1 Center for Cell and Molecular Signaling, Departments of Physiology and Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322; and 2 Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201-3098
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ABSTRACT |
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A mutation in the fifth
transmembrane domain of the cystic fibrosis transmembrane conductance
regulator (CFTR) Cl channel (V317E) resulted in whole
cell currents that exhibited marked outward rectification on expression
in Xenopus oocytes. However, the single-channel unitary
current (i)-voltage (V) relationship failed to
account for the rectification of whole cell currents. In excised
patches containing one to a few channels, the time-averaged single-channel current (I)-V relationship
(I = N × Po × i, where N is
the number of active channels and Po is open
probability) of V317E CFTR displayed outward rectification, whereas
that of wild-type CFTR was linear, indicating that the
Po of V317E CFTR is voltage dependent. The
decrease in Po at negative potentials was due to
both a decreased burst duration and a decreased opening rate that could
not be ameliorated by a 10-fold increase in ATP concentration. This
behavior appears to reflect a true voltage dependence of the gating
process because the Po-V relationship did not depend on the direction of Cl
movement. The
results are consistent with the introduction, by a point mutation, of a
novel voltage-dependent gating mode that may provide a useful tool for
probing the portions of the protein that move in response to
ATP-dependent gating.
cystic fibrosis transmembrane conductance regulator; chloride channel; voltage dependence
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INTRODUCTION |
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THE CYSTIC FIBROSIS
transmembrane conductance regulator (CFTR) is a member of the
ATP-binding cassette family (24) and functions as a plasma
membrane Cl channel that is expressed in many epithelial
tissues (1, 3). Channel activity in CFTR is controlled by
protein kinase A (PKA)-dependent phosphorylation of its regulatory
domain; subsequent interactions of ATP with the nucleotide binding
domains (NBDs) are coupled to channel gating. Neither gating nor
conductance of single wild-type (WT) CFTR channels is thought to be
voltage dependent. Similarly, macroscopic currents of WT CFTR are time independent.
We studied the impact on channel function of a mutation (V317E) at a site in the extracellular half of the fifth transmembrane domain (TM5). Mutations in this region of TM5 alter the selectivity properties of macroscopic currents (25). Mutations at a roughly homologous position in TM11 (S1118) confer voltage-jump relaxations of macroscopic current, consistent with voltage-dependent gating (31). V317E CFTR macroscopic currents display marked rectification. In principle, this behavior could result from either a change in conduction or a change in gating, but the mutation revealed only a minor effect on single-channel conductance. Instead, we found that the macroscopic rectification of V317E CFTR was due to a voltage dependence of the open probability (Po) of single channels. Hence, these results have implications for understanding the link between ATP-dependent gating and the conformational changes that gate the pore and suggest that V317 and S1118 may be at a key site of conformational change that is associated with channel gating. Portions of these data have been presented in abstract form (25, 30).
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MATERIALS AND METHODS |
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Oocyte preparation and cRNA injection.
The methods used were similar to those described previously (19,
21). Briefly, stage V-VI oocytes from Xenopus were
prepared as previously described (22) and were incubated
at 18°C in a modified Leibovitz's L-15 medium with the addition of
HEPES (pH 7.5), gentamicin, penicillin, and streptomycin. cRNA was
prepared from a construct carrying the full coding region of CFTR in
the pALTER vector (Promega, Madison, WI) for WT CFTR or in pBluescript (Stratagene, La Jolla, CA) for V317E and V317Q CFTR. For most experiments, oocytes were injected with ~5 ng of CFTR cRNA plus 0.6 ng of cRNA for the human 2-adrenergic receptor, which
allows activation of PKA-regulated currents by the addition of
isoproterenol (Iso) to the bath. Recordings were made 42-96 h
after cRNA injection.
