From the Departments of Medicine and
¶ Pharmacology and Physiology, University of Chicago, Chicago,
Illinois 60637 and the § Department of Cell Physiology,
Glaxo Wellcome Research Institute,
Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Although stimulation of insulin secretion by
glucose is regulated by coupled oscillations of membrane potential and
intracellular Ca2+ ([Ca2+]i),
the membrane events regulating these oscillations are incompletely
understood. In the presence of glucose and tetraethylammonium, transgenically derived -cells (
TC3-neo) exhibit coupled voltage and [Ca2+]i oscillations strikingly similar to
those observed in normal islets in response to glucose. Using these
cells as a model system, we investigated the membrane conductance
underlying these oscillations. Alterations in delayed
rectifier or Ca2+-activated K+ channels
were excluded as a source of the conductance oscillations, as they
are completely blocked by tetraethylammonium. ATP-sensitive K+ channels were also excluded, since the ATP-sensitive
K+ channel blocker tolbutamide substituted for glucose in
inducing [Ca2+]i oscillations. Thapsigargin,
which depletes intracellular Ca2+ stores, and maitotoxin,
an activator of nonselective cation channels, both converted the
glucose-dependent [Ca2+]i
oscillations into a sustained elevation. On the other hand, both SKF
96365, a blocker of Ca2+ store-operated channels, and
external Na+ removal suppressed the glucose-stimulated
[Ca2+]i oscillations. Maitotoxin activated a
nonselective cation current in
TC3 cells that was attenuated by
removal of extracellular Na+ and by SKF 96365, in the same
manner to a current activated in mouse
-cells following depletion of
intracellular Ca2+ stores. Currents similar to these are
produced by the mammalian trp-related channels, a gene
family that includes Ca2+ store-operated channels and
inositol 1,4,5-trisphosphate-activated channels. We found several of
the trp family genes were expressed in
TC3 cells by
reverse transcriptase polymerase chain reaction using specific primers,
but by Northern blot analysis, mtrp-4 was the predominant
message expressed. We conclude that a conductance underlying
glucose-stimulated oscillations in
-cells is provided by a
Ca2+ store depletion-activated nonselective cation current,
which is plausibly encoded by homologs of trp genes.
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INTRODUCTION |
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Pancreatic islet -cell secretion of insulin stimulated by
glucose is dependent upon elevations in intracellular calcium
concentration ([Ca2+]i)1
(1). Together, two voltage-dependent processes have been
proposed to constitute the glucose-stimulated rise in
[Ca2+]i: the influx of extracellular
Ca2+ through Ca2+ channels (2-4) and the
release of intracellular Ca2+ from the endoplasmic
reticulum (ER) (5-7). Glucose initiates changes in membrane potential
via an increase in cytosolic ATP, derived from the glycolytic reduction
of NAD+, that results in block of ATP-sensitive
K+ channels (KATP) (8, 9). Consequently, the membrane
potential slowly depolarizes from approximately
65 mV, whereupon
trains of action potentials occur (the active phase), interrupted by quiescent periods of hyperpolarization (the silent phase), both forming
the so-called electrical bursting behavior characteristic of
glucose-stimulated islets (10-12). Closely coupled with this bursting
activity are oscillations in [Ca2+]i that
ultimately determine the oscillatory nature of insulin secretion (5,
11-14). The mechanism underlying these oscillations in membrane
potential, [Ca2+]i, and insulin secretion remains
uncertain, but most likely stems from a cyclical activation of an ion
conductance governed indirectly by metabolism, changes in
[Ca2+]i, or some hitherto uncharacterized
intracellular mediator (15, 16).
Confounding an early resolution of this puzzle is the profusion of
membrane currents present in -cells. Thus, in addition to
ATP-sensitive K+ current (IKATP) and
voltage-dependent Ca2+
(ICa) and Na+ currents, delayed
rectifier K+ (IKDR),
Ca2+-activated K+
(IKCa), and G-protein-coupled inward rectifier
K+ currents have been described, all of which could
contribute to the regulation of the
-cell membrane potential by
glucose (17-20). More recently, a nonselective cation current whose
conductance is regulated by the Ca2+ content of the ER
(ICRAN; Ca2+ release-activated
nonselective cation current) was found in mouse
-cells (21). This
current could be activated indirectly by ER Ca2+ store
depletion or directly by maitotoxin (MTX) (12, 21). Carried primarily
by Na+, ICRAN produces membrane
depolarization and indirectly, an elevation in
[Ca2+]i, as a consequence of activation of
voltage-dependent ICa (21).
