Address correspondence to Cecilia M. Canessa, 333 Cedar Street, New Haven, CT 06520-8026. Fax: (203) 785-4951; E-mail: cecilia.canessa{at}yale.edu
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ABSTRACT |
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Key Words: ASIC calcium pH protons kinetics
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INTRODUCTION |
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ASIC1, -2, and -3 are gated by external protons whereas ASIC4 is not (Gründer et al., 2000). Gating by protons and expression in DRG have prompted the proposal that ASIC1 and ASIC3 may be involved in acid sensing (Waldmann and Lazdunski, 1998
; Sutherland et al., 2001
) and that they may constitute a class of nociceptors distinct from the vallinoid receptor (Caterina et al., 1997
). The role of the ASICs in the central nervous system has not been satisfactorily explained, although recently it was shown that mice with inactivation of the ASIC1 gene had a mild deficit in spatial memory and impaired eyeblink conditioning (Wemmie et al., 2002
). So far, a search for other potential agonists of the ASICs has revealed that the neuropeptide FMRFamide slows slightly the inactivation rate of ASIC1
(KD 33 µM) (Askwith et al., 2000
). Divalent cations such as Zn2+ were found to shift the pH dependence of heteromeric ASIC1
/ASIC2a channels, but not of homomeric ASIC1
(Baron et al., 2001
).
The purpose of this work is to characterize the unitary currents from homomeric ASICs and to identify properties that might be used to distinguish these channels in neurons from the peripheral and central nervous systems. This is an important issue because the main criterion for assigning a specific function to the ASICs has been localization in a particular subset of DRG neurons (nociceptor and/or mechanoreceptor). However, sensory neurons express more than one type of ASIC and the different ASICs can associate to form various types of heteromeric channels. At least in vitro, it has been shown that ASIC1 with ASIC2 (Waldmann and Lazdunski, 1998) and ASIC2 with ASIC3 (Babinski et al., 2000
; Zhang and Canessa, 2001
) form functional heteromeric channels. Moreover, most combinations of ASICs, independent of their putative function, mechanoreceptor versus nociceptor, are activated by low pHo. Together, these findings further indicate that measurements of whole-cell currents are insufficient to unequivocally distinguish the ASICs that generate proton-gated currents in neurons. To complicate the issue even more, species-specific differences have been found in the properties of the ASICs between rat, mouse, and human (Babinski et al., 2000
; Benson et al., 2002
).
Here we have used Xenopus oocytes injected with cRNAs from rat ASIC1, ASIC2a, or ASIC3 to examine the properties of the unitary and macroscopic currents from these three homomeric channels using the patch clamp technique in the outside-out configuration and whole-cell currents with the two-electrode voltage clamp.
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MATERIALS AND METHODS |
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Single-channel Recordings
Unitary currents were recorded using the outside-out configuration of the patch-clamp technique. Channels were activated by rapidly moving squared-glass tubes (ID 0.7 mm) delivering solutions of desired pHos in front of the tip of the patch pipette. The delivery device achieves complete solution changes within 20 ms (SF-77B, Perfusion Fast-Step; Warner Instrument Corp.). Pipettes were pulled from borosilicate glass (LG16; Dagan Corporation) using a micropipette puller (PP-83, Narishige; Scientific Instrument Lab) and fire polished to a final tip diameter of 1 µm. When pipettes were filled with solutions they had resistances of 510 M. Single-channel currents were recorded with an Axopatch-200B amplifier (Axon Instruments, Inc.) using a DigiData 1200 series interface and pClamp 8.0 software both from Axon Instruments, Inc. The data were collected at 10 kHz, filtered at 1 kHz, and stored on a computer for analysis. For display, data were filtered with a digital Gaussian filter to 0.5 kHz.
