From the Departments of Neuroscience and Physiology, and § Division of Endocrinology, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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The initiation of insulin release from rat islet
cells relies, in large part, on calcium influx through
dihydropyridine-sensitive (
1D) voltage-gated
calcium channels. Components of calcium-dependent insulin
secretion and whole cell calcium current, however, are resistant to
L-type channel blockade, as well as to
-conotoxin GVIA,
a potent inhibitor of
1B channels, suggesting the
expression of additional exocytotic calcium channels in the islet. We
used a reverse transcription-polymerase chain reaction-based strategy to ascertain at the molecular level whether the
1A
calcium channel isoform was also present. Results revealed two new
variants of the rat brain
1A channel in the islet with
divergence in a putative extracellular domain and in the carboxyl
terminus. Using antibodies and cRNA probes specific for
1A channels, we found that the majority of cells in rat
pancreatic islets were labeled, indicating expression of the
1A channels in
cells, the predominant islet cell
type. Electrophysiologic recording from isolated islet cells
demonstrated that the dihydropyridine-resistant current was sensitive
to the
1A channel blocker,
-agatoxin IVA. This toxin
also inhibited the dihydropyridine-resistant component of
glucose-stimulated insulin secretion, suggesting functional overlap
among calcium channel classes. These findings confirm the presence of
multiple high voltage-activated calcium channels in the rat islet and
implicate a physiologic role for
1A channels in
excitation-secretion coupling in
cells.
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INTRODUCTION |
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The metabolism of glucose in cells is linked to membrane
excitation through increases in the components that influence the ATP/ADP ratio (1, 2). A local increase in ATP relative to ADP inhibits
the ATP-sensitive K+ (KATP) channel,
giving rise to oscillations in KATP permeability and consequent fluctuations in membrane potential (3, 4). ATP-induced
depolarizations evoke insulin release through the activation of
voltage-dependent calcium channels, promoting calcium influx (3-6). If extracellular calcium is eliminated,
glucose-stimulated insulin secretion is abolished, highlighting the
important role of calcium channels in insulin homeostasis (7-11).
Of the six genes (A-E, S) encoding the pore-forming 1
subunits of high voltage-activated calcium channels (12), all are capable of coupling to exocytotic machinery, although differences in
tissue distribution and efficacy with which they stimulate secretion
vary among the channel classes (for review, see Ref. 13).
Pharmacological and heterologous expression studies have identified
selective antagonists for certain of these:
-agatoxin IV blocks
1A,
-conotoxin GVIA blocks
1B, and
1,4-dihydropyridines block
1C,
1D, and
1S (13). No selective antagonist has been identified to
date for
1E calcium channels, although they are sensitive to nonselective calcium channel antagonists (e.g.
-grammotoxin SIA and cadmium).
Previous work on pancreatic cells demonstrated that calcium
influx and depolarization-evoked insulin release are blocked 60-80%
by inhibitors of
1D channels (14-16). In addition,
-conotoxin GVIA blocks a portion of calcium current (17) and 27% of
the second phase insulin secretion from rat pancreatic
cells under conditions of maximal glucose stimulation (18). In the presence of both
-conotoxin GVIA and the dihydropyridine antagonist nifedipine, 25%
of Ca2+-dependent insulin secretion remains,
suggesting the involvement of another calcium channel type. Because
1A and
1B channels are known to
colocalize within mammalian central nervous system nerve terminals and
play prominent roles in neurosecretion (19-22), we sought to determine
whether the
1A isoform was also present in
cells and
responsible for the current and insulin release that are resistant to
dihydropyridines and
-conotoxin GVIA. Here we report the full-length
sequences of two unique
1A splice variants cloned from
rat pancreatic islets. We demonstrate their expression in
cells and
provide an initial characterization of their pharmacologic and
electrophysiologic properties using tight seal whole cell recording.
These channels play a role in calcium-dependent insulin secretion. Results may have significant implications for understanding, and perhaps treating,
cell disorders such as diabetes and
hyperinsulinemia.
