Nuclear KATP channels trigger nuclear Ca2+ transients that modulate nuclear function

Abstract

Glucose, the principal regulator of endocrine pancreas, has several effects on pancreatic beta cells, including the regulation of insulin release, cell proliferation, apoptosis, differentiation, and gene expression. Although the sequence of events linking glycemia with insulin release is well described, the mechanism whereby glucose regulates nuclear function is still largely unknown. Here, we have shown that an ATP-sensitive K(+) channel (K(ATP)) with similar properties to that found on the plasma membrane is also present on the nuclear envelope of pancreatic beta cells. In isolated nuclei, blockade of the K(ATP) channel with tolbutamide or diadenosine polyphosphates triggers nuclear Ca(2+) transients and induces phosphorylation of the transcription factor cAMP response element binding protein. In whole cells, fluorescence in situ hybridization revealed that these Ca(2+) signals may trigger c-myc expression. These results demonstrate a functional K(ATP) channel in nuclei linking glucose metabolism, nuclear Ca(2+) signals, and nuclear function.

Full text

Nuclear K
ATP
channels trigger nuclear Ca
2ⴙ
transients
that modulate nuclear function
Ivan Quesada*, Juan M. Rovira, Franz Martin, Enrique Roche, Angel Nadal, and Bernat Soria
†
Institute of Bioengineering, Miguel Herna´ ndez University, San Juan Campus, 03550 Alicante, Spain
Edited by Ramon Latorre, Center for Scientific Studies, Valdivia, Chile, and approved May 22, 2002 (received for review January 23, 2002)
Glucose, the principal regulator of endocrine pancreas, has several
effects on pancreatic beta cells, including the regulation of insulin
release, cell proliferation, apoptosis, differentiation, and gene
expression. Although the sequence of events linking glycemia with
insulin release is well described, the mechanism whereby glucose
regulates nuclear function is still largely unknown. Here, we have
shown that an ATP-sensitive K
ⴙ
channel (K
ATP
) with similar prop-
erties to that found on the plasma membrane is also present on the
nuclear envelope of pancreatic beta cells. In isolated nuclei, block-
ade of the K
ATP
channel with tolbutamide or diadenosine polypho-
sphates triggers nuclear Ca
2ⴙ
transients and induces phosphory-
lation of the transcription factor cAMP response element binding
protein. In whole cells, fluorescence in situ hybridization revealed
that these Ca
2ⴙ
signals may trigger c-myc expression. These results
demonstrate a functional K
ATP
channel in nuclei linking glucose
metabolism, nuclear Ca
2ⴙ
signals, and nuclear function.
Pancreatic beta cells play a critical role in maintaining a
steady-state level of glucose in the blood and tissues. In-
creased levels of glucose stimulate beta cells to secrete insulin
closing a well established feedback loop. Malfunction of beta
cells causes the widespread pathology, diabetes mellitus. The
signal transduction mechanism leading to insulin release involves
the closure of plasma membrane K
ATP
channels as a result of
glucose metabolism by increasing both the intracellular ATP兾
ADP ratio and diadenosine polyphosphates (DPs; refs. 1 and 2).
Channel closure leads to membrane depolarization and the
opening of voltage-activated Ca
2⫹
channels (3). The subsequent
cytosolic Ca
2⫹
signal, which is oscillatory (4), triggers a pulsatile
insulin secretion. In most cells, a single second messenger as
Ca
2⫹
is able to provoke different responses depending on its
route of entry, its localization, and a code of amplitude or
frequency of Ca
2⫹
oscillations (4, 5). In pancreatic beta cells,
Ca
2⫹
mediates not only insulin secretion but also a broad range
of other processes such as gene expression (6, 7). Although it is
well established that the nucleoplasmic concentration of free
Ca
2⫹
regulates nuclear function (5), the mechanism whereby
nuclear Ca
2⫹
signals are generated is still unclear. Here, we
report confocal measurements of nuclear Ca
2⫹
concentration
([Ca
2⫹
]
n
) in intact beta cells exposed to glucose. Experiments in
isolated nuclei revealed a K
ATP
channel present on the nuclear
envelope whose blockade results in a [Ca
2⫹
]
n
rise. This increased
[Ca
2⫹
]
n