Although membrane potential and [Ca2+]i
oscillations have been readily recorded from whole mouse islets and
-cell clusters (11-14, 20), single mouse
-cells normally do not
respond to glucose stimulation with regular oscillatory activity (16). We have previously reported that a stable transgenically derived murine
insulinoma cell line (
TC3-neo) also responds to glucose with large
amplitude oscillations in [Ca2+]i when in the
presence of 10-20 mM tetraethylammonium (TEA), a blocker
of delayed rectifier K+ channels (Kv) (20). We have thus
utilized this cell line to characterize and identify the membrane
conductance that underlies glucose-stimulated oscillatory activity.
In this article, we provide evidence that activation of
ICRAN in TC3-neo cells regulates
glucose-stimulated [Ca2+]i oscillations and
insulin secretion. Furthermore, to characterize the molecular identity
of ICRAN, we detected in insulin-secreting insulinoma cells and mouse islets the expression of multiple
trp (transient receptor potential) genes that are known to
encode intracellular Ca2+ store release-activated channels
in other mammalian and insect tissues (22-28). Our findings suggest
that the interaction between intracellular Ca2+ stores and
plasma membrane conductances is an important mechanism that controls
glucose-dependent stimulus response coupling in
-cells.
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MATERIALS AND METHODS |
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TC3 cells were cultured as described elsewhere (20, 29). A
clonal subline (
TC3-neo) was isolated after transfection with
pSV2-neo by electroporation (20) and maintained in the continued
presence of 1 mg/ml G418 (Life Technologies, Inc.). Cells were seeded
for 4-6 days in Dulbecco's modified Eagle's medium supplemented with
15% normal horse serum, 2.5% fetal calf serum, 50 units/ml
penicillin, 50 µg/ml streptomycin. Eighteen hours prior to
experiments, medium was changed to RPMI 1640 medium containing 15%
dialyzed horse serum, 2.5% dialyzed fetal calf serum, 1 mM
glucose, and antibiotics as above. MIN6 cells were cultured essentially
as described (30). Islets of Langerhans were isolated from
8-10-week-old C57BL/KsJ (+/+) mice (Jackson Laboratories) as described
(5, 9, 12).
Measurement of [Ca2+]i--
TC3-neo
cells were loaded with Fura-2 by a 25-min incubation at 37 °C in
Krebs-Ringer buffer containing (in mM): 119 NaCl, 4.7 KCl,
1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0 glucose, supplemented with 5 µM acetoxymethyl ester of Fura-2
(Molecular Probes Inc.). In some experiments, external NaCl was
replaced with equimolar amounts of
N-methyl-D-glucamine-Cl.
[Ca2+]i was expressed as the ratio change in
fluorescence (340/380) as detailed in full elsewhere (9, 20).
Patch Clamp Current Recordings--
Single TC3-neo cells were
voltage-clamped using perforated patch clamp techniques. Patch
electrodes contained 80 mM potassium aspartate, 50 mM KCl, 5 mM NaCl, 5 mM
MgCl2, 10 mM HEPES/KOH (pH 7.2), and 100-120
µg/ml nystatin (9, 20). The perfusion medium was a Krebs-Ringer
buffer solution as detailed above.