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MATERIALS AND METHODS |
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Islet Isolation-- Islets were isolated from male, 200-225 gm, Spraque-Dawley rats. Animals were anesthetized (Nembutol 50 mg/kg), and the pancreatic bile duct exposed, cannulated, and infused with 2 mg/ml collagenase (Boehringer Mannheim) in medium M199 (Life Technologies, Inc., with 5.6 gm/l HEPES, 2.2 gm/l NaHCO3). The infused pancreas was removed and incubated in a 50-ml tube submerged in a 37 °C water bath for 17 min. Tubes were shaken briefly to dissociate tissue, the digestion reaction was stopped by the addition of cooled M199 with 10% newborn calf serum (Life Technologies, Inc.), and the tissue was shaken again to further dissociate while washing free of collagenase. The tissue was spun for 30 s (speed 3) in an ICN table-top clinical centrifuge and washed in fresh medium M199 with serum. This procedure was repeated twice to remove collagenase. After a final spin (1 min. at speed 3) to pellet the tissue, the medium was discarded, and the tissue was suspended in 10 ml of Histopaque (Sigma) and overlaid with 10 ml of M199 (without serum). The tissue was centrifuged in this gradient at 900 × g in a swinging bucket rotor at 4 °C for 20 min. The supernatant containing the islets was removed and centrifuged (speed 4, ICN centrifuge) to pellet the islets. The supernatant was discarded and the islets washed with 10 ml of M199 (with serum) twice before use. They were used intact for insulin release assays, further dissociated into single cells for electrophysiology, or homogenized for isolation of RNA.
Cloning--
Primers were designed along the full-length of the
rat brain class A channel (rbA-1, GenBankTM accession no.
M64373). Regions of high homology to the rat brain 1B
channel (rbB) were avoided. Sense and antisense oligonucleotides ranged
in length from 18 to 24 bases beginning at the following 5' nucleotides
(nt)1 of the rbA-1 sequence:
20, 540, 1265, 1610, 2091, 2484, 2879, 3309, 3658, 4104, 4620, 5170,
5607, 5961, 6527, 7010, and 7125. Total RNA purified from homogenized
rat islets (Trizol, Life Technologies, Inc.) was DNase treated (Life
Technologies, Inc., amplification grade DNase) and reverse-transcribed
for 30 min at 42 °C (reverse transcriptase, Perkin Elmer). GC rich
regions were reverse transcribed at 50 °C (Superscript, Life
Technologies, Inc. or avian myeloblastosis virus reverse transcriptase,
Amersham Pharmacia Biotech) and 10% glycerol was used in the PCR
amplification. At least two separate PCR amplifications (Taq
polymerase, Perkin Elmer, Amersham Pharmacia Biotech) were performed
for each primer set and 2 sense and 2 antisense strands sequenced (ABI,
fluorescent-labeled automated sequencing) to ensure accuracy of
sequence. To verify regions detected to diverge from rbA-1 over a
~2-kilobase region, reverse transcription of islet total RNA was
performed at 50 °C for 1 h using avian myeloblastosis virus
reverse transcriptase (Amersham Pharmacia Biotech) and Superscript
(Life Technologies, Inc.) to generate longer products over GC rich
regions. For this "long" PCR, primers at nt 4620 and from the rbA-1
3' UT region were used and generated two ~2 kilobases alternatively
spliced carboxyl-terminal fragments as described below.
Generation of cRNA Probe-- Primers were designed to yield a 587-bp product from the 3'-end (5961-6548) of coding sequence of rbA-1 (23). Freshly isolated islet RNA for reverse transcription (Perkin Elmer reverse transcriptase), and Taq polymerase (Perkin Elmer) were used for cDNA amplification. Control samples, in which reverse transcriptase was omitted, were included to ensure that products had not been amplified from genomic DNA. Because the region contained a high percentage of GC-rich sequence, 10% glycerol was added to the reaction mix. The annealing temperature was 60 °C.
