(Received for publication, June 4, 1996, and in revised form, May 15, 1997)
From the Laboratory of Molecular Endocrinology, Howard Hughes Medical Institute, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114
Maitotoxin (MTX) activates a
Ca2+-dependent non-selective cation
current (ICa-NS) in insulinoma cells whose time course is
identical to non-selective cation currents activated by incretin
hormones such as glucagon-like peptide-1 (GLP-1), which stimulate
glucose-dependent insulin secretion by activating cAMP
signaling pathways. We investigated the mechanism of activation of
ICa-NS in insulinoma cells using specific pharmacological
reagents, and these studies further support an identity between MTX-
and GLP-1-activated currents. ICa-NS is inhibited by
extracellular application of genistein, econazole, and SKF 96365. This
inhibition by genistein suggests that tyrosine phophorylation may play
a role in the activation of ICa-NS. ICa-NS is
not inhibited by incubation of cells in glucose-free solution, by
extracellular tetrodotoxin, nimodipine, or tetraethylammonium, or by
intracellular dialysis with 4-aminopyridine, ATP, ryanodine, or
heparin. ICa-NS is also not significantly inhibited by
staurosporine, which does, however, partially inhibit the MTX-induced
rise of intracellular Ca2+ concentration. These effects of
staurosporine suggest that protein kinase C may not be involved in the
activation of ICa-NS but that it may regulate intracellular
Ca2+ release. Alternatively, ICa-NS may have a
small component that is carried through separate divalent
cation-selective channels that are inhibited by staurosporine.
ICa-NS is neither activated nor inhibited by dialysis with
KF, KF + AlF3 or GTPS (guanosine 5
-O-(3-thiotriphosphate)), suggesting that GTP-binding
proteins do not play a major role in the activation of this
current.
The consensus model of glucose-stimulated insulin secretion is
that closure of ATP-sensitive K+ channels
(K+ATP)1 permits
membrane depolarization, activation of voltage-dependent Ca2+ channels (VDCCs) and the influx of Ca2+
(1). Individual -cells, however, are often unresponsive to glucose
alone, but can become responsive by combined stimulation with glucose
and hormones, such as insulinotropic hormone glucagon-like peptide-1
(GLP-1; Ref. 2), that elevate intracellular cAMP levels (3-5). One
mechanism underlying this increased responsiveness is the enhanced
closure of K+ATP channels (2). A second
mechanism by which GLP-1, pituitary adenylate cyclase-activating
polypeptide (PACAP), and cAMP can induce
-cell depolarization is
through the activation of voltage-independent, non-selective cation
currents (6, 7). Similar cation currents are also activated by MTX, a
polyether toxin isolated from dinoflagellates that in
-cells has
been shown to stimulate insulin secretion and inositol trisphosphate
(Ins(1,4,5)P3) production (8) and to enhance the influx of
monovalent cations (9).
MTX-sensitive currents are activated by depletion of intracellular
Ca2+ stores (10), and GLP-1 enhances intracellular
Ca2+ mobilization through the potentiation of
ryanodine-sensitive Ca2+-induced Ca2+ release
in TC3 cells (11, 12). Increased cAMP levels stimulate Ca2+ release from secretory granules and reduce
mitochondrial Ca2+ uptake in
-cells (13, 14). These
observations raise the possibility that the activation of non-selective
cation currents by PACAP and GLP-1 may be a secondary consequence of
glucose- and cAMP-dependent intracellular Ca2+
release. The physiological role of Ca2+ release-activated
currents in
-cells remains controversial, but such currents have
been suggested to play a role in the cholinergic modulation of
electrical bursting activity (15), and may control the membrane
potential and intracellular Ca2+
([Ca2+]i) oscillations in response to nutrient
stimulation (10).
Both PACAP (16) and GLP-1 (17) are potent insulin secretagogues in the
presence of slightly elevated glucose levels. The activation of a
voltage-independent, non-selective cation current by these hormones
under conditions that stimulate insulin secretion suggests that this
current may play an important role in depolarizing -cells to
initiate insulin secretion. The aim of this study is to examine the
mechanism of activation of the MTX-sensitive current and to compare the
properties of ICa-NS with the current activated by GLP-1,
PACAP, and cAMP to determine whether these currents are likely to be
carried through the same channels.
