1 Institute of Neurobiology, University of Puerto Rico, San Juan 00901; and 2 Department of Biochemistry, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico 00936
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ABSTRACT |
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We investigated the contribution of sialic acid residues to the K+ currents involved in the repolarization of mouse ventricular myocytes. Ventricular K+ currents had a rapidly inactivating component followed by slowly decaying and sustained components. This current was produced by the summation of three distinct currents: Ito, which contributed to the transient component; Iss, which contributed to the sustained component; and IK,slow, which contributed to both components. Incubation of ventricular myocytes with the sialidase neuraminidase reduced the amplitude of Ito without altering IK,slow and Iss. We found that the reduction in Ito amplitude resulted from a depolarizing shift in the voltage of activation and a reduction in the conductance of Ito. Expression of Kv4.3 channels, a major contributor to Ito in the ventricle, in a sialylation-deficient Chinese hamster ovary cell line (lec2) mimicked the effects of neuraminidase on the ventricular Ito. Furthermore, we showed that sialylated glycolipids have little effect on the voltage dependence of Ito. Finally, consistent with its actions on Ito, neuraminidase produced an increase in the duration of the action potential of ventricular myocytes and the frequency of early afterdepolarizations. We conclude that sialylation of the proteins forming Kv4 channels is important in determining the voltage dependence and conductance of Ito and that incomplete glycosylation of these channels could lead to arrhythmias.
glycosylation; Kv4.3; arrhythmias; mouse ventricular myocytes; transient outward currents
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INTRODUCTION |
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IN THE MOUSE VENTRICLE, membrane depolarization activates a Ca2+-insensitive outward current that has a rapidly inactivating component followed by a slow inactivating sustained component. Three kinetically distinct currents have been proposed to underlie this current in the mouse ventricle (9, 34). The transient component is produced by a rapidly inactivating, transient outward current (Ito), while a noninactivating current (Iss) contributes to the sustained component (9, 34). A slowly inactivating current (IK,slow) is thought to contribute to both the transient and the sustained components of the ventricular outward K+ current.
Much progress has been made recently in determining the molecular identity of the channels underlying repolarizing K+ currents in the mouse heart. K+ channels of the Kv1 (Shaker), Kv3 (Shaw), and Kv4 (Shal) subfamilies, all of which produce rapidly inactivating currents, have been found in the ventricle of different species and were thus initially considered as possible contributors to Ito in this region of the heart (3, 6, 11, 12, 21-23, 37). In mouse ventricular myocytes both Kv4.3 and Kv4.2 are thought to produce the rapidly inactivating Ito found in these cells (35). Recent work has also provided clues on the molecular identity of the channels responsible for IK,slow and Iss in heart. In this regard Kv1.5 (20) and Kv2.1 channels (5, 33) are proposed to produce IK,slow in the mouse ventricle. While the search for a molecular correlate of the ventricular Iss has been less fruitful, a recent report suggests that Kv2.1 channels underlie Iss in mouse atria (5).
Reductions in the amplitude of repolarizing K+ currents have been found to increase action potential (AP) duration and the probability of arrhythmias (24). Changes in K+ current amplitude could occur due to a reduction in the number of functional channels in the membrane because of a lower number of Kv4 transcripts, as has been suggested during heart failure (17) and arrhythmias (38). However, K+ channel expression could also be controlled at the posttranscriptional level (25). Posttranscriptional modulation of glycoproteins includes incorporation of sugar residues to appropriate sites in the protein. K+ channels (31), like Na+ channels (4, 39), are heavily glycosylated proteins that contain unusually high levels of posttranslationally attached sialic acids on the external side of the channel. Work on human ether-à-go-go-related gene (HERG) (25) and Kv1.1 channels (31) suggests that K+ channel glycosylation could help to determine the voltage dependence and the surface expression of these channels. Although many of the K+ channels involved in the repolarization of the ventricle have consensus sites for protein glycosylation, the functional role of these negatively charged particles in the function of these channels is not completely understood.