Insulin Secretion Measurements--
TC3-neo cells were plated
at a density of 25 × 104/cm2 and cultured
overnight in RPMI 1640 medium as described above. Insulin secretion
measurements were made in the presence of 0 or 1 mM glucose
in the presence or absence of TEA, maitotoxin, or thapsigargin, as
described previously (20). The concentration of insulin was determined
using an SPA assay kit (Amersham Pharmacia Biotech) and calibrated
using rat insulin (Novo) as standard.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Identification and Amplification of Probes for trp-related Gene
Expression--
Mouse brain, TC3-neo, and MIN6 poly(A)+
RNA were prepared using an oligo(dT) binding method (Quickprep micro
mRNA kit, Amersham Pharmacia Biotech) and first strand cDNA was
reverse-transcribed using random primers (Superscript II transcriptase,
Life Technologies, Inc.). Transcripts of the six known members of the
murine trp (mtrp) family (accession number in
parentheses) were amplified by polymerase chain reaction (PCR)
using the following oligonucleotide primers: mtrp-1 (GenBank>U40980)
5'-GATTTTGGGAAATTTCTGGGAATG-3' (sense) and
5'-TTTATCCTCATGACTTGCTATCA-3' (antisense); mtrp-2 (U40981)
5'-GACATGATCCGGTTCATGTTC-3' (sense) and 5'-CATCAGCATCATCCTCGATCT-3' (antisense); mtrp-3 (U40982) 5'-GACATATTCAAGTTCATGCGTTCTC-3' (sense) and 5'-ACATCACTGTCATCCTCGATCTC-3' (antisense);
mtrp-4 (X90697) 5'-CTGCAGATATCTCTGGGAAGG-3' (sense) and
5'-GCTTTGTTCGAGCAAATTTCC-3' (antisense); mtrp-5 (U40984)
5'-TCTACTGCCTAGTACTACTGGC-3' (sense) and 5'-GTAGGAGTTATTCATCATGGCG-3'
(antisense); mtrp-6 (U40969) 5'-GATATCTTCAAATTCATGGTCATA-3'
(sense) and 5'-GTCCGCATCATCCTCAATTTC-3' (antisense). The PCR
protocol used consisted of an initial denaturation at 94 °C for 3 min; followed by five cycles of 94 °C for 1 min, 64 °C for
30 s; followed directly by 25 cycles of 94 °C for 1 min,
60 °C for 1 min, and 72 °C for 30 s.
Northern Blot Analysis--
For RNA isolation, TC3 cells were
grown to near confluence, washed three times with phosphate-buffered
saline, and lysed in solution containing 4 M guanidine
thiocyanate, 100 mM Tris-HCl (pH 7.5), 1%
-mercaptoethanol, and 0.5% lauryl sarcosinate. Total RNA was
purified by centrifugation through a 0.9-ml cushion of 5.7 M CsCl in a TLS-55 rotor at 53000 rpm for 4 h in the
Optima TL ultracentrifuge (Beckman, Palo Alto, CA). Mouse brain RNA was isolated using TRIzol (Life Technologies, Inc.) according to the manufacturer's protocol and purified by centrifugation through CsCl as
described above. RNA was size-fractionated in the presence of ethidium
bromide (50 µg/ml) in a 1% agarose-formaldehyde gel at 100 V and
transferred to Hybond N nylon membranes (Amersham Pharmacia Biotech).
The probes used were the subcloned PCR products described in the
previous section, except in the case of mtrp-4, for which a
full-length mtrp-4 clone derived from a
TC3 cDNA library was employed.2 Probes
were gel-purified and labeled with [
-32P]dCTP using
Prime-It random primer labeling kit (Stratagene, La Jolla, CA). After
overnight hybridization at 68 °C in ExpressHybe solution
(CLONTECH) blots were washed to a final stringency
of 0.1× SSC, 0.1% SDS at 60 °C and exposed to BioMax MS-1 film
with BioMax intensifying screen (Eastman Kodak Corp.) for 18 h to
1 week at
80 °C.
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RESULTS |
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In contrast to normal mouse islets, the murine insulinoma cell
line, TC3-neo, does not usually respond to a step increase in
glucose concentration with regular oscillatory increases in [Ca2+]i. Instead, a slow rise in
[Ca2+]i occurs with occasional intermittent
spikes (Fig. 1A). Exposure of
TC3-neo cells to 20 mM TEA, a blocker of delayed rectifier K+ channels, however, permitted the generation by
glucose of large regular oscillations in [Ca2+]i
of a type normally seen in mouse islets exposed to nutrients (5, 13,
20) (Fig. 1A). In the absence of glucose, TEA was without
effect (n = 5). This glucose dependence suggests that
the governing ionic conductance is carried by
IKATP as in the case of primary
-cells,
i.e. IKATP sets the resting membrane potential
(17, 18). Indeed, activation of IKATP with 250 µM diazoxide fully suppressed the TEA-activated
[Ca2+]i oscillations (Fig. 1B). The
[Ca2+]i oscillations were also immediately
abolished by exposure to either low Ca2+-containing
external solutions (50 µM EGTA, no added
Ca2+) or 1 µM nitrendipine, indicating their
dependence on depolarization-triggered Ca2+ influx through
voltage-dependent Ca2+ channels (VDCC) (Fig.
1C).
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It has been previously suggested that glucose-induced electrical and
[Ca2+]i oscillations result from oscillations in
metabolism (15, 31, 32). However, the experiments shown in Fig.