Mutagenesis. Site-directed mutations were engineered in one of two ways: 1) with a pSelect vector containing a 1.7-kb fragment of the amino end of CFTR (SacI-SphI) and the Altered Sites protocol (Promega) or 2) with a nested PCR strategy in which the mutation was designed into antiparallel oligomers that were first-round amplified with flanking oligomers (at AflII and SphI, respectively) and second-round amplified with the flanking oligomers alone. In both cases, the AflII-SphI fragment was subcloned back into a pBluescript vector (kindly provided by M. Drumm) containing the human WT CFTR from which the native AflII-SphI fragment had been separated. All mutations were confirmed by direct sequencing of the entire fragment before being subcloned.
Electrophysiology.
Standard two-electrode voltage-clamp (TEVC) techniques were used to
study whole cell currents. Borosilicate glass electrodes (Sutter
Instrument, Novato, CA) were pulled in four stages and filled with 3 M
KCl. Pipette resistances measured 0.4-0.9 M in the bath.
Macroscopic data were acquired at room temperature (~22°C) with a
GeneClamp 500 amplifier (Axon Instruments, Foster City, CA) and pCLAMP
software (Axon); macroscopic currents were filtered at 500 Hz and
acquired by the computer at 2 kHz. Bath solution (ND96) for most TEVC
experiments was nominally calcium free and contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES. The pH was adjusted to 7.5 with
NaOH. The oocytes were activated by superfusion of ND96 containing Iso
at a final concentration of 0.1-5.0 µM. To limit the activation
of endogenous calcium-activated Cl
channels, 1 mM
BaCl2 was added to all solutions used in the TEVC experiments. For characterization of permeation and rectification properties, macroscopic currents were elicited by stepping, for 75 ms,
from a holding potential of
30 mV to a series of test potentials
between
140 and +80 mV in +20-mV increments. Current-voltage plots
were constructed from initial data at each potential (average of the
first 5 ms) and from data at the end of the 75-ms pulse (average of the
final 10 ms) (31). Reversal potentials
(ER) were determined by linear regression around
the zero-current value. The macroscopic rectification ratio
(G+80/G
80) was determined by estimating the currents at ER +80
mV and ER
80 mV, using current-voltage
relationships provided by pCLAMP.
Kinetic analysis of gating.
Dwell-time analysis of open bursts was performed with Igor software.
Open bursts were defined as intervals separated by closings of 80 ms,
a value previously used in analysis of single-channel data in oocytes
(32) and established to discriminate between ATP-dependent
gating and intraburst blockade of CFTR in mammalian cells
(29). Some of the recordings used in the analysis of burst duration contained more than one simultaneous open level (up to three).
For the multiple-channel recordings, the mean burst duration was
estimated with the formula tn =
j · tj/n, where
tj is the time at which the j
channels were open at the same time and n is the total
number of transitions from an open burst to an interburst closed state.
Thus these multiple-channel open-burst events are transformed to
n single-channel open events with burst duration t
for each event. This method was first described by Fenwick and colleagues (7) and has been used in similar types of
analysis of muscarinic K+ channels (11) and,
more recently, CFTR (26, 32). Given the high degree of
filtering used for burst duration analysis, burst duration is
equivalent to open time in these experiments.
Variance analysis.
The mean time-averaged currents (I) and their variance
(2) were calculated from traces at least 15 s in
duration. The Po and the number of active
channels in the patch (N) were calculated from variance data
with the equations
2 = I × i(1
Po) and N = I/(i × Po), where
i is the unitary current. In our patches, we could
directly measure i and I; thus these equations
yield values for N and Po that are
independent of the number of open and closed states.
Statistics.
All reported values are presented as means ± SE unless otherwise
noted. Statistical analysis was performed with the t-test for paired or unpaired measurements (SigmaStat; Jandel Scientific, San
Rafael, CA) as appropriate for each set of experiments, with P 0.05 considered indicative of significance.
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RESULTS |
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Whole cell currents exhibit strong rectification.
