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J. Biol. Chem., Vol. 277, Issue 3, 2125-2131, January 18, 2002
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-Phosphate Linker in the Nucleotide Binding Domains of CFTR
Alter Channel Gating*
§,,
, and**
From the Howard Hughes Medical Institute, Departments
of Internal Medicine and Physiology and Biophysics, University of Iowa
College of Medicine, Iowa City, Iowa 52242, the ¶ Department of
Biological Sciences, Columbia University, New York, New York 10027, and
the Department of Physiology, University of Texas Southwestern
Medical Center, Dallas, Texas 75390
Received for publication, October 2, 2001, and in revised form, November 2, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The cystic fibrosis transmembrane conductance
regulator (CFTR) Cl The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is an epithelial Cl In CFTR, the membrane-spanning domains form an anion-selective pore
(4), and the activity of the cytoplasmic domains controls channel
opening and closing (5, 6). Channel activity requires phosphorylation
of the R domain. Then ATP binding and hydrolysis by the two NBDs
determine gating.
Previous studies have probed the structure and function of the CFTR
NBDs by examining the functional consequences of applying nucleotide
analogs and introducing site-directed mutations. Those studies have
revealed different roles for NBD1 and NBD2 in channel gating. Several
studies have also highlighted the role of ATP binding and hydrolysis in
CFTR gating (for reviews, see Refs. 5 and 6). Mutations of the
conserved Walker A lysines (Lys-464 in NBD1 and Lys-1250 in
NBD2) inhibit ATPase activity (7) and alter channel gating (5,
8-10). However, the effects of mutations in the two NBDs are not
symmetrical; the K1250A mutation dramatically prolongs the burst
duration, whereas the K464A mutation reduces the frequency of channel
opening but does not change burst duration. More recent studies have
suggested that ATP binding without hydrolysis may be sufficient to open
the channel (11-14). Thus, the molecular mechanisms by which the ATP-
and ADP-bound states of each of the two NBDs affect channel gating are
not well understood.
Recent work has described the three-dimensional crystal structures for
the isolated NBDs of ABC transporters in the presence of ATP (15), ADP
(16-19), or no nucleotide (17). These structures provide us with the
opportunity to model the amino acid contacts between the CFTR NBDs and
nucleotide and to evaluate structural models for channel gating. In
this study, we have tested specific predictions of such models using
site-directed mutagenesis to alter key residues and patch-clamp
electrophysiology to examine the functional consequences.
Materials--
The catalytic subunit of
cAMP-dependent protein kinase (PKA) was purchased from
Promega. Lipofectin was obtained from Invitrogen. All other
reagents were obtained from Sigma.
Site-directed Mutagenesis and Transfection--
CFTR mutants
were prepared in the pTM1-CFTR4 plasmid (20) (or QuikChangeTM,
Stratagene). Mutations were verified by sequencing around the site of
mutation. Wild type and mutant CFTR proteins were transiently expressed
in HeLa cells using the vaccinia virus/T7 expression system as
described previously (21). Cells were studied 6-24 h after
transfection, depending on the level of expression desired.
Electrophysiology--
Methods for excised, inside-out
patch-clamp recording were as described previously (22), except that
experiments were performed at room temperature (21-23 °C). The
pipette (extracellular) solution contained (in mM): 140 N-methyl-D-glucamine, 100 aspartic acid, 35.5 HCl, 5 CaCl2, 2 MgCl2, 10 Tricine, pH 7.3 with
1 N HCl. The bath (intracellular) solution contained (in
mM): 140 N-methyl-D-glucamine, 135.5 HCl, 3 MgCl2, 10 Tricine, 4 Cs(OH)2, 1 EGTA, 1 Na2ATP, pH 7.3 with 1 N HCl.