2 (A and B) would
indicate that direct modulation of plasma membrane conductances alone
is insufficient to induce Ca2+ oscillations. In the absence
of glucose, exposure of TC3-neo cells to 100 µM
tolbutamide to block IKATP induced
[Ca2+]i oscillations only in the presence of TEA
that were indistinguishable from those generated by glucose (Fig.
2A). Furthermore, membrane depolarization induced by 20 mM KCl was able to similarly substitute for glucose in
permitting TEA to induce [Ca2+]i oscillations
(Fig. 2B). Thus, a necessity for the conductance regulating
the [Ca2+]i oscillations to be coupled to
oscillations in KATP activity or glucose metabolism seems excluded.
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We were, however, able to measure marked alterations in the
[Ca2+]i oscillations stimulated by glucose
following exposure to thapsigargin, a selective inhibitor of the ER
Ca2+ ATPases (33, 34). Although thapsigargin caused no
detectable alteration in [Ca2+]i in the absence
of glucose, addition of thapsigargin following exposure to glucose and
TEA converted the resulting [Ca2+]i oscillations
into a sustained rise in [Ca2+]i (Fig.
3A). Previous studies in mouse
pancreatic islets and -cells have indicated that the effect of
maneuvers to deplete intracellular Ca2+ stores, as
exemplified by the action of thapsigargin, result from the activation
of ICRAN (12, 21, 34).
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The principal permeating ion carrying the depolarizing current through
ICRAN in mouse -cells is Na+
(21). To test whether ICRAN was involved in
regulating the glucose-dependent oscillations in
TC3-neo
cells, we therefore examined the effects of Na+
substitution. Reduction of external Na+ by substitution
with the non-permeant cation
N-methyl-D-glucamine largely suppressed the
[Ca2+]i oscillations induced in
TC3-neo cells
by glucose and TEA (Fig. 3B). Similarly, application of SKF
96365, a putative blocker of intracellular Ca2+ store
release-activated channels (35) reversibly abolished the
glucose-stimulated oscillations in [Ca2+]i (Fig.
3C). Nonspecific suppressive effects of SKF 96365 on
[Ca2+]i were ruled out by the
observation that KCl-induced elevations in
[Ca2+]i were unimpaired (Fig. 3D).
Application of 100 pM MTX, an agent that directly activates
nonselective cation channels in HL60 cells (36) and
ICRAN in mouse -cells (21), caused an
immediate sustained rise in [Ca2+]i, further
indicating the prominent role of this channel in regulating
[Ca2+]i in
TC3 cells (Fig.
4A). Similar to its effect on glucose-stimulated [Ca2+]i oscillations,
application of SKF 96365 also suppressed the MTX-dependent
elevation in [Ca2+]i in
TC3-neo cells (Fig.
4B). Finally, diazoxide application was able to completely
reverse the MTX-induced [Ca2+]i rise (Fig.
4C), indicating that ICRAN was a
relatively small current that could be overridden by concomitant
activation of IKATP.
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To confirm the actual presence of a nonselective cation channel in
TC3-neo cells, we carried out perforated patch recordings. Application of 1-100 pM MTX caused the activation of a
non-inactivating current with a reversal potential of
10 mV (Fig.
5A), properties identical to
ICRAN observed in mouse
-cells (21);
furthermore, both SKF 96365 and external Na+ removal were
able to reversibly attenuate the MTX-activated current (Fig. 5,
A and B). These findings suggest that the
increase in [Ca2+]i induced by MTX, and the
ablation of the glucose-stimulated [Ca2+]i
oscillations by SKF 96365 or external Na+ removal, were
secondary to activation and block of a Ca2+ store-operated,
nonselective cation channel, respectively.
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We also measured the effects of modulation of ICRAN on insulin secretion. As we described previously (20), in the presence of 20 mM TEA, 1 mM glucose gave a robust stimulation of insulin secretion (Fig. 6). We investigated the effect on insulin secretion of two mechanisms that would lead to the activation of ICRAN. Activation of the store-operated channel by inhibition of the ER Ca2+ ATPase with thapsigargin greatly potentiated glucose-stimulated insulin secretion in the presence of TEA (Fig. 6). Likewise, direct activation of ICRAN with 50 pM MTX also markedly increased glucose-stimulated insulin secretion (Fig. 6). Thus, indirect or direct activation of ICRAN leads to an amplification of glucose-stimulated insulin secretion, presumably by enhancing depolarization-driven influx of Ca2+ (21).