In Situ Hybridization-- The original 587-bp PCR product (nt 5961-6548) above was subcloned (vector pCR I, Invitrogen) and sequenced in both directions. Comparison of the sequences revealed 100% nucleotide identity to rbA-1. Using this subclone as a template, digoxigenin-labeled (Boehringer Mannheim) sense and antisense RNA probes were synthesized for in situ hybridization studies of rat pancreas. Paraffin sections were mounted on slides, cleared, and hydrated before treatment with proteinase K. This was followed by equilibration in 0.1 M triethanolamine, pH 8.0, to which acetic anhydride had been added. The probe was added at 4 ng/µl to the hybridization mix. Sections were incubated overnight at 60 °C in a humid chamber. Post hybridization, the slides were soaked in 2× SSC (300 mM sodium chloride, 30 mM sodium citrate), gently agitated in warm 50% formamide in 2× SSC, and transferred to 60 °C. The sections were then treated with RNase A buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, and RNase A at 20 µg/ml) to remove unhybridized probe. Slides were washed in 0.1× SSC at 65 °C. For color detection of labeled cells (Boehringer Mannheim Genius System), slides were blocked, washed, and incubated overnight at 4 °C with 200-500 ml of antidigoxigenin-alkaline phosphatase. The subsequent chromagen substrate (Sigma) consisted of nitroblue tetrazolium salt and X-phos (bromo-4-chloro-3-indoyl phosphate toluidinium salt).
Immunohistochemistry-- Experiments employed the antipeptide antibody, CNA-1 (generously provided by Drs. R. Westenbroek and W. A. Catterall). This antibody recognizes amino acids 865-881 in rbA-1, corresponding to nucleotides 2595-2643 (identical amino acids in the islet). 8-µm cryosections of rat pancreas were blocked with Tris-buffered saline, 1% bovine serum albumin, 0.1% Triton, washed, and incubated for 1 h at room temperature with CNA-1 primary antibody diluted 1:25 in Tris-buffered saline. Sections were washed, incubated with rhodamine-labeled anti-rabbit secondary antibody (Sigma, diluted 1:350 in Tris-buffered saline) for 20 min, washed again, then visualized and photographed through a Zeiss Axioplan Universal microscope.
Whole Cell Recording--
Islets were isolated as described
above, dissociated in trypsin/EDTA (0.05% trypsin, 0.53 mM
EDTA) and cultured 1-3 days in M199 (with serum) prior to the
experiments. Voltage-dependent calcium currents were
examined in the whole cell configuration using pipettes fabricated from
nonheparinized soda lime glass (VWR, 73811). Pipette resistances
averaged ~2 megaohms with an internal solution containing 120 mM N-methyl-D-glucamine, 20 mM tetraethylammonium-OH, 10 mM HEPES, 11 mM EGTA, 1 mM CaCl2, and 4 mM MgATP (pH 7.0) and an external solution containing 10 mM BaCl2, 120 mM NaCl, 20 mM triethanolamine, and 1.2 mM
MgCl2, 10 mM Na-HEPES, 1 µM
tetrodotoxin, 1 µM nimodipine (pH 7.4). -Agatoxin IVA
was applied by pressure ejection from a wide-bore (~2-3 µm) pipette positioned within 20 µm of the cell. The toxin was stored at
40 °C in 10-µl samples of 10 mM in distilled
deionized water and diluted to the final working concentration in
external solution immediately before use.