Data Analysis--
Data were collected at Potential Role of the
Based on this model, we examined the effect of two sets of mutations in
CFTR (Gln and Asn) that we predicted would have different effects on
the
We also mutated a highly conserved Asn (Asn-505 in NBD1 and Asn-1303 in
NBD2 of CFTR). These align with Asn-102 of LivG, which mediates
a set of hydrogen bonds with conserved residues in each conformation of
the
Thus, whereas the Gln and Asn mutations would not have precisely
opposite effects on the structure, these mutations might affect the
Mutating Gln-493 in NBD1 Reduced Channel Opening--
A trace of
wild type CFTR reveals characteristic bursts of activity separated by
closed interburst intervals (Fig. 2). In contrast, the Q493A variant exhibited a substantially reduced Po because of a prolonged interburst interval with little
change in the burst duration (Fig. 2). This result is consistent with the hypothesis that removal of the conserved Gln reduces either its
affinity for ATP or its ability to induce the conformational change in
the
In both HisP and Rad50, the conserved Gln side chain contacts the
The Gln side chain also contacts Mg2+ in Rad50 (23). In
HisP, which was crystallized in the absence of Mg2+, the
Gln side chain contacts a H2O molecule that was assumed to
take the place of Mg2+ (15). If the conserved Gln in CFTR
is a critical contact for the divalent cation, we expected that a Gln
mutation would alter the functional consequences of changing the
divalent cation. In wild type CFTR, the relative order of current
stimulation is CaATP > MgATP > Na2ATP (13, 24). Moreover,
mutation of the NBD2 Walker B Asp, which also contacts the
Mg2+, abolishes differential effects of Mg2+
and Ca2+ compared with ATP alone (13). Fig.
4 shows that varying the divalent cation
generated similar effects for wild type CFTR and the Q493A variant.
These results suggest that contact between the conserved Gln and the
divalent cation is not critical to this function.
ADP inhibits and pyrophosphate (PPi) stimulates CFTR
Cl Mutating Asn-505 in NBD1 Can Increase Channel Opening--
The
N505C mutation had an effect opposite to that of Q493A; it reduced the
interburst interval and increased Po (Fig.
7). As with Q493A, burst duration did not
change. CFTR-N505A had gating kinetics similar to those of wild type;
presumably the introduced Ala did not disrupt the structure to the same
extent as when Cys was substituted for Asn. The effect of the Cys
mutation can be explained by destabilization of the ADP conformation of
the loop promoting faster entry into the channel-activating ATP
conformation. Inhibition by ADP, stimulation by PPi, and
the EC50 for ATP stimulation were not altered by the N505C
or N505A mutations (data not shown). The opposite effects of the Q493A
and N505C mutations on gating are consistent with the expectation that
these mutations might have different effects on the Mutating Gln-1291 in NBD2 Reduced PPi Stimulation of
CFTR Channel Activity--
In contrast to the NBD1 Gln mutant, the
NBD2 Gln variant Q1291A showed gating much like wild type (Fig. 2).
These results indicate that the two NBDs in CFTR do not have equivalent
functions in controlling gating; this conclusion is consistent with
that drawn from earlier studies (9, 10, 13, 22). Previous studies have
also suggested that termination of an open burst depends upon ATP
binding and hydrolysis at NBD2. For example, the CFTR-K1250A mutant
markedly prolongs burst duration (9, 10, 13, 22). Thus the normal burst
duration in Q1291A suggests that mutation of the conserved Gln did not
disrupt hydrolysis.
Wild type CFTR and CFTR-Q1291A had the same apparent EC50
for ATP (Fig. 3) and the same response to variation of the divalent cation (Fig. 4). These results suggest that Gln-1291 is not a key
factor in ATP binding or hydrolysis.