All experiments were performed at room temperature. The composition of the solutions is given in mM. Incubation solution for oocytes: 96 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, adjusted to pH 7.4. Hyperosmolar solution to remove vitelline membrane: 220N-methyl-D-glucamine, 220 aspartic acid, 2 MgCl2, 10 EGTA, 10 HEPES, adjusted to pH 7.4 with KOH. Solution in the recording chamber: 150 NaCl, 1 CaCl2, 10 MES-TRIS, adjusted to pH 7.4. For activation of ASICs, outside-out patches were perfused with solutions of composition identical to the extracellular bath but buffered to a lower pH, either 6.0, 5.0, or 4.0 or as indicated in the experiment. To examine the effects of Ca2+, it was added only to the perfusion solutions to the desired concentrations. Pipette solution: 150 NaCl, 5 EDTA, HEPES, adjusted to pH 7.4 with KOH.
Whole-cell Recordings
For two-microelectrode recordings (TEVC), current and voltage electrodes were pulled from borosilicate glass and filled with 3 M KCl. The resistance was kept in the range of 0.5 to 1 M. Currents were recorded with an Oocyte Clamp OC-725B (Warner Instrument Corp.), digitized at sampling rate of 100 Hz (ITC-16; HEKA) and the data stored in a computer. The oocyte chamber (volume 400 µl) was continuously perfused at a rate of 12 ml/min. Composition of the standard bath solution (mM): 150 NaCl, 1 KCl, 1 CaCl2, 10 HEPES/MES adjusted to pH 7.4 or to 6.0, 5.0, or 4.0. To examine the apparent Ca2+ inhibition constant or EC50 for activation, the external solution contained variable CaCl2 (0, 0.1, 0.3, 1, 5, 20, 40, and 75). Each of the Ca2+ solutions was buffered at pH 7.4 and 5.0.
Data Analysis
Only patches containing a single channel were included in the analyses of the amplitude of subconductance states and of the modes of activity. All points amplitude histograms were binned into equally spaced bins and fitted with the sum of several Gaussian components using the maximal likelihood method of least-squares minimization. Lists of the duration of single-channel events were generated using the half-amplitude threshold criterion. Histograms of the distribution of open and closed dwell times were plotted as the logarithm of the dwell-time duration, and fitted with the sum of two exponential components.
Rates of desensitization were calculated by fitting the sum of unitary currents obtained from at least 1214 independent patches to Eq. 1:
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Normalized peak whole-cell currents elicited by changes in pHo from 7.4 to 5.0 were used to calculate the apparent Ca2+ EC50 according to Eq. 2:
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Apparent inhibition constant of Ca2+ was calculated with the following equation:
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The results are presented as mean ± SD of n independent experiments. For each experiment the n is given in the figure legend.
Online Supplemental Material
Additional representative examples of outside-out patches expressing ASIC1, ASIC2a, or ASIC3 are presented in this section to illustrate the presence and characteristics of subconductance states and Modes of activity of the ASICs. ASIC1
#1,2,3 and ASIC1
#4,5,6,7; ASIC2a#1,2,3,4,5 and ASIC2a#6,7,8,9,10; and ASIC3#1,2,3,4,5,6 show examples of outside-out patches from oocytes examined in 150 mM symmetrical Na+ and -60 mV membrane potential. Channels were activated by lowering the pHo as indicated by the bar above the traces. Modes of activity are indicated by bars under the traces and sublevels are indicated by arrows. Online supplemental figures are available at http://www.jgp.org/cgi/content/full/jgp.20028574/DC1.
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RESULTS |
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The amplitudes of the unitary currents of the three types of ASIC channels shown in Fig. 1 A are similar. To determine the conductance of these channels we generated I-V curves from unitary currents in the presence of symmetrical 150 mm Na+. The I-V curves from the three channels are linear and almost overlapping (Fig. 2). Cord conductances calculated from -100 mV to -20 mV are 23, 22.5, and 18.5 pS for ASIC1, ASIC2a, and ASIC3, respectively.