Exposure of oocytes expressing CFTR and 2-adrenergic
receptors to Iso resulted in the development of Cl
current that peaked within 6-7 min. On reaching peak stimulation, oocytes were voltage clamped to a series of potentials (from
140 to
+80 mV) to study properties of rectification and the time dependence of
macroscopic currents. Figure 1 shows
families of whole cell currents from TEVC experiments in WT and V317E
CFTR (Fig. 1, A and B, respectively). In this
series of experiments, whole cell current-voltage (I-V)
relationships were determined by monitoring the current during a 75-ms
voltage pulse. Consistent with previous results under identical
conditions (32), WT CFTR exhibited
time-independent currents over most of the voltage range, aside from a
brief relaxation visible at the most hyperpolarizing potentials.
I-V relationships for WT CFTR currents (Fig. 1C)
were linear for outward currents but showed significant rectification
at strongly hyperpolarizing potentials. As in previous studies, the
degree of rectification of WT CFTR macroscopic currents varied between
oocytes. Both the rectification and brief relaxation of inward currents
may have been due to blockage of the pore by constituents of the oocyte cytoplasm (19, 32). In contrast, V317E CFTR currents
exhibited time-dependent behavior at all voltages (Fig. 1B);
inward currents relaxed toward zero, while outward currents increased
in conductance through time. This behavior is evident in Fig.
1D, which shows the discrepancy between initial currents
(average of the first 5 ms after the voltage jump) and currents
recorded after 75 ms (late currents, average of the last 10 ms of the
voltage jump). The initial I-V relationship exhibited linear
currents at depolarizing potentials. In contrast, the I-V
relationship constructed from currents at the end of the 75-ms voltage
pulse exhibited pronounced outward rectification across the entire
voltage range. The outward current amplitude did not approach a
constant value during the short voltage pulse, indicating that the
underlying time- and voltage-dependent process may be extremely slow.
V317E CFTR also exhibited modest tail currents (Fig. 1B) not
seen in WT CFTR with this voltage protocol.
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Characterization of unitary currents.
To identify the mechanism responsible for the pronounced outward
rectification of V317E CFTR whole cell currents, single-channel studies
were performed. Unitary currents (i) were determined from all-points histograms (data not shown) of multiple closed-to-open transitions at a particular potential. In agreement with previous studies that utilized excised inside-out patches (2, 4), we found the i-V relationship of WT CFTR to be linear,
yielding a single-channel conductance (g) of 7.26 ± 0.09 pS (n = 13 patches) when measured over the range
of membrane potential (Vm) of 100 to +100 mV
in symmetrical Cl
(Fig. 2,
A and B). Single-channel current (i)
amplitude for WT CFTR was 0.73 ± 0.01 pA at +100 mV and 0.75 ± 0.01 pA at
100 mV (P > 0.7). In contrast, the
i-V relationship for V317E CFTR was found to exhibit slight
voltage dependence, with g = 7.65 ± 0.14 pS at
negative potentials and g = 6.34 ± 0.14 pS at
positive potentials (n = 7 patches; Fig. 2,
C and D). i Amplitude for V317E CFTR
was 0.64 ± 0.02 pA at +100 mV and 0.77 ± 0.03 pA at
100 mV (P < 0.02). Thus i at positive
potentials for V317E CFTR exhibited only 87% of the conductance of WT
CFTR. Most importantly, rectification of i was directed
inward, opposite to that of the whole cell currents.
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Characterization of rundown. To compare the time-averaged, steady-state currents in patches containing either WT or V317E CFTR at multiple holding potentials, it was necessary to control for differences in channel number (N) between patches. We constructed I-V relationships by normalizing currents at various potentials to currents at a single potential. This required accounting for time-dependent rundown of channel activity to ensure that all differences in I between WT and the mutant CFTR were due to voltage-dependent, not time-dependent, phenomena.