The goal of the present study was to investigate the role of sialic acid residues in the function of K+ channels involved in the repolarization of the mouse ventricle. We have presented evidence suggesting that the channels underlying Ito, Iss, and IK,slow in the mouse ventricle vary in regard to the number of sialic acid residues attached to them during posttranslational processing. Furthermore, we have shown that incorporation of sialic acid residues onto Kv4 channels during posttranslational processing of the channel protein is important in determining the voltage dependence and conductance of Ito. Finally, our data suggest that improper glycosylation of Kv4 channels could lead to arrhythmogenic Ito currents.
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METHODS |
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Isolation of cardiac myocytes. Adult animals (25 g) were euthanized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg) in strict accordance to the guidelines established by the Institutional Animal Care and Use Committee, which follow all applicable state and federal laws. Single mouse ventricular myocytes were isolated as previously described (27) and stored at room temperature (22-25°C) in Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical, St. Louis, MO) until used.
Cell culture and transfection.
Cultures of Chinese hamster ovary (CHO) clones k1 (American Type
Culture Collection, Manassas, VA) were maintained in nutrient mixture
F-12 Ham Kaighn's modification (Sigma) supplemented with fetal bovine
serum (10%; Life Technologies, Rockville, MD), L-glutamine (2 mM), and a streptomycin/penicillin (S/P; 1%) solution. Lec2 and
pro5 cells were maintained in minimum essential medium (alpha modification; Sigma) supplemented with fetal bovine serum (10%), L-glutamine (2 mM), and a 1% S/P solution. Cells were
transiently transfected with the pcDNA clones of the -subunit of the
rat Kv4.3 channel (Kv4.3; a generous gift from Dr. Jeanne Nerbonne) and
the enhanced green fluorescent protein (EGFP; Clontech Labs, Palo Alto,
CA) using Lipofectamine 2000 (Life Technologies) as suggested by the
manufacturer. For electrophysiological and imaging experiments, cells
were plated at low density on 25-mm coverslips the day before
experiments were performed.
Electrophysiology.
CHO cells expressing Kv4.3 channels were identified on the basis of
EGFP fluorescence. During experiments, cells were continuously superperfused with a solution (solution A) containing the
following constituents (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose; pH 7.4. Cells were patched
in this solution, and after a gigaohm seal was formed, a small amount
of negative pressure was applied through the patch pipette to break the
membrane and achieve the whole cell configuration of the patch-clamp
technique. To measure Kv4.3 currents in CHO cells, we filled patch
pipettes with a solution that had the following constituents (in mM):
110 K-aspartate, 30 KCl, 10 HEPES, 5 EGTA, and 4 Mg-ATP; pH 7.3. With this solution, the patch electrodes had resistances that ranged from
1.5 to 2.5 M. In the experiments involving cardiac myocytes, the
external solution (control solution) in which K+ currents
were measured had the following composition (in mM): 140 N-methyl-D-glucamine (NMG), 5.5 KCl, 0.0005 nifedipine, 10 HEPES, 0.1 CaCl2, 2 MgCl2, and
10 glucose; pH 7.4. To this solution we added 4-aminopyridine (4-AP; 50 µM) and/or tetraethylammonium chloride (TEA; 25 mM) to
pharmacologically isolate the K+ current(s) of interest.
After the 4-AP and/or TEA was added, the pH of the control solution was
verified to be 7.4.
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Cell fusion.