HIT-T15 cells were obtained
from the American Type Culture Collection. TC6 cells were obtained
from Dr. Shimon Efrat (Albert Einstein College of Medicine, New York,
NY). HIT-T15 cells (passages 67-75) were maintained in Ham's F-12
medium containing 10 mM glucose, 10% heat-inactivated
horse serum, and 2.5% fetal bovine serum.
TC6 cells (passages
24-59) were maintained in Dulbecco's modified Eagle's medium
containing 25 mM glucose, 15% horse serum, and 2.5% fetal
bovine serum. Culture media also contained 100 units/ml penicillin G
and 100 µg/ml streptomycin. Cells were plated onto glass coverslips
coated with 1 mg/ml of type V concanavalin A (Sigma), which facilitates
their adherence to glass. Cultures were maintained at 37 °C in a 5%
CO2 atmosphere incubator, and experiments were
conducted 1-5 days post-plating.
Cells were bathed in a standard
extracellular solution (SES) containing: 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES (295 mosM; pH adjusted to 7.4 with NaOH, approximately 4 mM), and 0.8 mM D-glucose unless
indicated as being different in the text. Na+-free,
N-methyl-D-glucamine (NMG) solutions were
prepared using 138 mM NMG substituted for NaCl and adjusted
to pH 7.4 with HCl. A Na+-free, 100 Ca solution was also
prepared containing: 100 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES (282 mosM; pH adjusted to 7.4 with KOH, approximately 6 mM) Other test solutions were prepared by substitution of
NaCl with 140 mM KCl, CsCl, LiCl, or choline chloride.
Ca2+-free solutions were prepared by substituting
MgCl2 or MnCl2 for CaCl2. Low
[Cl]o solution was prepared by substituting
NaCl with Na-aspartate.
Test solutions containing MTX were applied to individual cells by focal
application from micropipettes using a PicoSpritzer II pressure
ejection system (General Valve, Fairfield, NJ). A gravity-fed bath
superfusion system was used to exchange and refresh bath solutions.
Maitotoxin, econazole, staurosporine, nimodipine, and glyburide were
obtained from Sigma. Tetrodotoxin, SKF 96365, genistein, GTPS,
ryanodine, and heparin were obtained from Calbiochem. Tetraethylammonium chloride (TEA) and 4-aminopyridine (4AP) were obtained from Aldrich.
Cells were prepared for measurement of [Ca2+]i by incubation in fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes, Inc., Eugene, OR). Cells were loaded in SES supplemented with 2% fetal bovine serum, 0.03% pluronic F-127, and 1 µM fura-2 AM for 15 min at room temperature (20-22 °C). Coverslips with fura-loaded adherent cells formed the base of a recording chamber mounted on a temperature-controlled stage (Micro Devices, Jenkintown, PA). Cells were visualized using a Zeiss IM35 microscope equipped with a Nikon UVF100 100× objective. Measurements of [Ca2+]i were performed at 1-s intervals from the average of 10 video frames using a dual excitation wavelength video imaging system (IonOptix Corp., Milton, MA). Experiments were conducted at 32 °C. [Ca2+]i was estimated from the ratio of 510 nm emission fluorescences due to excitation by 350 nm and 380 nm wavelength light from the following equation (18).
![]() |
(Eq. 1) |
Cell resting potentials and membrane currents were measured under current clamp or voltage clamp using either whole-cell or perforated patch configurations (19, 20). Patch pipettes pulled from borosilicate glass (Kimax-51, tip resistance 2-4 megohms) were fire polished and tip-dipped in K- or Cs-pipette solution containing: 95 mM K2SO4 (or Cs2SO4), 7 mM MgCl2, 5 mM HEPES (pH adjusted to 7.4 with NaOH; final concentration of Na+ approximately 2 mM). Pipettes were then back-filled with the same solution, to which nystatin (240 µg/ml) was added for perforated patch recording.