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Activation and Desensitization of the ASICs by External Protons
All three ASICs are activated by external protons, but the EC50 varies according to the composition of the channel. Fig. 4 shows the responses of ASIC1, -2a, and -3 to increasing concentrations of external protons measured with TEVC. The protocol shown for ASIC1
consists of exposure to a low pHo test solution followed by return to pHo 7.4 for 25 s after each test solution to allow complete recovery of the channels from desensitization. The apparent pH50 was calculated by measuring peak currents obtained at each pHo and fitting the normalized data to the value obtained at pHo 4.0 with Eq. 1 (insets in Fig. 4). The calculated pH50 for ASIC1
was 5.9 ± 0.3. In the lower trace from Fig. 4 A, the oocyte was exposed sequentially to pHo 6.0 and 5.0. Under this condition, ASIC1
fails to activate, indicating that the channels are refractory to activation by protons.
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Peak currents of ASIC2a at each pHo were normalized to the value obtained at pHo 4.0 to calculate the apparent pH50, 5.0 ± 0.12, mean ± SD of five independent oocytes.
Fig. 4 C shows whole-cell current traces from an oocyte expressing ASIC3. These channels inactivate rapidly and completely in the continual exposure to protons, and they are sensitive to the preconditioning pHo as indicated by the second part of the experiments, after the dotted line. The calculated apparent pH50 value for ASIC3 was 5.4 ± 0.13, mean ± SD of six independent oocytes.
The rates of desensitization of the three types of ASIC channels were calculated by adding the unitary currents of 1214 patches and fitting the resulting curves to a single exponential. The solid lines on the curves in Fig. 5 represent the fit of the data with calculated values for the desensitization rates of 2.0, 0.045, and 4.5 s-1 for ASIC1, ASIC2a, and ASIC3, respectively. For each type of channel we show three of the patches included in the calculations. Notice the different time scale for ASIC2a, 1,000 ms versus 200 ms. In the three examples, ASIC2a channels remain open for the 5-s period shown in Fig. 5, whereas ASIC1
and ASIC3 channels desensitize much faster. These desensitization rates were obtained at pHo 5.0; lower pHo results in faster desensitization.
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Two kinetic modes were readily identified in ASIC1 based on Po, frequency, and duration of closures and the presence of subconductance states. Fig. 6 A shows traces from three independent single channels activated by pHo 5.0 that illustrate the modes of activity. In the top and bottom examples, immediately after lowering the pHo, channels open to a high Po mode rarely interrupted by complete closures (Mode 1). Closures in Mode 1 are partial, they correspond mainly to level S3, and less frequently to S1 and S2. The average Po of Mode 1 is high, 0.92 ± 0.07. In these two examples the channels suddenly changed kinetics to a second mode (Mode 2) that has a flickering appearance due to the presence of frequent and short closures. The frequent closures give to Mode 2 a lower Po, 0.47 ± 0.08, than Mode 1. Subconductance states are also seen in Mode 2, primarily S1 and S2, that are prominent at the end of the burst of activity as indicated in the examples. The middle trace shows a channel that opens and remains in Mode 2 during the whole period of activity.
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Although not all activity from ASIC1 channel could be classified according to the modes, at least one mode was identified in most of the ASIC1
records. Out of 40 single channel patches examined for modes, three patches exhibited only Mode 1 and two patches only Mode 2. The large majority of channels had mixtures of more than a single mode (35/40). The most frequent sequence was a progression from Mode 1 to Mode 2 as indicated by the examples of Fig. 5 A, whereas we reverse sequence was not observed.
ASIC2a also exhibits sudden changes in kinetics that can be classified according to Po and kinetics into Mode 1 and Mode 2. Fig. 6 C shows three examples of ASIC2a channels activated by pHo 5.0 with bars underneath the traces indicating the modes of activity (Mode 1 and Mode 2). Activity in Mode 1 has a Po of 0.74 ± 0.12 and long open events, whereas Mode 2 has much lower Po, 0.073 ± 0.025, short opening and long closures. Dwell time histograms constructed from segments displaying high or low Po revealed the presence of two opened and two closed states in each of the modes (Fig. 6 D). In Mode 1, one of the open states exhibits long duration, 58.07 ms (0.517), whereas the other open state is much shorter, 3.86 ms (0.483). The mean duration of the closed states in Mode 1 are 17.95 ms (0.25) and 0.99 ms (0.75). The open states in Mode 2 are short with mean duration of 6.32 ms (0.124) and 1.45 ms (0.876). The closed states in Mode 2 are 159 ms (0.432) and 1.2 ms (56.8).