In the ADP-bound state, the conserved Gln of LivG MJ1267 (Gln-89), LolD
MJ0796 (Gln-90), and TAP1 (Gln-586) does not have the opportunity to
contact a
PPi and AMP-PNP applied with ATP lock channels open by
prolonging the burst duration (22, 26, 30-32). It is thought that PPi and AMP-PNP bind tightly in an open-channel
configuration and that the channel closes with dissociation of
PPi or the non-hydrolyzable nucleotide. Several NBD2
mutations, but not equivalent NBD1 mutations, abolish PPi
stimulation (9).2 This effect
may be mediated at NBD2, although a recent report showed that the
nucleotide base of AMP-PNP interacts with NBD1, suggesting more
complicated explanations (33). Despite the lack of effect of the Q1291A
mutation on other aspects of gating, it dramatically reduced
PPi stimulation (Fig. 6). This result suggests that an
interaction with the conserved NBD2 Gln is critical to either
stabilizing the bound polyphosphate or transducing the signal generated
by its binding.
Mutating Asn-1303 in NBD2 Increased the Burst Duration and
Decreased the Opening Rate--
In addition to its role in determining
structure, we were interested in Asn-1303 mutations because they are
associated with CF (34). The single-channel gating of CFTR-N1303K, a
relatively frequent CF-associated mutation, showed a long burst
duration and a long interburst interval (Fig.
8). We observed similar kinetic effects
when Asn-1303 was changed to another CF-associated mutation (N1303H), a
sequenced mutation with undetermined clinical consequences (N1303I),
and an Ala (N1303A) (Fig. 8).
These functional consequences are similar to those observed with
mutations of Lys-1250, which disrupt ATP hydrolysis and probably binding (7, 13, 22). To further examine similarities between CFTR-N1303K and CFTR-K1250A, we examined the effect of PPi
and ADP. The N1303K mutation prevented
PPi-dependent stimulation of current and
eliminated ADP-dependent current inhibition (Fig. 9). Pyrophosphate caused only a 26.9 ± 18.6% (n = 4) increase in current for the
CFTR-N1303K channel, and ADP caused only a 3.2 ± 6.8%
(n = 3) inhibition in current. However, in contrast to
the reduced EC50 for ATP observed for mutations at Lys-1250 (21), the apparent EC50 for CFTR-N1303A (253 ± 62 µM, n = 5) was not different from that of
wild type CFTR (176 ± 67 µM, n = 5).
We hypothesized that amino acid contacts with the NBD
Mutations of the conserved Gln and Asn did not alter the
EC50 for ATP-dependent current stimulation.
These results suggest that disrupting the interaction of the
The conclusions that the Gln and Asn mutations did not inhibit ATP
binding or abolish hydrolysis are consistent with other studies. In
HisP, the Q100L mutation showed normal ATP binding although the rate of
histidine transport was reduced (36). In P-glycoprotein (Pgp), mutation
of the conserved glutamine in either NBD1 or NBD2 caused no change in
the apparent affinity for ATP, the Mg2+ requirement, or
transition state trapping. Moreover, these mutations only modestly
reduced the ATPase rate (37). In LivG, mutation of the equivalent Gln
residue did not change the Km for ATP and decreased
hydrolysis only modestly.3
Thus, the An ATP-induced change in the position of the Our finding that mutation of equivalent residues in the two NBDs
produced different gating patterns is consistent with earlier work
indicating that the two NBDs have different functions in gating CFTR
(9, 10, 22, 35) and in the function of some other ABC transporters
(38-40). For example, in CFTR movement of the Based on the structural differences in ATP-bound, ADP-bound, and
nucleotide-free NBDs, Karpowich et al. (17) proposed
that an ATP-induced alteration in the affinity between the two NBDs controls their interaction, thereby converting chemical into mechanical energy. Their analysis suggested that the position of the Some of the mutations we studied have been observed in patients with
CF. N1303K is a frequent CF-associated mutation that has been shown to
affect channel processing (42). Our data indicate that this mutation
also affects channel gating and reduces Po. N1303H and
N1303I also alter channel gating although their effect on
Po was minor. Because these are rare variations, it is not clear whether or not they cause CF. Two relatively uncommon missense mutations have been described at Gln-1291, Q1291H and Q1291R
(43),4 and a single
chromosome was reported to encode a variation at Gln-493,
Q493R.4 The relatively mild gating abnormalities we
observed for these variants suggest that if they cause CF, altered
processing or some other abnormality may be the cause for the loss of
CFTR function.