Insulin Secretion Evaluated by RIA--
Islets were isolated
from exocrine pancreas as described above. They were selected for
uniform size (150-200 µm), washed in Krebs-Ringer buffer (KRB,
equilibrated with O2 for 30 min), evenly divided into KRB
with 2.8 mM glucose (with or without 500 nM
-agatoxin IVA) and allowed to recover from isolation. The islets
were incubated at 37 °C (95% O2, 5% CO2)
during equilibration. After 90 min of preincubation in low glucose KRB,
the islets were divided into groups of 10 islets/vial in 0.5 ml of
fresh KRB, three vials for each condition (2.8 or 11 mM
glucose with or without
-agatoxin IVA). All samples were incubated
in a 37 °C water bath for 1 h to promote glucose-stimulated
insulin secretion. Samples were centrifuged at 700 rpm, and 300 µl of
supernatant was removed and viewed under a dissecting microscope to
ensure no islet tissue was present. This sample was assayed for insulin
content using ICN insulin radioimmunoassay reagents. Briefly, 100 µl
of each sample or standards were added to duplicate anti-insulin
antibody-coated tubes followed by 900 µl of 125I-labeled
insulin diluted into buffer. In addition, control samples of toxin
alone were included to ensure that the
-agatoxin IVA did not
interfere with insulin antibody binding. The samples and standards were
incubated for 18 h at room temperature. The supernatant was
removed, and tubes were washed with double-distilled H2O
and counted in a gamma counter. Sample counts were compared with the standard curve for determination of insulin concentrations. Results are
reported as mean ± S.E. of the mean, and statistical significance was evaluated by Student's t test.
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RESULTS |
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Cloning of Islet 1A Splice Variants--
Given our
goal of identifying the class A (
1A) variants in
pancreatic islets, a PCR strategy was followed using primers designed along the full-length of the cloned
1A sequence from rat
brain, rbA-1 (23, 24). Each product was generated twice, and both the
sense and antisense strands were sequenced to ensure accuracy. Reverse
transcriptase was eliminated from control samples to ensure that
amplification products were from islet mRNA and not of genomic origin. The entire coding sequence was determined. From the 5'-UT to
base 4806, the sequence was 100% identical to the type "a" splice
variant (
1A-a) of rbA-1 (25). At nt 4807, in a putative extracellular domain, there is a six base in-frame insertion in both
variants described below cloned from rat islets. This insertion results
in the addition of amino acids asparagine and proline (Figs.
1A and
2), as previously reported for
1A in rat kidney cortex (26) and
1A-b in
rat brain (25). In addition, two carboxyl-terminal variants are present
in the islet that have not been reported elsewhere. One, riA-1,
diverges over the bases 5385-5477, altering 10 amino acids throughout
the region (Figs. 1 and 2). Further 3', the bases corresponding to
6160-6195 (in rbA-1) are deleted in riA-1. The riA-1 variant is also
significantly longer than rbA-1 due to a 5-base (GCCAG) insertion
before the stop codon, resulting in a frameshift (Figs. 1 and 2). This
same insertion has also been described in human brain
1A
channels (27). The contiguity of the observed variations in riA-1 was confirmed by generating and sequencing >2 kilobase fragments using primers at 4620 and in the 3'-untranslated region. Although
combinations of these carboxyl-terminal modifications have been
described in human brain partial cDNA clones (27), riA-1 is unique
among known full-length clones.
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Cellular Localization of Rat Islet 1A
Channels--
In situ hybridization methods were employed
to examine the cellular localization of
1A mRNA in
islets. A riboprobe was synthesized by PCR of reverse transcribed islet
total RNA with class A calcium channel primers, generating a 587-bp
carboxyl-terminal fragment (bp 5961-6548). No product was obtained if
reverse transcriptase was omitted. This fragment, 100% identical to
rbA-1 (23), was subcloned and labeled with digoxigenin-UTP to generate
both sense and antisense RNA probes. The majority of the cells,
representing the islet core, were labeled with the antisense probe.
This demonstrates that the
1A calcium channel mRNA
is present in
cells, which comprise the core and ~80% of islet
cells (Fig. 3A).
Islets remained unlabeled when the digoxigenin-sense probe was used
(Fig. 3B).
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Whole Cell Recording of Cell Calcium Channel Currents--
To
test whether the rat islet class A transcripts give rise to functional
channels in
cells, we recorded from dissociated islet cells using
tight seal whole cell methods. Calcium channel currents were isolated
with standard intra- and extracellular solutions (30, 31). Nimodipine
(1 µM) was added to all extracellular solutions in order
to suppress dihydropyridine-sensitive (class C/D) currents.