Lec2 and k1 cells were fused using methods similar to those described
by Hoppe and colleagues (14) but with some minor
modifications. Briefly, the day of experiments, k1 cells expressing
Kv4.3 and GFP were dissociated into single cells and plated at very low densities in 25-mm coverslips. One hour after the k1 cells were plated,
lec2 cells were stained with the potentiometric dye 8-di-ANEPPS (Molecular Probes, Eugene, OR). To stain lec2 cells with
ANEPPS, we incubated these cells in a solution containing a 5 µM
concentration of the dye for 30 min. Once lec2 cells had been labeled
with ANEPPS, they were dissociated into single cells, resuspended in
culture medium, and laid onto k1 cells at a ratio of 1 k1 to 5 lec2
cells per coverslip. One hour after lec2 cells had been added, the
culture medium was removed and substituted with a 50% polyethylene
glycol (PEG; molecular weight 1500) solution (Roche
Diagnostic) in which cells were maintained for 4 min. At this point the
PEG was removed and substituted with a high-K+ solution
with the following constituents (in mM): 125 K-aspartate, 25 KCl, 10 HEPES, 10 glucose, 1 MgCl2, and 1 EGTA; pH 7.4. Cells were
maintained in this high-K+ solution for 10 min and then
returned to their normal medium.
Confocal microscopy. Confocal images were collected with a Bio-Rad MRC 600 confocal system coupled to an inverted Nikon diaphot microscope (Nikon, Melville, NY) equipped with a Nikon ×60 oil-immersion lens (NA 1.4). The confocal microscope was operated using a personal computer running COMOS software. Images were analyzed using custom software written by one of the authors (L. F. Santana) in IDL language (RSI, Boulder, CO). EGFP and ANEPPS were excited with the 488-nm line of a krypton-argon laser. The light emitted by EGFP (520 nm) and ANEPPS (>565 nm) was separated with appropriate filter cubes, and each was acquired through one of the two acquisition channels.
Desialylation of ventricular K+ channels. Enzymatic removal of sialic acid residues from glycoproteins in mouse ventricular myocytes was performed by using a protocol similar to that described by Yee and colleagues (36). Briefly, freshly dissociated mouse ventricular myocytes were incubated for 3 h in a DMEM solution to which 0.3 U/ml neuraminidase (Fluka, St. Louis, MO) was added. A similar protocol has been shown to desialylate Ca2+ (36), Na+ (39), and Kv1.1 (31) channels.
Statistics. Data are presented as means ± SE. Two-sample comparisons were performed using Student's t-test. When multigroup comparisons were necessary, they were made using a one-way analysis of variance (ANOVA) followed by Tukey's test. In all statistical tests a P value <0.05 was considered an indicator of a significant difference.
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RESULTS |
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Sialidase neuraminidase reduces Ito, but not IK,slow and Iss, in mouse ventricular myocytes. To investigate the role of sialic acid residues in the function of K+ channels in the ventricle, we incubated freshly dissociated mouse ventricular myocytes in the presence of 0.3 U/ml sialidase neuraminidase for 3 h at room temperature (22-25°C). Neuraminidase has been shown to hydrolyze sialic acid residues from glycoproteins and has been used by others to study the role of glycosylation in Ca2+ (36), Na+ (4, 39), and Kv1.1 channel (31) function. After ventricular myocytes were incubated with neuraminidase, cells were used for either electrophysiological experiments or biochemical analysis of Kv4 channels (see below).
Our first set of experiments examined the properties of K+ currents in control and neuraminidase-treated (NT) mouse ventricular myocytes (Fig. 1). K+ currents were evoked in these cells by step depolarizations (1.2 s) from the holding potential (HP) of
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Neuraminidase reduces Ito by reducing its conductance
and shifting its voltage dependence of activation in ventricular
myocytes.
Next we investigated the possibility that neuraminidase reduces
Ito in ventricular myocytes because it modifies
the voltage dependencies of activation and/or inactivation of this
current. Because Iss and
IK,slow are unaffected by neuraminidase, in
these experiments these currents were blocked by a combination of 50 µM 4-AP and 25 mM TEA+, which blocks
IK,slow and Iss but has
little effect on Ito (9). Figure
3 shows two representative families of
Ito currents from a control and NT ventricular
myocyte under these experimental conditions. Again, note that the NT
cell had much smaller Ito than control cells.