After excision of a patch from an oocyte expressing WT CFTR, the addition of ATP in the absence of PKA resulted in minimal channel activity, suggesting low phosphorylation (data not shown). Application of PKA (50 U/ml) and ATP (1 mM) resulted in an increase in I that reached a steady state of 15.20 ± 2.17 pA in the patch shown in Fig. 3A. With variance analysis, the Po and N values were estimated to be 0.59 and 34, respectively, for the record shown. Washout of ATP resulted in a dramatic reduction in channel activity that was apparently due to a marked reduction in the opening rate (Fig. 3B). On readdition of 1 mM ATP in the continuing presence of PKA, the steady-state I was 7.59 ± 2.01 pA (Fig. 3C), indicating rundown approaching 50%. Variance analysis of this current yielded Po and N values of 0.29 and 35, consistent with the decrease in current being due to a decline in Po rather than a change in N. The effect of a second washout of ATP was very similar to that of the first (Fig. 3D). A third application of 1 mM ATP (with PKA) resulted in a steady-state I of 7.72 ± 2.04 pA (Fig. 3E). Variance analysis of this current yielded a Po of 0.30 and an N of 34. These results suggest that in our system and for a given patch 1) the initial decline in CFTR steady-state activity observed in the presence of 1 mM ATP (Fig. 3, A vs. C) was due to a decline in the Po and not in N, and 2) after PKA application, phosphorylation levels were fairly constant within a given patch over several minutes after the initial rundown of activity. To ensure consistent phosphorylation levels in the experiments described in Steady-state I-V relationships, 50 U/ml of PKA were present in all excised-patch experiments. Furthermore, all recordings were performed after the initial rundown in activity. This is important because alteration in phosphorylation levels can affect CFTR gating (5, 12, 17, 23, 27). To account for differences in phosphorylation levels between patches, data at multiple potentials in each patch were referenced to data at
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Steady-state I-V relationships.
Computed steady-state time-averaged current (I) values were
compared in excised inside-out patches containing multiple WT or mutant
channels. WT CFTR exhibited a linear I-V relationship, with
a ER of ~0 mV in the presence of symmetrical
Cl (Fig. 4A).
Observation of WT CFTR channel activity at both positive and negative
potentials revealed currents of similar magnitude in a given patch
(Fig. 4B). However, this was not the case for V317E CFTR.
The I-V relationship for this mutant was markedly rectified
in the outward direction (Fig. 4C) due to the reduced Po at negative membrane potentials (Fig.
4D). Outward currents exhibited a linear I-V
relationship. Hence the I-V relationship from single
channels recapitulated the rectification seen in the true macroscopic
I-V relationship from whole cell recording (Fig. 1D).
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Mechanism of the voltage dependence of V317E CFTR.
Comparison of V317E and WT CFTR revealed markedly different
single-channel kinetics (Fig.
5A). Despite the fact that WT
CFTR macroscopic currents showed no voltage dependence, we found that WT CFTR single channels exhibited significantly longer burst durations at negative membrane potentials than at positive membrane potentials (P < 0.05). We suspect that this prolongation of the
open-burst duration at negative potentials was due to the
voltage-dependent intraburst flicker (not evident in Figs. 2, 4, and 5
because of filtering) that has been previously reported (8, 9,
17, 19, 28) and likely reflects blockage of the pore by the pH buffer TES in our intracellular solutions (13). If this
intraburst flickery behavior is truly indicative of a blocking
substrate occluding the pore, then prolongation of the burst duration
is to be expected according to the concepts of classic open-channel block (see Ref. 6). However, because the individual dwell
time for each blocking event is quite brief (typically <0.25 ms at 100 mV) (19), no significant difference was observed in
the magnitude of time-averaged currents at positive and negative
potentials (Fig. 4A). This result is not considered further
here. For V317E CFTR, burst durations were significantly reduced
compared with WT CFTR over the entire voltage range (Fig.
5B). However, the difference between V317E and WT CFTR burst
durations cannot explain the voltage dependence of
Po observed in the mutant, because the burst
durations of V317E CFTR at negative potentials were not significantly
shorter than the burst durations at positive potentials.
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Characterization of i and I in asymmetrical Cl.