Ba2+ (10 mM) was used as a charge carrier to
enhance calcium channel currents over those observed in 1.2 mM Ca2+ (the normal extracellular
concentration).
cells could be identified by the presence of small
processes, as has been reported elsewhere (32).
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A Portion of Insulin Secretion Is Inhibited by -Agatoxin
IVA--
The presence of significant class A current in
cells
suggested the possibility that these channels play a role in
exocytosis. To test this, we employed a radioimmunoassay to measure
glucose-stimulated insulin release from islets, and evaluated the
involvement of class A calcium channels using
-agatoxin IVA. Islets
were equilibrated for 90 min at 37 °C in KRB ± 500 nM
-agatoxin IVA. Addition of 11 mM glucose
evoked insulin release to a level of 1127% of that seen in basal
glucose (2.8 mM) during a 60-min incubation period.
-Agatoxin IVA (500 nM) diminished this
glucose-stimulated insulin release to 811% of basal secretion (Fig.
6A). The inclusion of nimodipine (1 µM) in all solutions to block
dihydropyridine-sensitive calcium channels reduced 11 mM
glucose-evoked insulin release to a level 140% of that seen in basal
glucose (2.8 mM). Thus, a portion of glucose-stimulated
insulin release is dihydropyridine-resistant. All of the latter is
mediated by Ca2+ influx through class A channels, since it
is eliminated by 500 nM
-agatoxin IVA (Fig.
6B). Control samples without islets, with
-agatoxin IVA
alone, had no counts above background, indicating the toxin does not
interact with anti-insulin antibodies. These results demonstrate that
class A calcium channels are effective co-regulators of insulin
secretion.
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DISCUSSION |
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Our results demonstrate two new variants of the class A calcium
channel in pancreatic islets and establish the presence of both
mRNA and protein in cells. We have begun to characterize the
electrophysiology and demonstrate that currents through these channels
resemble currents recorded through class A channels in other cells
(33-38). Additionally, we show that, as in the nervous system, calcium
influx through islet class A channels is coupled to exocytosis. Despite
identification of several
1A splice variants and
missense mutations (described below), little is known about the
biophysical properties conferred by these changes. With the molecular
information described here, we can begin to ascertain how specific
amino acid changes influence electrical and functional properties of
1A channels.
Multiple 1A Isoforms--
Certain of the variable
regions reported here occur in clones described elsewhere, but the
islet clones as a whole are unique. Zhuchenko et al. (27)
demonstrated five partial human clones from brain containing the GCCAG
insertion, producing a frameshift and a longer carboxyl terminus.
Unlike the islet clones described here, the human variants contain a
CAG repeat in the extended carboxyl terminus that is responsible for a
form of familial ataxia, SCA6 (spinocerebellar ataxia). This repeat is
not expressed in the riA-1 carboxyl terminus (Fig. 2). Two partial
clones from human had the 36-base pair deletion, and two others had the
94-base pair divergent sequence present in riA-1 (27). In addition, the
6-base insertion at nt 4607 in both islet isoforms has been described
in a
1A PCR cDNA fragment from rat kidney (26) and in rat brain
1A-b (25). The two deletions in riA-2 (nt
4922-4987 and 5256-5480) have not been described previously.
Interestingly, the deletion beginning at nt 5256 in riA-2 corresponds
to the 94-bp COOH-terminal region of divergence found in riA-1 and in two of the partial human clones. This region has homology to EF hand
motifs in dihydropyridine-sensitive calcium channels demonstrated to
bind calcium and promote inactivation (25, 28), but also see Ref. 39.
Rabbit BI-I
1A calcium channels are 92% identical to
riA-1 but differ substantially in the intracellular loop between repeats II and III. Of note, there are also several 3 and 6 base insertions in BI-I
1A isoform not seen in riA-1
(40).