In contrast to ASIC1, which first opens to a high Po mode, ASIC2a switches from high to low Po modes throughout the period of activity.
Single channel kinetics of ASIC3 are different from ASIC1 and ASIC2a primarily because the bursts of activity induced by low pHo are shorter and the openings are very rarely interrupted by closures. These properties are illustrated in the examples from Fig. 1 A.
Requirement of External Ca2+ for Activation of the ASICs
Previous publications have reported stimulatory and inhibitory effects of external Ca2+ on the activity of the ASICs. For instance, de Weilli reported increase in ASIC1 activity with concentrations of Ca2+ up to 5 mM and inhibition at higher concentrations (de Weille and Bassilana, 2001
). In contrast, Waldmann indicated that external Ca2+ was a blocker (KD
1.3 mM), and Berdiev and colleagues, in studies performed with ASIC1
reconstituted in lipid bilayers, reported that µM concentrations of external Ca2+ inhibited ASIC1
, an effect that was released by protons (Berdiev et al., 2001
).
We first examined the requirement of external Ca2+ for proton-gating of the ASICs measuring whole-cell currents of oocytes expressing each of the homomeric ASIC channels with the TEVC. The external solution contained 150 mM Na+ and either 1 or 0 mM Ca2+. Oocytes were voltage clamped at -60 mM and channels were activated by a rapid change of the pHo in the bath from 7.4 to 5.0. Fig. 7 shows activation of ASIC1 and ASIC2a with 0 mM Ca2+ solutions but not of ASIC3, which required external Ca2+ with an apparent KD of 0.28 mM (inset Fig. 6 C).
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Inhibitory Effects of Ca2+ on ASIC1
The previous experiments indicate that external Ca2+ inhibits ASIC1 by decreasing the amplitude of the unitary currents. However, the magnitude of the block is not enough to account for a KD of
1.3 mM (Waldmann et al., 1997a
), suggesting that other mechanisms may participate in the Ca2+-induced inhibition of ASIC1
. To further investigate this issue we examined the effect of Ca2+ on the kinetics of single channels. The top trace of Fig. 10 A shows a patch containing three channels (O1, O2, and O3) activated by pHo 5.0 in the presence of 0 mM. The bottom trace is from the same patch activated in the presence of 10 mM external Ca2+. Besides reducing the conductance, Ca2+ also affects the kinetics of ASIC1
. In this example, three channels open in 0 mM Ca2+, whereas only one channel opens in 10 mM Ca2+ and it remains active for a shorter time. Although ASIC1
exhibits usage-dependent inactivation, the effect shown in Fig. 10 A was not due to this phenomenon because performing the experiment in the opposite order, 10 mM Ca2+ first followed by 0 mM Ca2+, produced the same result. The number of activated channels were reduced when the concentration of Ca2+ was progressively increased, from a mean of 2.6 ± 0.4 in 0 mM Ca2+ to 2.2 ± 0.3 and 1.9 ± 0.3 in 5 and 10 mM Ca2+, respectively.
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To evaluate the overall effect of the various inhibitory mechanisms of Ca2+ on ASIC1 activity, we measured the apparent KD of whole-cell currents with the TEVC. The external solution contained 150 mM Na+ and increasing concentrations of Ca2+. For each Ca2+ concentration, oocytes were preconditioned at pHo 7.4 and activated by identical solutions buffered at pHo 5.0. Peak currents were normalized to the value obtained with 0 mM Ca2+. The line in Fig. 10 C represents the fit of the data with Eq. 2 and KD value of 9.8 ± 2 mM. For comparison with the original report (Waldmann et al., 1997a
) the plot is drawn in a linear scale. Even at very high Ca2+ concentrations the currents are not completely blocked.