To determine whether the voltage dependence of
Po in V317E CFTR was influenced by an anion
gradient, we repeated our experiments using a low-Cl
(15 mM) solution in the pipette. Comparing WT CFTR channel behavior at +100
and
100 mV (Fig. 7A,
top and bottom, respectively), one can see a
reduced i at +100 mV, due to the decreased driving force for
inward Cl
movement, but no drastic difference in the
overall Po. Examination of the i-V
for WT CFTR revealed a 50-mV rightward shift in the ER (59.8 mV after correction for junction
potential) in the presence of the low-Cl
pipette solution
(Fig. 7B). As expected for recordings with low Cl
concentration ([Cl
]) in the pipette,
the time-averaged I-V for WT CFTR in our asymmetrical Cl
solutions exhibited slight inward rectification.
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NPo vs. voltage relationship.
We determined the product of N and Po
(NPo) in all of our patches, and because both
N and Po varied greatly from patch to patch (but were consistent within a patch after allowing for rundown), we normalized these values to the NPo at 100
mV in each patch. In symmetrical Cl
there is an obvious
voltage dependence in NPo for V317E CFTR (Fig.
8A) that was most pronounced
at Vm values more positive than 0 mV,
where net Cl
movement is inward. To determine whether
this voltage-dependent increase in NPo could be
due to some form of open-channel block at negative
Vm, we also performed this analysis on
recordings obtained with low-[Cl
] pipette solutions. By
shifting the ER for Cl
flow ~50
mV to the right, we dissociated Vm from the
direction of anion flow between 0 and approximately +50 mV. With
symmetrical Cl
(Fig. 8A), currents measured
between Vm = 0 and +50 mV reflected anion
entry from the pipette. With reduced bath Cl
(Fig.
8B), currents in the same voltage range reflected anion exit
into the pipette. As shown in Fig. 8B, the increase in
NPo was still observed at positive potentials
(Vm = +10-40 mV), where net
Cl
movement is in the same direction (outward) as
Cl
movement at
100 mV. This insensitivity (to the
direction of ion movement) of the voltage dependence of
NPo for V317E CFTR argues strongly against
voltage-dependent blockade of the pore.
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Voltage-jump relaxations indicate behavior similar to voltage-gated
channels.
The single-channel experiments indicated that the rectification of
macroscopic V317E CFTR currents was due to voltage-dependent kinetics
of opening and closing. However, the change in macroscopic rectification and the slow relaxations during a voltage step, as shown
in Fig. 1, indicate that the voltage-dependent alteration of
Po is a time-dependent process as it is in
voltage-gated channels of excitable cells. Because our single-channel
recordings were made several seconds after the membrane voltage was
changed, they do not reflect the time dependence of this gating
process. To determine whether the process evident in V317E CFTR was
similar to voltage-dependent gating mechanisms observed in other
channels, we asked whether the kinetics of the relaxations at
hyperpolarizing potentials were sensitive to Vm.
Oocytes were held at 30 mV, pulsed to +50 mV for 100 ms, and then
stepped for 200 ms to potentials ranging from
120 to
60 mV (data
not shown). Relaxations of inward currents were fitted with a
second-order exponential in V317E CFTR, whereas only a first-order
exponential was required for WT CFTR currents. Time constants for these
relaxations in V317E CFTR were strongly dependent on voltage (values
for the longer component,
2, were 157.0 ± 10.7 ms
at
120 mV and 294.2 ± 18.2 ms at
60 mV; n = 5 patches; P < 0.001 by paired t-test). The kinetic behavior of V317Q-CFTR macroscopic currents did not differ from
WT CFTR (data not shown).
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DISCUSSION |
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Channel gating in WT CFTR is traditionally thought to be voltage
independent. The results presented here demonstrate that it is possible
to induce significant voltage dependence in the gating mechanism of the
Cl channel by introducing negative charge at one position
in TM5. The voltage-dependent gating of V317E CFTR was evident both in relaxations of macroscopic currents and in the altered gating of single
channels. Thus voltage-dependent gating is the mechanism underlying the
strong outward rectification of macroscopic currents carried by this channel.