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To characterize the molecular identity of ICRAN,
we used RT-PCR and Southern blots of TC3-neo and MIN6 insulinoma
cells and mouse islets to identify transcript expression and prepare
probes for Northern blot analysis. Primers (see "Materials and
Methods") were designed to amplify the six known mouse trp
genes (26, 27). Products from all six trp genes were
detected in
TC3-neo cells and mouse brain. In MIN-6 cells,
trp-1, trp-2, trp-4, and trp-6 were expressed, whereas trp-3 and
trp-5 were not reproducibly observed. In mouse islets all of
the trp gene products were evident except trp-5.
RT-PCR experiments with mock cDNA synthesis in the absence of
reverse transcriptase were done in parallel, and no bands were
observed, excluding amplification of genomic DNA (data not shown). All
six trp PCR products from the brain and
TC3
amplifications were cloned and sequenced, revealing identity to those
reported previously (26, 27). We then characterized the expression of
trp genes using Northern blots comparing total RNA prepared from mouse brain and
TC3 cells (Fig.
7). This showed that, despite amplification of most of the trp genes by RT-PCR, only
mtrp-4 message was readily detectable in
TC3 cells, while
five of six trp transcripts (the exception being
trp-2, a putative pseudogene; Ref. 24) were visualized in
brain RNA. Prolonged exposures (>1 week) of several blots revealed
very faint bands in
TC3 RNA corresponding to trp-1 and
trp-3 (data not shown).
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DISCUSSION |
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We have employed a -cell model, the
TC3-neo cell, to study
the underlying mechanisms that regulate glucose-induced oscillations of
[Ca2+]i and insulin secretion. Previous studies
in insulinoma cells and islets have demonstrated a close coupling
between glucose-stimulated oscillations in membrane potential and the
consequent oscillations in [Ca2+]i and insulin
secretion (14, 15, 20). However, precise identification of the
signaling events underlying oscillations has remained elusive. In this
study, we have demonstrated the importance of a nonselective cation
current activated following depletion of intracellular Ca2+
stores in the regulation of glucose-dependent oscillations
of [Ca2+]i and show that activation of
ICRAN potentiates insulin secretion. Our
findings suggest that this ionic conductance generated by the discharge
and refilling of intracellular calcium stores plays a critical role in
regulating glucose-induced oscillatory signaling in
-cells.
Similar to the responses elicited in mouse primary -cell clusters
and islets, glucose stimulation of insulin-secreting
TC3-neo cells
results in synchronous oscillations in membrane potential and
[Ca2+]i (20). However, activation of the
oscillations required the additional presence of TEA. In this respect,
the
TC3 cells more closely resemble rat islets, which also do not
respond to glucose with large amplitude oscillations in
[Ca2+]i, but can be induced to do so by TEA (20).
This probably reflects subtle differences between the expression levels
of K+ channels between
-cells of different species.
Recently, we determined that the glucose-dependent effects
of TEA on
TC3-neo cells stemmed from inhibition of delayed rectifier
K+ channels (KDR) (20).
TC3-neo cells have a
similar complement of KDR subtypes to normal rodent
-cells, and express Shab (Kv2.1)- and Shaw
(Kv3.2)-related transcripts (18, 20). Under conditions where
IKDR is blocked,
TC3-neo cells display
glucose-dependent oscillatory responses of the types seen
in intact mouse islets or
-cell clusters. This suggests that the
TC3-neo cell line will prove useful as a relevant model to examine
the ion conductances underlying glucose-stimulated oscillatory behavior
in islets.
Several models have been advanced to explain islet bursting activity
induced by glucose. One of the earliest proposed that Ca2+
influx during the active phase caused a slow rise in
[Ca2+]i, which activated
IKCa that in turn repolarized the membrane
potential and inactivated ICa (37). Our data are
not consistent with this model, since oscillations were observed in the
presence of 10-20 mM TEA (20), conditions under which
IKCa should be completely suppressed by TEA
(Kd, 140 µM) (17). A more recent model
suggests that cyclic inhibition of IKATP, or a
direct coupling between cellular fuel metabolism and electrical activity in -cells acts as the
-cell membrane potential
oscillator (15, 32). Our findings that in the presence of complete
block of IKATP with tolbutamide,
[Ca2+]i oscillations identical in nature to those
induced by glucose are produced would rule out a requirement for
oscillations in IKATP, and by inference
metabolism, being responsible. Although cyclic variations in KATP
activity or glucose metabolism were not a prerequisite for the
induction and maintenance of [Ca2+]i
oscillations, glucose was nonetheless an important activator of
oscillatory behavior. This permissive effect of glucose and tolbutamide
is most likely related to blockade of the repolarizing influence of
IKATP, the predominant ionic current in
pancreatic
-cells. In support of this contention, direct overriding
of IKATP using depolarizing concentrations of
KCl was similarly able to substitute for both glucose or tolbutamide in
permitting the induction of [Ca2+]i oscillations.