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DISCUSSION |
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Subconductance States of the ASICs
Here, we report and characterize the presence of subconductance states in homomeric ASIC1 and ASIC2a channels but not in ASIC3. ASIC1
exhibits three subconductance states (S1, S2, and S3) and a fully open state, whereas ASIC2a exhibits only one sublevel (S1). The frequency of sublevels was higher in ASIC1a than in ASIC2a, thus making this property a useful feature to distinguish ASIC1
channels.
It is possible that a larger number of sublevels exist, but we could not resolve them with the resolution of our system. Evidence supporting the interpretation that the sublevels represent partially open states and not independent channels is that the smaller conductances were never seen superimposed on the fully open state in <80 single channel patches examined in this work. An alternative explanation for the sublevels is that they represent partial blocking events induced by Ca2+ or Mg2+. This possibility is unlikely because the sublevels remained when divalent cations were removed and EDTA was added to the perfusate. An additional argument against the blocking mechanism was the absence of voltage dependence in the frequency or duration of the sublevels.
Sublevels were present through the burst of activity, including the initial opening and final closure; therefore, we do not believe they represent intermediate steps in the activation or inactivation processes (Zheng and Sigworth, 1998). It is tempting to postulate that the sublevels in ASIC1
reflect a conformational state from each of the subunits reflecting a tetrameric structure. The notion is consistent with the proposed stoichiometry for other members of the ENaC/DEG family (Coscoy et al., 1998
; Firsov et al., 1998
).
Single Channel Kinetics of the ASICs
Inspection of single channel records revealed marked differences in kinetics among the ASICs. The most significant and readily recognizable differences were the rate of desensitization and the overall appearance of the kinetics of single channels. The rates of desensitization were faster in the order ASIC3>ASIC1>> ASIC2a. Most significant, the rate of desensitization of ASIC2a was not only slower but also incomplete, leaving active channels on the continual exposure to low pHo. In contrast, protons rapidly and completely desensitized ASIC1
and ASIC3, making the patches quiescent a few seconds after the change to low pHo.
Openings of ASIC1 and ASIC2a were frequently interrupted by closures, and ASIC1
and ASIC2a exhibit sudden changes in kinetics given rise to modes of activity with different Po and kinetics. According to these criteria, two modes of activity were identified: Mode 1 and Mode 2. We do not know the mechanisms underlying the modes of activity; although, they likely reflect entering of channels into a particular subset of states from the overall kinetic scheme. The progressive decrease in Po and the faster kinetics from Mode 1 to Mode 2 observed in ASIC1
suggest that the modes may be linked to the desensitization process of this channel. However, the same interpretation cannot be applied to ASIC2a because this channel exhibits sudden shifts from Mode 1 to Mode 2 and back to Mode 1 throughout the period of activity.
External Ca2+ Elicits Multiple Effects on ASICs
Elucidation of the effects of external Ca2+ is paramount to understand the consequences of ASIC activation in the nervous system. In particular, it is important to determine Ca2+ permeability because of the roles of Ca2+ on triggering cell death, synaptic plasticity, and long-term potentiation. The first publication after the cloning of ASIC1 reported that the channel was highly permeable to Ca2+ (Waldmann et al., 1997a
). Later publications have not been able to confirm this result (Bässler et al., 2001
; de Weille and Bassilana, 2001
). Since Waldmann also reported a high degree of block of Na+ currents by Ca2+ (KD 1.8 mM), we sought to investigate the possibility of Na+ and Ca2+ interaction in the channel pore as an explanation to these two apparently contradictory results. However, we did not find evidence to support ion interaction in the pore, but our findings instead indicated that Ca2+ permeability is very small. Moreover, addition of 10 mM Ca2+ in the bath did not change the reversal potential of unitary ASIC1
currents. On the other hand, we found that Ca2+ inhibits ASIC1
by several mechanisms: reduction in the amplitude of unitary currents, decrease in the number of proton-gated channels, and increase in the desensitization rate. In spite of all these effects that together suppress ASIC1
activity, the KD for inhibition of macroscopic currents was found to be 9.2 ± 2 mM, a value that is too high for considering Ca2+ as a physiological modulator of ASIC1
. In this regard, our result is consistent with a report by de Weille and Bassilana who found 50% inhibition with 12 mM Ca2+ (de Weille and Bassilana, 2001
).