The induction of voltage-dependent gating by substituting a charged residue, but not by substituting a neutral residue, suggests that this site resides in a portion of the protein that is conformationally mobile such that it can move in response to a change in the membrane electric field. The data presented are not sufficient to reveal whether this apparent movement is related to the normal gating cycle of the protein or whether, instead, it reflects a novel motion that would only be seen in this altered protein molecule. In either case, however, the results suggest that it may be possible to use the introduction of voltage-dependent gating as a tool to probe the mobility of segments in the protein and, perhaps, to identify those parts that move in response to events initiated by the hydrolysis of ATP. In the latter case, one might expect a strong interaction between ATP-dependent and voltage-dependent gating events such that, for example, the sensitivity of the channel to activating conditions (PKA + ATP) would become voltage dependent.
Other glutamate substitutions in TM domains 1, 5, 6, and 12 have been investigated [G91E, G314E, and K335E (16); S341E and T1134E (20)], but none of these exhibited voltage-dependent gating (McCarty and Dawson, unpublished observations). Although these mutants have not yet been studied at the single-channel level, no voltage-jump relaxations indicative of voltage-dependent gating were apparent in macroscopic recordings. Hence this phenomenon is thus far specific to the introduction of glutamate at position 317 in TM5. Introduction of a similarly sized but uncharged amino acid side chain at this position (V317Q CFTR) did not affect gating, indicating that it is the charge, not the bulkiness of the side chain, that confers the voltage-dependent behavior.
The voltage dependence of the whole cell I-V relationship is almost purely a gating effect, aside from a minor change in single-channel conductance at positive potentials. The small change in single-channel conductance was found for both V317E and V317Q-CFTR, indicating that it does not result from electrostatic repulsion due to introduction of the negative charge. Hence, this residue apparently has little or no impact on ion permeation, indicating that it does not lie in the permeation pathway. Rather, introduction of a bulky amino acid (glutamic acid or glutamine) at this position may induce rectification in single-channel conductance by altering protein conformation.
The voltage dependence induced by the V317E mutation impacts the CFTR gating scheme at a point distal to the binding and hydrolysis of ATP at the NBDs, as evidenced by the insensitivity of the opening rate to increased [ATP]. Alternatively, it is possible that the mutation resulted in a conformational change that altered exposure of a phosphorylation site in a voltage-dependent manner. However, the kinetic differences were observed immediately upon switching between positive and negative potentials and were observed to be immediately reversible, making it unlikely that rapid changes in phosphorylation had occurred. It also seems unlikely that a mutation in TM5 would alter the structure of the regulatory domain, which includes most of the sites of PKA-dependent phosphorylation in the CFTR. Hence, it appears that the conformational change that is induced by ATP binding and hydrolysis at the NBDs is inhibited at negative Vm in this mutant. It has recently been suggested that the pore of CFTR is not conformationally static in that permeation properties depend on gating status (13, 15, 18, 31). Recent work by Kogan and colleagues (14) shows that inhibitors of permeation, such as diphenylamine-2-carboxylate, affect the rate of ATP hydrolysis, suggesting that bidirectional communication between the pore domain and cytosolic gating domains occurs. It is thus possible that the pore of the WT channel undergoes a conformational change at or in the vicinity of position 317, and we have introduced voltage dependence to this process by introduction of a charged amino acid at position 317. To date, the region that experiences this conformational change (i.e., the gate) has not been identified in CFTR. The ability to confer voltage dependence to the gating mechanism, as seen with the V317E mutation, may provide a means to localize the structures that comprise the gate.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. C. Hwang and Dr. C. Hartzell for insightful and helpful comments.
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FOOTNOTES |
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* Z.-R. Zhang and S. Zeltwanger contributed equally to this work.
This work was supported by American Heart Association Grant 9820032SE and by National Science Foundation Grant MCB-0077575. S. Zeltwanger was supported by National Institute of Diabetes and Digestive and Kidney Diseases 5T32-DK-07656 and by Cystic Fibrosis Foundation Fellowship ZELTWA00F0.
Address for reprint requests and other correspondence: N. A. McCarty, School of Biology, Georgia Inst. of Technology, 310 Ferst Dr., Atlanta, GA 30332 (E-mail: nael.mccarty{at}biology.gatech.edu).
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.
Received 22 February 2001; accepted in final form 24 August 2001.
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