The patch pipette was connected to an Heka Electronik EPC-9 patch clamp amplifier (Instrutech Corp., Mineola, NY) interfaced with a Macintosh Quadra 840AV computer running Pulse version 8.0 software (Instrutech Corp.). The series resistance (Rs) and cell capacitance (Cm) were monitored following seal formation, and experiments were conducted when Rs declined to <35 megohms. In voltage clamp experiments, Rs was compensated for by 60%.
Values are given as mean ± S.E. Statistical significance was determined using Student's t test computed using Lotus 1-2-3 spreadsheets.
MTX activates a non-selective cation current in TC6 cells (Fig.
1) that has a reversal potential of -7.8 ± 1.0 mV
(n = 72) in SES (142 mM
[Na+]o). This current was observed in all cells
tested, and its reversal potential becomes more negative as
[Na+]o is reduced (Fig. 1, Table
I), although the shift is less than predicted from the
Nernst potential for Na+. ICa-NS is also
observed in solutions where extracellular Na+ is replaced
by Cs+, K+, Li+,
choline+, or NMG+ and in cells dialyzed with
K+, Cs+, Na+, choline+,
or NMG+ with Na+ in the bathing solution (Table
II), confirming the non-selective nature of this
current. Changing from normal [Cl
]o to low
[Cl
]o extracellular solution did not affect the
amplitude or reversal potential of the current (data not shown),
confirming the cation selectivity of ICa-NS.
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It is reported that Ca2+ is impermeant through
MTX-activated channels in mouse -cells (10) in contrast with a
report that Ca2+ is permeant through such channels in mouse
L-cells (21). We therefore decided to re-examine the Ca2+
permeability of ICa-NS. Ca2+ influx through
MTX-sensitive channels in HIT-T15 cells is suggested by an increase of
[Ca2+]i when hyperpolarizing voltage steps are
applied following activation of the MTX-sensitive current and by the
rapid and reversible fall of [Ca2+]i when
extracellular Ca2+ is removed (Figs. 2 and
3A). Fig. 2 shows that a hyperpolarizing voltage clamp step
from
70 mV to
100 mV had no effect on [Ca2+]i
in a HIT-T15 cell before stimulation with MTX, but that similar voltage
steps applied after activation of ICa-NS resulted in a
pronounced increase of [Ca2+]i that was
reversibly abolished by removal of extracellular Ca2+.
Hyperpolarizing pulses applied following activation of non-selective cation currents by GLP-1 (22) or 8-bromo-cAMP (7) also increased [Ca2+]i.
We further tested the effects of removal of extracellular
Ca2+ to confirm the activation of MTX-sensitive currents by
Ca2+-free solutions (10) and to determine the effects of
activation of the current on [Ca2+]i in the
absence of extracellular Ca2+. In 15/18 cells tested, bath
perfusion with a Ca2+-free solution containing 50 µM EGTA (the Ca2+ concentration in this
solution was measured (using K5 fura-2) to be 150 nM) before stimulation with MTX caused a small, reversible increase of the membrane current and a fall of
[Ca2+]i (Fig. 3A).
Holding currents at 70 mV increased from
0.96 ± 0.16 pA/pF in
SES to
1.64 ± 0.30 pA/pF (p = 0.005) and [Ca2+]i decreased from 117 ± 15 nM to 99 ± 15 nM on removal of
extracellular Ca2+ (not statistically significant,
p = 0.4). In 3/18 cells the holding current showed a
much larger increase on transfer to Ca2+-free solution
(Fig. 3B). The holding current in these cells increased by
21.8 ± 4.3 pA/pF and [Ca2+]i reduced from
75 ± 15 nM to 37 ± 4 nM
(p = 0.1). This current inactivates rapidly, and
[Ca2+]i is transiently elevated when SES (2.6 mM Ca2+) is reintroduced to the bath (Fig.