channel is an ATP-binding
cassette transporter that contains conserved nucleotide-binding
domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and
close the channel. Crystal structures of related NBDs suggest a
structural model with an important signaling role for a 
-phosphate
linker peptide that couples bound nucleotide to movement of an
-helical subdomain. We mutated two residues in CFTR that the
structural model predicts will uncouple effects of nucleotide binding
from movement of the -helical subdomain. These residues are Gln-493
and Gln-1291, which may directly connect the ATP -phosphate to the
-phosphate linker, and residues Asn-505 and Asn-1303, which may form
hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced
the frequency of channel opening, suggesting a role for this residue in
coupling ATP binding to channel opening. In contrast, N505C increased
the frequency of channel opening, consistent with a role for Asn-505 in
stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased
the effects of pyrophosphate without altering other functions.
Mutations of Asn-1303 decreased the rate of channel opening and
closing, suggesting an important role for NBD2 in controlling channel
burst duration. These findings are consistent with both the bacterial
NBD structural model and gating models for CFTR. Our results
extend models of nucleotide-induced structural changes from
bacterial NBDs to a functional mammalian ATP-binding cassette transporter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel that
belongs to the ATP-binding cassette (ABC)
transporter family (1-3). The conserved features of this family are
two membrane-spanning domains and two cytoplasmic nucleotide-binding
domains (NBD). CFTR also contains a unique regulatory (R) domain. The
membrane-spanning domains of CFTR show topological similarity to those
of other ABC transporters, but there is little sequence similarity. In contrast, the NBDs show significant sequence conservation throughout the family.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 or 80 mV,
filtered at 1 kHz using a variable eight-pole Bessel filter (Frequency
Devices Inc., Haverhill, MA), and digitized at 5 kHz. Open state
probability was determined from patches containing one to three
channels. Single-channel data were analyzed with a burst delimiter of
20 ms. Data analysis was done using the pClamp 6.0 software package (Axon Instruments Inc., Foster City, CA). Data are shown as means ± S.E. Statistical significance was determined as indicated in the
figure legends. p values of < 0.05 were considered
statistically significant. The apparent EC50 for
ATP-stimulated CFTR activity was determined by fitting data to the
equation A=M·[ATP]/(EC50 +[ATP]) where A is the
ATP-induced Cl current and M is the fitted value for the
current at saturating ATP concentrations. Fitting was done using a
least squares method with IgorPro 2.04 software (WaveMetrics, Inc.,
Lake Oswego, OR).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Phosphate Linker in
NBD-dependent Gating of CFTR--
Karpowich et
al. (17) compared the crystal structure of NBDs in the ATP- or
AMP-PNP-bound state (HisP and Rad50 in Refs. 15 and 23) with that of
the ADP-bound or nucleotide-free NBD (LivG (MJ1267), LolD (MJ0796),
MalK, and TAP1, in Refs. 16-19). In the ATP-bound state, a conserved
Gln (Gln-100 in HisP) contacts the -phosphate of ATP
(Fig.1). Gln-100 lies at the
amino-terminal end of a short segment of amino acids called the
-phosphate linker. With ATP present, the -phosphate linker
of the NBD stabilizes an -helical subdomain in a fixed relationship
to the ATP-binding core subdomain. In contrast, in the absence of
nucleotides or with ADP bound (for example in LivG), the loss of
contact with the -phosphate allows a conformational change to occur
in the -phosphate linker, which facilitates outward rotation of the -helical subdomain away from the ATP-binding core subdomain. Thus
the presence or absence of the ATP -phosphate determines the
position of the conserved Gln and hence the position of the -helical subdomain via the -phosphate linker. As we note
below, other structural interactions may also influence the position of
the -phosphate linker.
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Fig. 1.