Indeed, at 60 mV, Ito was 35 ± 5% smaller in NT than in control cells. We found that this neuraminidase-induced reduction in amplitude of Ito was accompanied by
an ~18-mV depolarizing shift of the conductance
(G/Gmax)-voltage relationship of NT
cells (V1/2 = 19.51 ± 1.11 mV,
n = 9; P < 0.001) relative to control (V1/2 = 1.14 ± 0.37 mV,
n = 7), where V1/2 is the
voltage at which 50% of the current was observed (Fig. 3).
Note also that neuraminidase reduced the conductance of
Ito. In fact, the conductance of
Ito at 60 mV was nearly 40 ± 4% lower in
NT cells than in control cells (P < 0.05).
Furthermore, the steady-state activation of Ito
was shifted in the depolarizing direction by neuraminidase. The
V1/2 of the voltage dependence of the
steady-state inactivation of Ito was,
respectively, 43.82 ± 0.67 (n = 7) and
33.32 ± 0.45 mV (n = 9) in control and NT
myocytes (P < 0.001).
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Voltage dependence of activation and steady-state inactivation of Kv4.3 channels expressed in a sialylation-deficient cell line are shifted toward more positive potentials. Previous studies have shown that the ventricular Ito is produced by channels of the Kv4 (Shal) subfamily (11). Therefore, we investigated whether the effects neuraminidase had on the ventricular Ito could be reproduced by the removal of sialic acid residues from Kv4 channels. To test this hypothesis, we expressed the Kv4.3 channel in CHO-k1 (k1) and the sialylation-deficient CHO cell line lec2. The lec2 cells have extremely low levels (<2%) of CMP-sialic acid transport into the trans-Golgi compartments, which results in a dramatic reduction in sialylation during posttranslational processing of glycoproteins (8, 29, 30). As an additional control we also expressed Kv4.3 in CHO-pro5 cells (pro5), the nonmutant parental cell line from which lec2 cells were cloned.
Figure 4 shows three representative families of Kv4.3 currents that were evoked in k1, pro5, and lec2 cells by the protocol described. Similar to ventricular myocytes, there was no significant difference (P = 0.42) in the rate of inactivation of transient outward currents in lec2 (
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Voltage dependence of activation of sialic acid-deficient Kv4.3
channels is less sensitive to external
Ca2+ concentration.
To test the hypothesis that sialic acid-deficient Kv4.3 channels are
less sensitive to changes in external Ca2+ concentration
([Ca2+]o) than control channels, we recorded
Kv4.3 I-V relationships in the presence of varied
[Ca2+]o (2, 5, 10, and 20 mM). The results of
these experiments are summarized in Fig.
5. We found that increasing external
Ca2+ concentration shifts the conductance-voltage
dependence of Kv4.3 toward depolarized potentials in k1, pro5, and lec2
cells. However, the shift was much less pronounced in lec2 cells, such
that an order of magnitude in [Ca2+]o caused
a 39- and 38-mV shift in pro5 and k1 cells, respectively, and only a
28-mV shift in lec2 cells. It is also interesting to note that as the
[Ca2+]o reaches 10 mM or higher, the voltage
dependencies of activation of Kv4.3 currents in k1, lec2, and pro5
cells have a similar V1/2 of activation.
These findings are consistent with the surface potential theory
discussed above. Furthermore, our data suggest that glycosylation of
Kv4.3 channels is an important step in the posttranslational processing
of the channel protein.
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Sialic acids on proteins forming Kv4.3 channels have a much larger impact on the voltage dependence of Ito than sialic acids on glycolipids. In the experiments described above, we used two strategies to investigate the role of sialic acid in the voltage dependence of Ito. With one strategy, ventricular myocytes were treated with the sialidase neuraminidase, and with the other, Kv4.3 channels were expressed in a cell line (lec2) unable to incorporate sialic acid residues into the channel proteins. However, one difficulty with these experiments is that both the channel and glycolipids could have been deficient in sialic acids. This raises the following question: What is the contribution of sialic acids in glycolipids to the voltage dependence of Kv4.3 currents? The next series of experiments was designed to address this important issue.