3B). Elevation of [Ca2+]i and
activation of a non-selective cation current were also observed in a
similar subset of cells following stimulation with thapsigargin (2-10
µM, data not shown), as reported previously (10).
Bath perfusion with Ca2+-free solution after the activation
of ICa-NS results in a rapid, reversible fall of
[Ca2+]i but has only a small effect on the
current amplitude (Figs. 2 and 3A). The reversal potential
of the MTX-activated current in Ca2+-free solution is
10.1 ± 2.8 mV (n = 12), not significantly
different (p = 0.43) from the reversal potential
measured in SES. These data suggest that Ca2+-influx
carries only a small proportion of the current through MTX-sensitive
channels in the presence of Na+. However, the rapid and
reversible effects of removing extracellular Ca2+ on
[Ca2+]i suggests that ICa-NS does
permit Ca2+ entry.
Further evidence for the influx of divalent cations through MTX-sensitive channels is suggested by increased Mn2+ quenching of intracellular fura-2 fluorescence following MTX-stimulation (Fig. 3C). Fig. 3C (i and ii) shows the raw fura-2 fluorescence emission values, application of a 60-s pulse of Ca2+-free, Mn2+-substituted solution before stimulation with MTX produces a gradual quenching of the fluorescence signals, a small increase in the holding current (Fig. 3C, iii), and little or no change in the 350 nm/380 nm fluorescence ratio (Fig. 3C, iv). Following activation of the inward current and rise in [Ca2+]i, a pulse of Ca2+-free, Mn2+-substituted solution produces rapid quenching of fura-2 fluorescence (Fig. 3C, i and ii) with little or no effect on membrane current (Fig. 3C, iii) and reduces the fluorescence ratio (Fig. 3C, iv), consistent with a decrease in [Ca2+]i. Similar activation of Mn2+ quenching has been observed following stimulation of insulinoma cells with PACAP (6), cAMP (22), and thapsigargin (23, 24).
A role for Ca2+ influx through MTX-sensitive channels is
further supported by the effects of applying high
[Ca2+]o solutions. A Na+-free test
solution containing 100 mM Ca2+ was applied to
a TC6 cell prior to stimulation with MTX and produces a small
increase of [Ca2+]i (Fig. 4),
similar to the effects of applying elevated [Ca2+]o solutions to mouse islets (25).
Application of 100 mM [Ca2+]o
solution following stimulation with MTX produces a pronounced
inhibition of ICa-NS (Fig. 4) and a negative shift in the
reversal potential of the current (Table I). This inhibition of
ICa-NS by the 100 mM Ca2+ solution
is accompanied by a rise of [Ca2+]i (Fig. 4).
These data could be explained by Ca2+ influx through a
single class of non-selective cation channel with a lower permeability
to Ca2+ than to Na+ under these experimental
conditions. Alternatively, ICa-NS may have two (or more)
components, one component being monovalent cation selective, and the
other, smaller, component being selective for divalent cations.
The non-selective cation current activated by 8-bromo-cAMP in HIT-T15
cells is inhibited by whole cell dialysis with Ca2+-free,
EGTA-buffered solutions or by loading the cells with BAPTA (1,2-bis(2-aminophenoxy)ethane N,N,N,N
-tetraacetic acid),
a Ca2+ chelator (7) leading us to test the dependence of
ICa-NS activation on [Ca2+]i (Fig.
5). Whole cell recordings from HIT-T15 cells were
performed with either normal K-pipette solution (nominally Ca2+-free) or the same solution with 5 mM EGTA
added (Ca2+-free). Whole cell dialysis with
Ca2+-free intracellular solution inhibited activation of
ICa-NS compared with control cells from the same platings
dialyzed with nominally Ca2+-free solution or compared with
cells in perforated patch voltage clamp (Fig. 5). These observations
suggest that physiological [Ca2+]i levels are
required for activation of the current. It is notable that dialysis of
the cells with Ca2+-free solution does not induce
activation of the current alone, whereas dialysis with this solution
might be expected to deplete intracellular Ca2+ stores and
thus activate store-operated currents.