Structural models of HisP, LivG, and CFTR
NBDs. The figure depicts the relationship between the
nucleotide, conserved glutamine residue, -phosphate linker, and
-helical subdomain for HisP (top) and LivG
(bottom). In the HisP structure, the side chain of Gln-100
contacts the -phosphate of ATP via a water molecule
(red). The extended conformation of the -phosphate linker
backbone (pink) is stabilized by hydrogen bonds with Arg-166
and Asn-113. In the ADP-bound LivG structure, the -phosphate loop
has a less extended conformation, perhaps stabilized by hydrogen
bonding between Arg-166 and Asn-102. Named amino acids and nucleotides
are shown as ball-and-stick structures. Other aspects of
protein structure are shown diagrammatically. The CFTR amino acid
homologues appear in parentheses below the name of each
bacterial protein residue.
-phosphate linker and hence the position of the -helical
subdomain. We mutated the conserved Gln (Gln-493 in NBD1 and Gln-1291
in NBD2) that connects the -phosphate linker to the -phosphate of
ATP (Fig. 1). We hypothesized that these mutations should destabilize
the ATP-bound conformation of the NBD. They might alter the affinity of
ATP binding, thereby reducing channel activity. The mutations might
disrupt ATP hydrolysis, thereby altering durations of the open and
closed states. Or by interfering with the ability to "sense" the
ATP -phosphate, they might disrupt ATP-dependent
movement of the -phosphate linker and the -helical subdomain,
thereby uncoupling the structural consequences of ATP binding and hydrolysis.
-phosphate linker. In the ATP-bound state, this conserved Asn
(Asn-113 in HisP) connects via hydrogen bonds to the -phosphate
linker. However, with ADP bound, hydrogen bond contacts with the
-phosphate linker are disrupted, and hydrogen bonds form between a
conserved Arg (Arg-166 in LivG) and the side chain of the Asn (Asn-102
in LivG) (17). These putative changes in bonding may favor an ADP-bound
conformation in which the -phosphate linker is withdrawn from the
nucleotide binding site (Fig. 1). We hypothesized that mutation of this
conserved Asn would be more destabilizing to the ADP conformation and
therefore favor the ATP conformation. In this case, the Asn mutations
would be expected to reduce the rate of entry into the ADP conformation
and subsequent nucleotide dissociation.
-phosphate linker position in different ways. We introduced the
mutations at the indicated sites, expressed the variant CFTR in HeLa
cells, and examined CFTR channel activity in excised, inside-out
patches of membrane. Because the two NBDs have different functions in
CFTR gating (reviewed in Refs. 5 and 6), we first show the effect of
these mutations at NBD1 and then NBD2. Earlier models have suggested
that ATP binding and/or hydrolysis at NBD1 are important for initiating
channel activation and that ATP binding and hydrolysis at NBD2 are
important in the opening and closing in a normal gating cycle.
-phosphate linker required for channel activation, resulting in
a lower frequency of channel opening. It could also be explained by a
reduced rate of ATP hydrolysis (see "Discussion").
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Fig. 2.
Single-channel gating of wild type CFTR,
CFTR-Q493A, and CFTR-Q1291A. A, examples of current
from excised inside-out membrane patches containing single CFTR
channels in the presence of 1 mM ATP and 75 nM
PKA. Membrane potential was clamped at 80 mV. B, data from
multiple patches. Asterisk indicates p < 0.05; n = 7 for WT, n = 4 for
CFTR-Q493A, and n = 3 for CFTR-Q1291A.
-phosphate of the bound nucleotide through a water molecule (15,
23). This arrangement suggested that mutating Gln might impair ATP
binding. Because ATP binding may open the CFTR Clchannel
(13, 14), thereby reducing the interburst interval, we asked if the
prolonged interburst interval in Q493A was consistent with attenuated
binding. To test this, we examined the effect of varying the ATP
concentration. Wild type CFTR and CFTR-Q493A had the same apparent
EC50 for ATP (Fig. 3). These
data suggest that the conserved Gln is not critical for ATP
binding.
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Fig. 3.