As will be discussed further, the inhibition of
IKATP allows smaller conductance(s) to exert
significant effects on the membrane potential and, indirectly, on
[Ca2+]i.
In addition to stimulating the influx of Ca2+ as a result
of depolarization-induced opening of VDCCs, it has been recently
suggested that glucose mobilizes intracellular Ca2+ from
the ER (5-7). As a corollary of this triggered release, glucose has
been demonstrated by a number of groups to stimulate the sequestration
of Ca2+ in the ER (34, 38, 39). The state of filling of the
ER with Ca2+ in turn seems to regulate a plasma membrane
nonselective cation channel in pancreatic -cells, akin to the
"capacitative" Ca2+ entry system described in
non-excitable cells (40). This pathway permits the refilling of
depleted intracellular Ca2+ stores by activation of a
voltage-independent Ca2+ current called
ICRAC (Ca2+ release-activated
Ca2+ current), that directly supplies Ca2+ to
the depleted organelle (41). By contrast, in
-cells
ICRAN enhances the entry of Ca2+
through ICa by causing a sustained membrane
depolarization (21). We have confirmed the presence of
ICRAN in
TC3 cells using patch clamp
techniques, whose properties are indistinguishable from the current
previously described in primary mouse
-cells (21). Thus, the current
is activated by exposure to MTX and the principal permeating ion seems
to be Na+; the reversal potential of around
10 mV
confirms the nonselective nature of the channel.
Since in other cell systems the mobilization of intracellular
Ca2+ stores is often oscillatory in nature (42), this
suggests that a conductance activated by the state of filling of
intracellular Ca2+ stores (e.g.
ICRAN) would similarly oscillate and could therefore underlie the membrane potential oscillations seen in islets and -cells exposed to glucose. It was therefore of great interest to
observe that pharmacological manipulation of
ICRAN resulted in alterations in
[Ca2+]i oscillations in
TC3-neo cells that
were consistent with such a role for the channel. Thus, depletion of
intracellular Ca2+ stores with thapsigargin to induce
activation of ICRAN resulted in a conversion of
oscillatory alterations in [Ca2+]i to a sustained
rise. That this was due to continuous entry of Ca2+ through
ICa, activated as a consequence of
ICRAN-dependent membrane depolarization, was demonstrated by the ablative effects of the L-type
Ca2+ channel inhibitor
nitrendipine.3 Consistent
with its effects on glucose-stimulated alterations in
[Ca2+]i, thapsigargin potentiated the insulin
secretion response of
TC3-neo cells elicited by glucose and TEA.
Direct activation of ICRAN following exposure to
MTX induced a similar, nitrendipine-sensitive sustained elevation in
[Ca2+]i. MTX also stimulated insulin secretion in
the presence of glucose alone, to the same extent as that produced by
the combined administration of glucose, TEA and thapsigargin.
Interestingly, in the absence of glucose, MTX was without effect on
insulin secretion,3 consistent with the proposal that the
MTX-activated current is of small magnitude and unable to override
IKATP, which would be activated under these
conditions. Suppression of current flow through
ICRAN had the opposite effect on the
glucose-stimulated [Ca2+]i oscillations in
TC3-neo cells. Thus, SKF 96365, a blocker of intracellular
Ca2+ store release-activated channels (35) completely
suppressed glucose-induced [Ca2+]i oscillations.
Although this compound has also been shown to block L-type calcium
channels at high concentrations, the demonstration that KCl-induced
depolarization raised [Ca2+]i in the presence of
SKF 96365 suggests that voltage-dependent calcium channels
were not inhibited. Furthermore, reduction of extracellular
Na+, the principal permeating ion through
ICRAN, similarly ablated the glucose stimulated
oscillations.