On the other hand, Berdiev had suggested that activation of ASIC1 by protons is mediated by the release of Ca2+ inhibition that normally occurs at pHo 7.4 (Berdiev et al., 2001
). Our results from experiments performed with EDTA in the external solution did not confirm this mechanism; although the differences in results may be attributed to the different experimental systems: patches from the plasma membrane of X. oocytes injected with cRNAs versus extraction and reconstitution of ASIC1
in lipid bilayers.
The effects of external Ca2+, however, are not equal in the three types of ASIC channels. We found that ASIC3 requires small concentrations of external Ca2+ to be gated by protons. Whether Ca2+ is important in the physiological modulation of ASIC3 activity is an open question. The range of Ca2+ concentrations (EC50 0.28 mM) that affect the activation of this channel is low in comparison to the free Ca2+ concentration in plasma or the cerebrospinal fluid (1.8 mM). It is conceivable that local acidosis and chelation of Ca2+ by lactate may decrease the concentration of free Ca2+ sufficiently to differentially modulate ASIC3 activity and not of ASIC1 or ASIC2a. These results are different from the ones reported by Immke and McCleskey (2001)
, who found that lactate increases ASIC3 currents by a mechanism consistent with chelation of divalent ions, Ca2+ and/or Mg2+. However, the currents they showed as belonging to ASIC3 did not conform to the rapid desensitization and the low apparent pH50 characteristic of the rat ASIC3 channel. It is possible that in neurons from DRG other ASIC currents, perhaps from herteromeric channels, were responsible for the lactate response.
How to Distinguish the ASICs
Northern blot analysis and in situ hybridization studies have demonstrated the presence of mRNA from several of the ASICs in the neurons from the nervous system, indicating that many cells express more than one type of ASIC. It is therefore desirable to distinguish the various proton-gated currents arisen from these proteins. Our analysis of the single channel properties of homomeric ASIC1, ASIC2a, and ASIC3 provides some functional criteria to differentiate these channels.
ASIC1 and ASIC3 share several properties, mainly a fast and complete desensitization after exposure to external protons. These channels are therefore refractory to stimulation by a new pulse of low pHo. Although the desensitization rate is faster in ASIC3 than in ASIC1
, the difference is not large enough to readily distinguish the two populations of channels expressed in the same cell. However, these channels can be distinguished by measuring proton-gated currents in the presence or absence of small concentrations of Ca2+ in the activating solution (
1 mM Ca2+). This simple maneuver silences ASIC3 without affecting ASIC1
channels. Additionally, ASIC1
and ASIC3 can be distinguished at the single channel level by their kinetics. As we indicated, ASIC3 does not exhibit closures, sublevels, or modes of activity.
Identification of ASIC2a poses fewer difficulties because these channels desensitize at a much slower rate and exhibit a component of sustained current at low pHo that is absent in ASIC1 and ASIC3. A particularly useful property is the low sensitivity to preconditioning pHo; therefore, exposing the patch first to pHo 6.0 inactivates ASIC1
and ASIC3, leaving only ASIC2a channels for activation with further decreases in pHo. Table I summarizes the properties of the homomeric ASIC channels that distinguish them.
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FOOTNOTES |
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* Abbreviations used in this paper: ASIC, acid-sensing ion channel; DRG, dorsal root ganglia.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health (DK54062 and P50HL55007) to C.M. Canessa.
Submitted: 7 February 2002
Revised: 12 August 2002
Accepted: 12 August 2002
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REFERENCES |
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