Wild type CFTR, CFTR-Q493A, and CFTR-Q1291A
had similar dose response curves for ATP-stimulated Cl
current. CFTR was first phosphorylated with 75 nM PKA
in the presence of 1 mM ATP. Patches were then exposed to
varying concentrations of ATP at 40 mV, and the ATP-stimulated
current was measured. Data were normalized to the current measured with
1 mM ATP. The apparent EC50 was 176 ± 67 µM for wild type CFTR, 217 ± 55 µM
for CFTR-Q493A, and 159 ± 70 µM for CFTR-Q1291A.
n = 4- 5 for each data point.
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Fig. 4.
Wild type CFTR, CFTR-Q493A, and CFTR-Q1291A
showed similar cation requirements for Cl channel
activity. A, examples of current from patches
containing multiple CFTR channels. Membrane potential was clamped at
40 mV. Patches were incubated with 1 mM MgATP,
Na2ATP, and CaATP during the times indicated by
bars. Without divalent cations the solution contained 1 mM EDTA and 1 mM EGTA to eliminate excess
Mg2+ and Ca2+. B, data from multiple
patches. There was no difference between the three constructs in the
response to divalent cations.
current (9, 21, 25-27). These effects
appear to be mediated predominantly through an interaction at NBD2. The
Q493A mutation did not alter the response to either agent (Figs.
5 and
6).
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Fig. 5.
Wild type CFTR, CFTR-Q493A, and CFTR-Q1291A
were all inhibited by ADP. A, examples of patches
incubated with ATP (1 mM) and ADP (1 mM) during
the times indicated. B, data from multiple patches show that
wild type CFTR, CFTR-Q493A, and CFTR-Q1291A currents were inhibited by
ADP to a similar extent. n = 3.
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Fig. 6.
CFTR-Q1291A was stimulated only weakly by
PPi. A, examples of patches containing
multiple CFTR channels. The membrane potential was clamped at 40 mV.
Patches were incubated with 1 mM ATP and 4 mM
PPi during the times indicated. B, data from
multiple patches show that 4 mM PPi stimulated
less Cl current in CFTR-Q1291A (n = 6)
than in wild type CFTR (n = 4) or CFTR-Q493A
(n = 4). Asterisk indicates
p < 0.05 relative to wild type CFTR.
-phosphate
linker.
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Fig. 7.
CFTR-Asn-505 mutations did not change the
burst duration but altered the interburst interval. A,
examples of single-channel traces recorded in the presence of 1 mM ATP and PKA. B, data from multiple patches.
Asterisk indicates p < 0.05 compared with
wild type CFTR; n = 4 for CFTR-N505A and 6 for
CFTR-N505C.
-phosphate and makes no direct contacts with the
-phosphate of ADP (17-19). In CFTR, ADP inhibits channel activity,
at least in part by competitively inhibiting ATP binding (27-29). This
effect is abolished by some NBD2 mutations (21). The fact that the Gln
does not interact with ADP bound to the model bacterial NBDs, coupled
with our finding that the Gln mutations did not appear to inhibit ATP
binding, predicted that the Gln mutations would not alter
ADP-dependent inhibition of current. Fig. 5 shows that this
was the case.
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Fig. 8.
Comparison of single-channel kinetics of
CFTR-N1303K, CFTR-N1303H, CFTR-N1303I, and CFTR-N1303A.
A, examples of current from indicated variants in the
presence of 1 mM ATP and PKA. Membrane potential was 80
mV. B, data from multiple patches. Asterisks
indicate p < 0.05 compared with wild type CFTR;
n = 3-6 for each mutant.
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Fig. 9.
Pyrophosphate and ADP did not affect
CFTR-N1303K. Data are tracings in the presence of 4 mM
PPi or 1 mM ADP as indicated. 1 mM
ATP and PKA were present throughout. Addition of 4 mM
PPi increased the burst duration of wild type CFTR but did
not alter the burst duration of CFTR-N1303K. The addition of 1 mM ADP increased the interburst interval of CFTR but did
not affect CFTR-N1303K.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate linker are an important determinant of NBD function. By combining knowledge of the structure of related NBDs with measurements of CFTR gating, our results provide insight into the relationship between structure and function of CFTR and other ABC transporters in
the region of the -phosphate linker.