To investigate the role of sialylation of glycolipids on the voltage dependence of Kv4.3 channel, we expressed EGFP and Kv4.3 channels in k1 cells and then fused them to lec2 cells. To differentiate lec2 from k1 cells, we stained the surface membrane of lec2 cells with the potentiometric dye ANEPPS. Figure 6A shows a set of confocal images taken from a k1 cell expressing EGFP and Kv4.3 and from a lec2 cell stained with ANEPPS before fusion. We chose these fluorescent markers for these experiments because they would be found in different regions of the cells (i.e., cytosol vs. surface membrane) and because of their spectral properties, which allow good separation of the emission signals. Note that in the k1 cell, the EGFP fluorescence is homogeneously distributed in cytosol of the cell, with virtually no signal detected in the ANEPPS acquisition channel. The ANEPPS-labeled lec2 cell, in turn, had the "donut-like" confocal fluorescent pattern typical of a cell that has a fluorophore located in its surface membrane. Note that there is very little (<10%) ANEPPS light "spilling over" into the EGFP channel.
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Neuraminidase increases AP duration in ventricular myocytes.
One testable prediction of the experiments presented above is that the
smaller Ito produced by incompletely
glycosylated Kv4 channels would lead to longer APs in the mouse
ventricle. To test this hypothesis, we recorded APs from control and NT
ventricular myocytes (see METHODS). We found that NT cells
had an AP that was significantly longer than control cells at both 50 and 90% repolarization (APD50 and APD90; Fig.
7). Indeed, in control and NT cells,
APD50 was 12.89 ± 2.24 ms (n = 25)
and 30.69 ± 7.03 ms (n = 20) (P = 0.01), respectively. Neuraminidase increased APD90 from
42.53 ± 8.96 ms (control; n = 25) to 199.26 ± 23.86 ms (NT; n = 20) (P < 0.001).
Because AP prolongation has been found to be a major cause for
arrhythmias, we investigated whether the increase in AP detected in NT
cells was accompanied by a higher probability of arrhythmogenic voltage
fluctuations, such as early afterdepolarizations (EADs), in these
cells. For these experiments, NT and control cells were stimulated at a
low frequency (1 Hz), to increase the likelihood of arrhythmias, for a
period of 5 min. Indeed, we found that cells exposed to neuraminidase
were more likely to develop EADs than control cells. While EADs were
observed in only 1 ± 1% (n = 25) of the control
cells examined, 35 ± 6% (n = 20) of the NT cells
had EADs (P < 0.001).
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DISCUSSION |
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In this paper we have examined the role of sialic acids in the function of the K+ currents involved in the repolarization of the mouse ventricle. We found that the sialidase neuraminidase modified Ito but left IK,slow and Iss unaltered. The lack of effect of neuraminidase on IK,slow and Iss suggests that sialic acids are not linked to these channels during posttranslational processing. In parallel experiments we showed that the effects of neuraminidase on Ito could be reproduced if Kv4.3, a major contributor to Ito in the ventricle, was expressed in a sialylation-deficient cell line. These experiments also showed that sialic acid incorporation onto the K+ channel protein during posttranslational processing is not an absolute requirement for the surface expression of functional K+ channels. Furthermore, we found that sialic acids linked to glycolipids contribute little to the voltage dependence of Kv4 channels. Finally, we found that sialic acid removal produces arrhythmogenic changes in Ito.
Differential contribution of sialic acids to the function of Ito, IK,slow, and Iss in the mouse ventricle. A series of recent papers have suggested that glycosylation is an important step in the processing of certain K+ channels (25). However, these studies have been carried out with heterologously expressed K+ channels only. These previous investigations have not directly examined the functional consequences (see below) of poor or deficient K+ channel glycosylation on the native cell function. The experiments presented in this paper addressed these specific issues.