Ca2+-activated non-selective cation (Ca-NS) channels are
expressed in -cells that are activated at cytosolic
[Ca2+] > 10
4 M and are blocked
by 1 mM ATP and also by 10 mM 4AP when applied to the cytosolic face of isolated, inside-out patches (26, 27). Activation of ICa-NS is observed with physiological
[Ca2+]i levels but inhibition of the current by
dialysis of cells with Ca2+-free solutions suggests that it
may be carried through Ca-NS channels. To test this possibility,
TC6
cells were bathed in glucose-free SES with 5 mM TEA and 10 nM glyburide added (to block Ca2+-activated
K+ channels and K+ATP channels) and
dialyzed in the whole cell recording mode with K-pipette solution and
10 mM 4AP either with or without 2 mM ATP. MTX-induced currents in cells dialyzed without ATP had a mean peak
amplitude of
23.6 ± 7.9 pA/pF (n = 6), and
cells from the same platings dialyzed with 2 mM ATP had
peak amplitudes of
22.5 ± 7.5 pA/pF (n = 6, not
significantly different, p = 0.9). These data indicate
that activation of ICa-NS is not
glucose-dependent, consistent with previous reports of
MTX-stimulated, glucose-independent insulin secretion (28). These
differences in the sensitivity of ICa-NS to
[Ca2+]i and to block by ATP and 4AP in whole cell
recordings compared with inside-out patches may indicate that
ICa-NS is not carried through the Ca-NS channels reported
previously (26, 27) or may reflect the different recording
configurations.
The non-selective cation current activated by PACAP in TC6 cells is
inhibited by SKF 96365 (22), a blocker of depletion-activated currents
(29) that inhibits MTX-induced Ca2+ influx and insulin
secretion (30). Fig. 6 shows that application of 50 µM SKF 96365 reversibly inhibits ICa-NS in
TC6 cells, similar to its effect on the PACAP-induced current in
these cells (22), further supporting the suggestion that these currents
are carried by the same channel type.
The activation of Mn2+ quenching of intracellular fura-2
fluorescence by MTX (Fig. 3C) is similar to that observed
following thapsigargin treatment of insulinoma cells (23, 24).
Thapsigargin-sensitive Ca2+ pools can also be depleted by
econazole, which inhibits Ca2+-ATPases (31) and thereby
elevates [Ca2+]i and also inhibits
Mn2+ quenching in HIT cells (23). We therefore tested the
effect of econazole on the activation of ICa-NS. Fig.
7A shows that 10 µM econazole
significantly reduces the amplitude of ICa-NS compared with
control cells from the same platings. The basal (pre-MTX) [Ca2+]i rose from 52 ± 7 nM to
182 ± 41 nM (n = 5, p = 0.01) in the presence of econazole and the peak amplitude of
ICa-NS decreased from 12.6 ± 2.3 pA/pF to
3.6 ± 1.1 pA/pF (n = 5, p = 0.01). The rise of [Ca2+]i above basal during the
MTX response was reduced from 449 ± 206 nM to 49 ± 14 nM (p = 0.09) by econazole.
The effect of genistein, a tyrosine kinase inhibitor, on the activation
of ICa-NS was tested as capacitative Ca2+
influx activated by thapsigargin can also be inhibited by genistein (Ref. 32, Fig. 7B). The peak amplitude of ICa-NS
reduces from 20.2 ± 3.3 pA/pF (n = 6) in
control cells to
8.2 ± 2.6 pA/pF (n = 8, p = 0.01) following exposure of cells from the same
platings to 100 µM genistein. Basal
[Ca2+]i (pre-MTX) is not significantly affected
by genistein (88 ± 12 nM in control cells, 118 ± 19 nM in genistein, p = 0.2), while the
peak rise of [Ca2+]i during MTX responses reduced
from 1026 ± 388 nM (control) to 313 ± 92 nM (genistein, p = 0.06).