Heterogeneous Cell Responses--
The density of
-agatoxin
IVA-sensitive current in
cells varied considerably from cell to
cell. The majority of the cells expressed detectable toxin-sensitive
currents (as well as
1A mRNA and protein detected by
in situ and immunochemical methods); in those sensitive to
toxin blockade, the inhibition varied from 15 to 80%. As
-agatoxin
IVA has been demonstrated to inhibit a variety of
1A
calcium channels, both in primary and heterologous cells, the
heterogeneity found for the
cells likely reflects variations in
expression levels for both class A channel types. Quantitating this
relationship will require single-cell comparisons between the mRNA
or protein levels for the calcium channel subunits and amplitude of
-agatoxin IVA-sensitive currents. In undifferentiated PC12 cells,
both the sensitivity of the calcium current to
-agatoxin-IVA and the
rate of toxin-induced inhibition vary from cell to cell, as observed
for neurons (37, 38), raising the possibility that pharmacological
differences between class A channel variants might underlie the
heterogeneity of
cell responses to
-agatoxin IVA.
Class A Channels in Other Tissues-- Exocytosis from mammalian central neurons relies heavily on class A and B channels and very little on dihydropyridine-sensitive class C/D channels (13). By contrast, secretion from neuroendocrine cells is largely dihydropyridine-sensitive. Our results demonstrate that such differences are not absolute. That is, although the majority of insulin release is dependent on dihydropyridine-sensitive channels, a portion is mediated through class A channels. Class A and B channels have been demonstrated by pharmacology and electrophysiology to be present in adrenal chromaffin cells, (50-52), carcinoid cells of the gut (53), pituitary corticotropes (54), the pituitary cell line AtT20 (55), GH4C1 pituitary cells (56), and in RINm5f and HIT insulinoma cells (57, 58). Dihydropyridine-resistant channels play a role in exocytosis for certain of these cells (e.g. carcinoid and HIT cells) (51, 58) but not all. Release of adrenocorticotrophic hormone from AtT20 cells, for example, is evoked entirely through dihydropyridine-sensitive channels, despite the fact that >50% of the calcium current in the cells is resistant to blockade by dihydropyridines (55). The mechanisms that determine specificity of coupling between calcium channels and the exocytotic machinery remain to be defined.
Significance of 1A Calcium Channels in the
Islet--
Recent statistical analyses suggest that more than 15 million people in the United States suffer from diabetes (59). A
population-based retrospective study indicates the age-adjusted
prevalence of diabetes cases rose 65% for men and 37% for women
between 1970 and 1990 (60), and a "massive increase" in the
prevalence of noninsulin-dependent diabetes is predicted
globally, as countries succumb to the "Westernization" of diet and
activity patterns (61). It is important, therefore, to continue to
expand our understanding of the mechanisms by which the
cell
controls the secretion of insulin. Knowing the molecular structure of
the
1A isoforms present in the islet and the roles that
they play within the context of the functioning islet may allow the
development of clinically useful agents to modulate calcium entry into
cells through these channels, providing another tool to regulate
insulin release. In addition, defining the entire sequence of splice
variants will facilitate investigations of the ways in which changes in
primary structure alter the pharmacology, biophysical, and/or
exocytotic properties of class A voltage-gated calcium channels.
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ACKNOWLEDGEMENTS |
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We are grateful to L. Moss for support of this project, Joe Dillon and Xudong Liang for assistance with automated sequencing, Jeff Tatro and Jack Leahy for advice on radioimmunoassays, W.A. Catterall and R. Westenbroek for CNA-1 antibody, Ron Lechan and Barbara Corkey for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health MCSDA KO8 NS01923 (to B. L.), and National Institutes of Health Grants DK34447 (to A. E. B., III and Larry Moss), NS16483 (to K. D., Jacob Javits Award), and National Institutes of Health GRASP Center Grant DK34928.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.
To whom correspondence should be addressed: Dept. of Neuroscience,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel.: 617-636-6938; Fax: 617-636-0576.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF051526, AF051527.
1 The abbreviations used are: nt, nucleotide(s); PCR, polymerase chain reaction; bp, base pair(s); KRB, Krebs-Ringer buffer.
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REFERENCES |
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