The observation that neuraminidase decreased the amplitude of Ito, but not Iss and IK,slow, in mouse ventricular myocytes suggests that the proteins forming the channels underlying these currents are differentially glycosylated during posttranslational processing. Thus the number of sugar residues linked to these channels differs, at least with respect to their sialic acid content. This situation is not unique for K+ channels. In heart, it has been shown that neuraminidase modifies T-type Ca2+ current without affecting L-type Ca2+ current (36). Indeed, it would be interesting to investigate whether variations in the level of glycosylation between K+ channels could account, at least in part, for differences in their voltage dependence. In addition, our results suggest that sialylation of K+ channels is not an absolute requirement for the surface expression of functional K+ channels. However, it was recently shown that in HERG channels, complete elimination of N-linked glycosylation either through pharmacological (i.e., tunicamicin) or molecular (mutation of potential N-linked glycosylation sites) strategies prevented the surface expression of this channel (25). Thus it may be that the degree of glycosylation of the channel determines surface membrane expression.Sialic acids linked to Kv4 channels contribute to the voltage
dependence of Ito.
Although incorporation of sialic acid residues onto the Kv4 channels is
not required for their surface expression, our results show that these
negatively charged particles do contribute significantly to the voltage
dependence of these channels. We found that sialic acid-deficient Kv4
channels produced currents that had depolarized voltage dependencies of
activation and steady-state inactivation. However, we note that
neuraminidase, in addition to shifting the G/Gmax-voltage relationship of
Ito toward depolarized potentials, reduces the
conductance of Ito in mouse ventricular
myocytes, which suggests a reduction in the total number of activatable channels over a physiological range of voltages or a reduction in Kv4
single-channel conductance. These effects of neuraminidase on Kv4
channels would have the effect of reducing the amplitude of
Ito under physiological conditions. The positive
shift in the steady-state inactivation of Ito
would not compensate the shift in activation and smaller conductance of
this current because at the resting potential of these ventricular
myocytes (approximately 80 mV), nearly 100% of the channels are
available for activation.
Sialic acid deficiency produces arrhythmogenic changes in native Ito in mouse ventricular myocytes. The positive shift in the voltage dependence of activation of Ito would have the consequence of reducing the magnitude of this current. At 60 mV, Ito in NT cells was ~35% smaller than in control myocytes. This reduction in amplitude of Ito induced by K+ channel glycosylation contributed to a significant increase in the duration of the AP of ventricular myocytes. The increase in AP duration was evident at APD50 and APD90. Although a reduction in Ito could account to a large extent for the observed increases in AP duration, it is important to note that neuraminidase treatment is known to modify the function of other channels, including T-type Ca2+ channels (18), Na+ channels (7, 39), and several K+ channels (19, 28). It also is possible that in these experiments neuraminidase could have deglycosylated a transporter such as the Na+/Ca2+ exchanger (15), which could have altered its function in ways that prolong the AP. Thus the changes we detected in the AP in ventricular myocytes reflect changes in Ito and these other currents that are affected by neuraminidase. It is important to note that the increase we observed in AP duration was accompanied by an increase in the number of EADs observed. This suggests that proper glycosylation of K+ channels is crucial for the normal electrical functioning of the heart.
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ACKNOWLEDGEMENTS |
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We express gratitude to Luis E. Quiñones and Sonia Ramírez for technical assistance and to Dr. Richard Orkand for reading earlier versions of this paper.
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FOOTNOTES |
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This project was funded by National Institute of Neurological Disorders and Stroke Grants 1-U54 NS-39405-02 (L. F. Santana) and RO1 NS-38770 (D. J. Baro), National Heart, Lung, and Blood Institute Grant HL-67927 (L. F. Santana), a National Science Foundation Experimental Program to Stimulate Competitive Research Award (L. F. Santana and D. J. Baro), and National Center for Research Resources Research Centers in Minority Institutions-University of Puerto Rico Grant G12 RR-03051 (L. F. Santana and D. J. Baro).
Address for reprint requests and other correspondence: L. F. Santana, Institute of Neurobiology, Univ. of Puerto Rico, 201 Blvd. Del Valle, San Juan, PR 00901 (E-mail: lsantana{at}neurobio.upr.clu.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 29 March 2001.
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