Protein kinase C (PKC) activates capacitative Ca2+ entry in
rat insulinoma (RINm5F) cells, and this activation of Ca2+
entry can be blocked by 1 µM staurosporine (24). We
therefore tested the effects of 1 µM staurosporine on
membrane currents and on the rise of [Ca2+]i
following MTX stimulation of TC6 cells (Fig. 7C). Staurosporine had no significant effect on ICa-NS; the mean
peak amplitude of control currents was
54.2 ± 19.5 pA/pF
(n = 5) compared with
51.1 ± 7.7 pA/pF
(n = 6, p = 0.9) in cells from the same platings after addition of 1 µM staurosporine to the
bathing solution. The pre-MTX basal [Ca2+]i in
control cells was 61 ± 11 nM and 96 ± 15 nM in cells bathed in staurosporine (p = 0.1), whereas the peak rise (increase above basal levels) of
[Ca2+]i was reduced from 1653 ± 252 nM to 142 ± 28 nM (p < 0.001) by staurosporine.
Activation of some types of non-selective cation current has been shown
to be mediated by GTP-binding proteins (33). We therefore examined the
potential role of GTP-binding proteins in the activation of
ICa-NS by dialysis of TC6 cells with Cs-pipette solution
supplemented with 10 mM 4AP, 2 mM
Na2ATP, and either 10 mM KF, 10 mM
KF + 100 µM AlF3, or 100 µM
GTP
S. The bath contained SES with 5 mM TEA and 1 µM TTX. Whole cell dialysis of cells for 15-20 min with
KF (n = 4), KF + AlF3 (n = 5), or GTP
S (n = 5) failed to activate inward
currents in cells that all subsequently responded to stimulation with
MTX (data not shown).
MTX stimulates an increase in Ins(1,4,5)P3 levels in
-cell lines (8), and this increase might activate ICa-NS
through an intracellular Ca2+ release mechanism. Heparin is
a specific blocker of Ins(1,4,5)P3 receptors that should
inhibit activation of ICa-NS if
Ins(1,4,5)P3-gated Ca2+ stores play an
important role.
TC6 cells were dialyzed in whole cell recordings
with Cs-pipette solution plus 10 mM 4AP, 2 mM Na2ATP, and 0.5 mg/ml heparin for 3-4 min before
stimulation with MTX. Activation of ICa-NS was not
inhibited in cells dialyzed with heparin. The amplitude of the currents
was not significantly different from that in control cells from the
same platings, and the reversal potential of the currents was
7.0 ± 2.0 mV (n = 9), not significantly
different from control values (p = 0.96).
GLP-1 enhances intracellular Ca2+ mobilization from
ryanodine-sensitive stores in TC3 cells, and ryanodine reduces the
amplitude of [Ca2+]i spikes produced by
depolarizing voltage clamp steps within 1 or 2 min (11). We introduced
100 µM ryanodine into
TC6 cells by whole cell dialysis
in the Cs/4AP/ATP-pipette solution (as above) and allowed 3-4 min
dialysis before stimulation with MTX. Ryanodine failed to prevent
activation of ICa-NS under these conditions, and the
current amplitude and reversal potential (-8.0 ± 1.9 mV,
n = 5) are not significantly different from control cells (p = 0.97).
We propose that MTX activates the same non-selective cation
current as stimulation with the peptide hormones GLP-1 and PACAP (6, 7,
22). These hormones couple through GTP-binding proteins (G-proteins) to
activate adenylyl cyclase and elevate intracellular cAMP in -cells
(3-5), and cAMP analogs can also activate these non-selective cation
currents. However, activation of G-proteins, by dialysis of cells with
KF, KF + AlF3, or GTP
S (compounds that stimulate
G-protein mediated activation of non-selective cation currents in
epithelial cells (33) and activate K+ATP
channels in RINm5F and HIT-T15 insulinoma cells (34, 35)), neither
activated nor inhibited the MTX-sensitive current.