We initially characterized the putative molecular identity of
ICRAN using RT-PCR with Southern blot analysis,
and identified trp genes (trp1-6) expressed
differentially in two mouse insulinoma cells and islets of Langerhans.
These findings are in partial agreement with recent studies in which
the expression of mouse trp-1 was reported in MIN6 cells
(43). However, upon Northern blot analysis, trp-4 was the
only detectable trp transcript expressed in TC3
insulinoma cells, whereas five of the six genes were readily identified
in mouse brain (Fig. 7).
Drosophila trp and trp-like (trpl)
genes encode Ca2+ store-operated Ca2+ channels
and inositol 1,4,5-trisphosphate-activated nonselective cation
channels, respectively (22, 23). Recent evidence has suggested that
mammalian trp family channels may have several activation
mechanisms, with functional channels encoded by multiple subunits (44).
Mammalian trp genes are unlikely to direct the expression of
ICRAC, as none of the expressed trp
genes to date are able to reproduce the Ca2+ selectivity or
single channel conductance (0.03 picosiemens) of the native channel
(44). For example, htrp-1, also termed hTrpC1, encodes a
nonselective channel activated by depletion of Ca2+ stores
with a single channel conductance of 16 picosiemens (45). Both TrpC1
and TrpC3 can increase calcium entry following depletion of
intracellular stores (27). When transfected into murine
L(tk) cells, a mixture of plasmids containing antisense
cDNAs for six mouse trp genes eliminated the
thapsigargin-induced increase in [Ca2+]i (27),
and a similar transfection with a 1.2-kilobase pair antisense fragment
to mtrp4 alone also eliminated receptor-stimulated calcium
entry (46). mtrp6 was likewise found to increase
Ca2+ entry when stimulated by activation of a cotransfected
muscarinic receptor, an effect that was blocked by SKF 96365, an agent
we employed here to also block store-operated currents (47). The similarity of ICRAN to trp family
channels is further supported by the finding of a single channel
conductance of 25 picosiemens for the MTX-sensitive nonselective cation
channel activated by ER Ca2+ store depletion in
fibroblasts, within the range reported for htrp-1 (48). Our
findings thus support the likelihood that trp-related genes
encode not only the current we have termed
ICRAN, but also the
Na+-dependent inward currents that cause rises
in [Ca2+]i in
-cells following
Ca2+ store depletion with carbachol, acetylcholine,
pituitary adenylate cyclase activating polypeptide, and glucagon-like
peptide-1 (49-52).
Fig. 8 depicts a model in which
ICRAN regulates glucose-stimulated
oscillations of membrane potential and [Ca2+]i.
This model provides an explanation for the oscillatory behavior of
TC3-neo cells in the presence of glucose, and may also be applicable
to islet responses following nutrient stimulation. An experimental
model centered around ICRAN modulation has also recently been advocated to explain the effects of carbachol on [Ca2+]i and membrane potential (49). In
conclusion, our data suggest that ICRAN
modulation plays a critical role in controlling nutrient-dependent electrical bursting behavior in islets,
and also demonstrates that alterations in ICRAN
activity modulate insulin secretion. Since defective ER
Ca2+ sequestration has been demonstrated in several animal
models of non-insulin-dependent diabetes mellitus (53, 54),
it is possible that alterations in ICRAN
regulation may play a role in aberrant insulin secretion responses in
some forms of non-insulin-dependent diabetes mellitus.
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FOOTNOTES |
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* 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. E-mail:
id22327{at}glaxowellcome.com.
1 The abbreviations used are: [Ca2+]i, intracellular free Ca2+ concentration; CRNSC, calcium release-activated nonselective cation channel; ER, endoplasmic reticulum; ICa, calcium current; ICRAC, Ca2+ release-activated Ca2+ current; ICRAN, calcium release-activated nonselective cation current; IKATP, ATP-sensitive K+ current; IKCa, calcium-activated K+ current; IKDR, delayed rectifier K+ current; KATP, ATP-sensitive K+ channel; Kv, delayed rectifier K+ channel; mtrp, murine trp; MTX, maitotoxin; PCR, polymerase chain reaction; RT, reverse transcriptase; TEA, tetraethylammonium; trp; transient receptor potential gene; VDCC, voltage-dependent Ca2+ channel.
2 F. Qian and L. H. Philipson, manuscript in preparation.
3 M. W. Roe, A. A. Mittal, L. H. Philipson, and I. D. Dukes, unpublished observations.
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