes of a 2-s test pulse at -80 mV, acquired at 10-s intervals. The channel open probability did not change 4 min after addition of 20 µl of RDP to the trans (extracellular) solution (B). Further addition of RDP to the cis (intracellular) solution resulted in complete closure of the CFTR channel (C). Marks to the right of the traces indicate base-line level (zero current, closed channel).
Figure 4:
Lack of effects of R domain protein on
other ion channels in T84 cells. With the same recording solution (200
mM KCl (cis)/50 mM KCl (trans)),
two other types of ion channels were also encountered in the microsomal
vesicles isolated from T84 cells. The traces shown in A were
taken from a small conductance, diphenylcarboxylate-insensitive
Cl channel. The channel had slope conductance of 3.2
± 0.8 pS, with reversal potential of 24.5 ± 2.8 mV (data
not shown). Open probability of this small conductance chloride channel
did not change when 20 µl of RDP was added to both sides of the
channel. The traces shown in B were obtained from a large
conductance K
channel. This channel is insensitive to
inhibition by RDP (20 µl added to the cis solution). The marks on the right of each panel indicate
the base-line levels. The records in A were filtered at 40 Hz
frequency, and those in B were filtered at 300 Hz
frequency.
Figure 5: Preventing phosphorylation of RDP causes sustained CFTR channel block. PKI was added to the intracellular side of a single CFTR channel to a final concentration of 10 µg/ml, approximately 5 times the concentration necessary to prevent phosphorylation of RDP by PKA determined in separate assays. Channel open probabilities (Po) were calculated as the fractional time occupied by the open state during the 5-s test pulse to -80 mV. The diary plot (upper panel; see Fig. 2legend) shows that addition of PKI did not affect Po of the control channel, but it prevented the recovery of channel activity closed by RDP. In the presence of PKI, the blocking effect of RDP became essentially irreversible. The channel remained closed during the whole period of the experiment. Selected single channel traces are shown for the control condition (A), 5 min after addition of PKI (B), and 13 min after addition of RDP (C). Base line (zero current, closed channel) is indicated for each line by marks on the left. This experiment is representative of four others.
Figure 6: Prephosphorylation of R domain protein eliminates its inhibitory activity. The RDP was prephosphorylated in a test tube with 100 units/ml catalytic subunit of PKA and 5 mM Mg-ATP before addition to the CFTR channel, which was captured in a vesicle from HEK293 cells. Under control conditions, the bilayer contained at least three active CFTR channels (A), which remain stable over a period of 12 min. Panel B illustrates channel activity following addition of the phosphorylated RDP. No inhibition was seen over a period of 12 min. Subsequent addition of PKI and unphosphorylated RDP resulted in progressive inhibition of CFTR channel activity. The traces shown in C were taken 12 min after addition of PKI and unphosphorylated RDP. The residual channel activity remaining could be completely blocked by adding 3 mM diphenylcarboxylate to the trans solution (data not shown). Base line (zero current, closed channels) is shown by a dashed line. This experiment is representative of three others.
The results indicate that phosphorylation plays a key role in mediating the binding of RDP to the CFTR molecule; only the unphosphorylated form of RDP is capable of blocking the CFTR channel.
The large intracellular R domain is a special feature of the
CFTR molecule, alone among members of the ABC family. In the presence
of adequate concentrations of intracellular ATP, phosphorylation of any
one of four main phosphorylation sites (18, 19) is
sufficient for full opening of the channel, and indeed, small increases
in channel open probability can be detected in response to PKA
phosphorylation even in native CFTR molecules with all consensus
phosphorylation sites converted to alanines(20) . However,
CFTRR-S660A, a molecule with amino acids 708-835 deleted (to
render it constitutively open) with the remaining consensus PKA site
mutated, is insensitive to activation by PKA(9) , suggesting
that activation by phosphorylation takes place in the R domain. Our
results show that the exogenous RDP interacts specifically on the
intracellular side of the CFTR channel. Inhibition of channel activity
is abolished by phosphorylation of RDP.
This inhibitory activity is specific for the R domain, since an anonymous protein of comparable molecular weight translated under the same conditions fails to block the channel, and inhibition appears to be specific for CFTR, since the small linear chloride channel is not blocked, nor is a large cation channel identified in the T84 vesicle preparations. The inhibition depends on the phosphorylation state of the R domain protein, however. Prephosphorylated R domain protein fails to block the channel, and when R domain protein is added under conditions where phosphorylation has been inhibited, the channel is closed indefinitely. However, when unphosphorylated R domain protein is added to the bilayer system in the presence of PKA and ATP, there is an initial block, followed by slow reversal as (presumably) the R domain protein undergoes phosphorylation during the course of the experiment. A second round of block and reversal can be demonstrated by adding fresh, unphosphorylated R domain protein. These experiments demonstrate that block by the R domain protein is not due to destruction of the CFTR channel, since the channel recovers with time (and phosphorylation of the R domain protein), and recovery of the channel is not accompanied by insensitivity to R domain block, since addition of fresh R domain protein restores the block.
These data are consistent with the hypothesis that the R domain protein binds reversibly to the CFTR channel in its open state, probably in the region of the pore. In the conditions in which PKA and ATP are still available on the intracellular side of the CFTR molecule, unbound R domain protein is gradually phosphorylated. As the R domain protein disengages from CFTR, there is, over time, less and less R domain protein remaining unphosphorylated to interact with CFTR. Eventually, the concentration of unphosphorylated R domain protein falls below that required to bind to and block the channel. When the R domain protein is prephosphorylated, there is no interaction. When the R domain protein is protected from phosphorylation by inclusion of PKI on the intracellular side of CFTR, there is always sufficient unphosphorylated R domain protein to interact with the channel. Since phosphorylation at even a single site in CFTR in the native molecule is sufficient to open the CFTR channel(9, 18, 19) , we speculate that binding of the R domain to the pore requires an unphosphorylated R domain protein molecule. An alternative hypothesis is that the exogenous R domain binds to a site in the closed state of the CFTR molecule, exerting steric hindrance to channel opening. In this case, one would expect that the phosphorylated R domain protein would be a better blocker than the unphosphorylated protein, because in the native molecule, stabilization of the open state must occur when the R domain is phosphorylated. Therefore, our data are less consistent with this hypothesis.
The reconstitution system presented here demonstrates specific interaction between the R domain and CFTR, which has functional consequences, and should be useful for further investigation of the specific amino acid residues involved in the binding sites on both RDP and CFTR itself.