Depletion of intracellular Ca2+ stores activates a
MTX-sensitive current in mouse -cells (10), and the stimulation of
Ins(1,4,5)P3 production by MTX (8) raises the possibility
that Ins(1,4,5)P3-gated stores may play a role in the
activation of this current. Parasympathetic, cholinergic stimulation of
-cells stimulates Ins(1,4,5)P3 production, potentiates
glucose-induced insulin secretion (36), and also activates a
TTX-insensitive Na+-dependent depolarizing
current (37) that may also be carried through Ca2+
release-activated non-selective cation channels (15). The role of
intracellular Ca2+ release in the activation of this
current has, however, been disputed, and cholinergic activation of this
Na+ current is reported to be mediated by
M3-type muscarinic receptors being coupled to
Na+ channels (38). We observed that dialysis with heparin,
a blocker of Ins(1,4,5)P3 receptors, failed to inhibit
activation of ICa-NS, suggesting that Ca2+
release from these stores may not be critical.
The presence of a Ca2+ store depletion-activated current in
pancreatic -cells was proposed from studies showing: 1) that the state of filling of endoplasmic reticulum stores could regulate the
membrane potential in mouse
-cells (39), 2) that thapsigargin can
activate Mn2+ quenching of intracellular fura-2 in RINm5F
cells (24), and 3) that the Mn2+ quenching pathway is
inhibited by econazole in HIT-T15 cells (23). Activation of
ICa-NS by thapsigargin and its inhibition by both econazole
and SKF 96365 are consistent with the suggestion that
ICa-NS may represent a Ca2+ release-activated
current (10, 31).
A role for PKC in the activation of store-operated Ca2+ entry in RINm5F cells was suggested from observations that the sustained [Ca2+]i rise in response to combined stimulation with PKC-activating phorbol esters and thapsigargin is inhibited by staurosporine (24). We observed that staurosporine reduced the MTX-induced [Ca2+]i rise but did not significantly inhibit the amplitude of ICa-NS, suggesting that PKC may not play a direct role in the activation of ICa-NS but does regulate [Ca2+]i responses. Such effects could be mediated through inhibition of Ca2+ release from intracellular Ca2+ stores, or could reflect the inhibition of a small, divalent cation selective component of ICa-NS carried through a distinct set of channels other than the non-selective cation channels. The high concentration of staurosporine (1 µM) used in these experiments would also be expected to inhibit cAMP-dependent protein kinase, and further studies are required to elucidate the role(s) of PKC and cAMP-dependent protein kinase in the pathway(s) leading to the activation of ICa-NS and regulation of [Ca2+]i changes.
Inhibition of both ICa-NS and the MTX-induced [Ca2+]i rise by genistein, an inhibitor of tyrosine kinases (40) that blocks thapsigargin- and carbachol-induced Ca2+ entry (32, 41), suggests a role for tyrosine phosphorylation in the activation pathway of ICa-NS. However, the role of tyrosine phosphorylation remains ambiguous as only certain tyrosine kinase inhibitors (including genistein) effectively block capacitative Ca2+ entry (42). Thapsigargin-induced Ca2+ entry can also occur in the absence of detectable tyrosine phosphorylation but is still inhibited by tyrosine kinase inhibitors (43), and, therefore, the role of tyrosine kinases in activation of ICa-NS remains to be clarified.
Ca-NS channels are expressed in -cells that are activated at
cytosolic [Ca2+] > 10
4 M (26,
27). Activation of ICa-NS is observed at physiological [Ca2+]i (approximately 100 nM) and is
inhibited by dialysis of cells with Ca2+-free solution,
similar to the inhibition of cAMP-activated currents (7) and suggesting
a Ca2+-dependent step in the activation pathway
of these channels. Ca-NS channels are also expressed in pancreatic
acinar cells, and these channels are activated at much lower cytosolic
Ca2+ concentrations in whole cell records than in isolated
patches (44); a similar difference in Ca2+ sensitivity
seems likely to occur for Ca-NS channels in
-cells. Ca-NS channels
are blocked by 1 mM ATP and by 10 mM 4AP when
applied to the intracellular face of isolated membrane patches (27); however, these two compounds did not inhibit ICa-NS when
introduced into the cytosol by whole cell dialysis (at 2 mM
and 10 mM, respectively). It remains to be determined
whether these differences in sensitivity to
[Ca2+]i, 4AP, and ATP are a consequence of the
different recording conditions or suggest that Ca-NS channels do not
carry ICa-NS.