-phosphate linker with ATP is not critical for ATP binding. Whether
the Gln and Asn mutations disrupt hydrolysis by CFTR NBDs is not known.
We can, however, compare the gating of the Gln and Asn mutants with the
behavior of channels with Walker A lysine mutations, which are known to disrupt hydrolysis (7). The Asn-1303 mutations prolonged the burst
duration and increased the interburst interval, gating changes similar
to mutations of the NBD2 Walker A lysine (9, 10, 22, 35). The Gln-493
mutation increased the interburst interval without affecting the burst
duration, similar to the behavior of channels with mutation of the NBD1
Walker A lysine (9, 10, 22, 35). However, mutations of Asn-505 and
Gln-1291 were not consistent with the gating effects expected of
mutations that disrupt hydrolysis. Therefore, we surmise that the Gln
and Asn mutations probably have, at most, modest effects on hydrolysis.
-phosphate linker is probably not critical for either ATP
binding or hydrolysis.
-phosphate linker may
be a key event, or one of several key events, that couples ATP binding
and hydrolysis to gating of the channel. This mechanism has been
proposed for Pgp, for which the conserved Gln residue coupled ATP
binding and hydrolysis to transmembrane transport (37). In CFTR, the
requirement for Gln-1291 to mediate the effects of an activating
polyphosphate supports this hypothesis. We hypothesize that the
-phosphate of ATP contacts the side chain of Gln-1291 (possibly
through a water molecule), influencing the position of the
-phosphate linker and -helical subdomain, and thereby maintaining
the channel in an open state.
-phosphate linker in
NBD1 may be part of an "activating" mechanism that allows NBD2 to
play the predominant role in gating the channel (13). Although the
details of how conformational changes in the NBDs cause opening and
closing of the channel remain untested, the very recent crystal
structure of the Escherichia coli MsbA transporter indicates
that the NBDs are likely to transmit a conformational signal to the
membrane-spanning domains, and are unlikely to interact directly with
the channel pore (41).
-helical subdomain contributes to NBD-NBD interactions and hence the activity of
the transporter. Specifically, when ATP is bound, inward rotation of
the -helical subdomain may promote NBD-NBD interactions that activate the transporter. Such a model is also consistent with a
structural relationship between the transmembrane domain and NBD of
MsbA (41).
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ACKNOWLEDGEMENTS |
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We thank Pary Weber, Phil Karp, Tamara Nesselhauf, Theresa Mayhew, and Rosie Smith for excellent assistance and laboratory colleagues for helpful discussions.
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FOOTNOTES |
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* This work was supported by NHLBI, National Institutes of Health (NIH) Grant HL29851; the Cystic Fibrosis Foundation; the Howard Hughes Medical Institute; the Diabetes and Endocrine Research Center, Grant NIH DK25295; and the In Vitro Models Cell Culture Core, Grant NIH HL51670 and CFF.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Associate of the Howard Hughes Medical Institute.
** Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail: mjwelsh@blue.weeg.uiowa.edu.
Published, JBC Papers in Press, November 5, 2001, DOI 10.1074/jbc.M09539200
2 M. R. Carson, A. L. Berger, and M. J. Welsh, unpublished results.
3 L. Millen, J. F. Hunt, and P. J. Thomas, manuscript in preparation.
4 www.genet.sickkids.on.ca.
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ABBREVIATIONS |
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The abbreviations used are:
CFTR, cystic
fibrosis transmembrane conductance regulator;
ABC transporter, ATP-binding cassette transporter;
CF, cystic fibrosis;
NBD, nucleotide-binding domain;
Pgp, P-glycoprotein;
PKA, cAMP-dependent
protein kinase;
AMP-PNP, adenosine 5'-(-imino)triphosphate.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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