Reducing extracellular [Cl] has no effect on
ICa-NS amplitude or on its reversal potential, confirming
the cation selectivity of the channel. Depletion of intracellular ATP
levels through bathing cells in glucose-free media and dialyzing with
ATP-free solutions or dialysis of cells with 2 mM ATP has
no effect on ICa-NS amplitudes. This further distinguishes
the MTX-induced current from the non-selective anion current described
in insulin-secreting cells that increases in amplitude upon dialysis
with 2 mM ATP (45).
Elevation of [Ca2+]i is observed following the activation of non-selective cation currents by MTX, GLP-1, or PACAP in voltage clamped cells where activation of voltage-dependent Ca2+ channels is prevented (6, 7, 22). This rise of [Ca2+]i is reversed by removal of extracellular Ca2+, suggesting that Ca2+ influx is associated with the non-selective current, although it remains to be determined whether a single class of channel is permeant to both monovalent and divalent cations, or if two (or more) distinct conductances are involved. The physiological role of Ca2+ influx associated with ICa-NS in the stimulation of insulin secretion remains to be determined. It has been reported that sustained Ca2+ influx through L-type VDCCs is strongly coupled to insulin secretion from HIT-T15 cells, whereas more transient Ca2+ influx through N-type Ca2+ channels is only weakly coupled (46). The Ca2+ influx associated with ICa-NS is prolonged and may, therefore, be able to contribute to the sustained [Ca2+]i elevation that triggers secretion. However, the magnitude of this Ca2+ influx is likely to be small compared with that through L-type channels as, under physiological conditions, the cells will depolarize to a value close to that for the reversal potential of ICa-NS, and also influx through L-type VDCCs raises [Ca2+]i very rapidly, whereas the [Ca2+]i increase associated with ICa-NS develops much more slowly. This slow time course of the rise in [Ca2+]i is consistent with a small amplitude Ca2+ influx and would explain why it is difficult to resolve a decrease in ICa-NS amplitude on changing to Ca2+-free solution with normal extracellular Na+ concentrations. It therefore seems that the main physiological role for these non-selective cation currents in the control of insulin secretion will be to depolarize the membrane potential and activate VDCCs.
The currents activated by GLP-1, PACAP, cAMP analogs, and MTX are
Ca2+-dependent non-selective cation currents
that activate over tens of seconds and persist for extended periods
following removal of the stimulus. These currents are all insensitive
to TTX, L-type Ca2+ channel blockers, TEA, and ryanodine
but are inhibited by NMG, SKF96365, and La3+. Based upon
these similarities between the temporal properties of the currents, and
their associated [Ca2+]i changes, and the
pharmacology of the currents, we propose that these agents activate the
same non-selective cation channels. The precise mechanism(s)
controlling the activation of these non-selective cation channels
remains to be determined, but a role for tyrosine kinase-induced
phosphorylation is suggested by the effects of genistein. Activation of
ICa-NS may also be controlled by the state of filling of
intracellular Ca2+ stores, or may be partly due to
Ca2+ release from intracellular stores raising cytosolic
Ca2+ levels. We propose that activation of MTX-sensitive
non-selective cation channels may play an important role in
depolarizing -cells in response to stimulation by GLP-1 and PACAP
during feeding to initiate insulin secretion without large elevations
of blood glucose. We also suggest that Ca2+ is permeant
through the MTX-sensitive channels and suggest that spontaneous
activity of these channels may form the depolarizing, non-selective
background conductance that permits Ca2+ influx (25) and
opposes the activity of ATP-sensitive K+ channels in
regulating the resting potential of
-cells under both basal
conditions and in response to hormonal stimulation.
We thank Maurice Castonguay for maintenance of cell cultures.