induces phosphorylation of the transcription factor
cAMP response element binding protein (CREB). We further
demonstrate that [Ca
2⫹
]
n
elevation may result in c-myc expres-
sion in whole cells.
Materials and Methods
Cell Isolation, Culture, and Permeabilization. Islets from adult (8–10
weeks old) Swiss albino male mice (OF1) killed by cer vical
dislocation were isolated and then dispersed into single cells
after a published procedure (3). Isolated cells were cultured in
RPMI medium 1640 for 24 h. Cell permeabilization was per-
formed as described (8).
Spot Confocal Microscopy. Cells were loaded with 2
␮
M Calcium
Green-1兾AM (Molecular Probes) for 60 min at room temper-
ature (RT) and then were perfused in a modified Krebs–Ringer
buffer [119 mM NaCl兾4.7 mM KCl兾1.2 mM MgSO
4
兾1.2 mM
KH
2
PO
4
兾25 mM NaHCO
3
兾2.5 mM CaCl
2
兾3 mM glucose bub-
bled constantly with a mixture of 95% O
2
兾5% CO
2
(pH ⫽7.4)].
Ca
2⫹
was measured by using spot confocal microscopy, which
excels in measuring minute Ca
2⫹
transients because of its high
signal-to-noise ratio. The spot illumination-detection configura-
tion has been described (9, 10). Briefly, a laser-illuminated
pinhole (10
␮
m) was focused onto a spot through the objective
on a nucleus equatorial plane. Thus, the predicted detection
volume is about 0.6 ⫻0.6 ⫻1.1
␮
m
3
. Changes in fluorescence
in this nuclear volume were precisely detected with a photodiode
(HR008; United Detector Technology) which was connected to
an Axopatch-200A amplifier (50 G⍀feedback; Axon Instru-
ments, Foster City, CA). The laser illumination time was 30 ms.
Ca
2⫹
-induced fluorescence intensity ratio was plotted as a
function of time (F
t
兾F
0
).
Nuclei Isolation. While dispersed, islet cells were suspended in a
buffer that mimicked the intracellular medium (125 mM KCl兾2
mM K
2
P0
4
兾40 mM Hepes兾0.1 mM MgCl
2
,pH7.2兾100 nM Ca
2⫹
,
with 10.2 mM EGTA and 1.65 mM CaCl
2
). The cell membrane
and cytoskeleton were disrupted by brief sonication. Single
isolated nuclei were separated by centrifugation, resuspended in
the intracellular medium, and then allowed to attach onto glass
chambers (11). Ethidium homodimer-1 labeling (Molecular
Probes) indicated that the suspension was 90% enriched in
nuclei. This probe also was applied to identify nuclei after each
electrophysiological and imaging experiment. An antibody to
calnexin allowed an assessment of contamination of the nuclei
preparation with endoplasmic reticulum (ER) fragments (12).
We further performed confocal images and three-dimensional
(3D) reconstruction of nuclei labeled with rhodamine B hexyl
ester to detect the existence of some debris attached to the
surface of some nuclei (10%), which was associated with ER
contamination (13, 14). As this ER debris was completely
detectable by transmitted light or fluorescence microscopy, we
only performed experiments in intact and clean nuclei (free of
debris).
Glibenclamide-Dipyrrometheneboron Difluoride (BODIPY) Staining.
Cultured isolated islet cells were labeled with 40 nM of green-
fluorescent glibenclamide-BODIPY-FL (30 min at 4°C; Molec-
ular Probes) and stained with anti-insulin antibodies (15). Im-
munofluorescence for insulin revealed more than 80% of
pancreatic beta cells in the cultures. Isolated nuclei also were
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: KATP, ATP-sensitive K⫹channel; [Ca2⫹]n, nuclear Ca2⫹concentration; CREB,
cAMP response element binding protein; RT, room temperature; BODIPY, dipyr-
rometheneboron difluoride; nKATP, nuclear KATP channel; ER, endoplasmic reticulum;
ER兾SR, endo-sarcoplasmic reticulum; RyR, ryanodine receptors.
*Present address: Department of Bioengineering, University of Washington, Seattle, WA
98195.
†To whom reprint requests should be addressed at: Institute of Bioengineering, Miguel
Herna´ ndez University, San Juan Campus, Carretera Alicante-Valencia Km 87, 03550 Ali-
cante, Spain. E-mail: bernat.soria@umh.es.
9544–9549
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PNAS
兩
July 9, 2002
兩
vol. 99
兩
no. 14 www.pnas.org兾cgi兾doi兾10.1073兾pnas.142039299

stained with glibenclamide-BODIPY-FL following the same
protocol. Nuclear envelope was identified with 1
␮
M of rhoda-
mine B hexyl ester (5 min at RT; Molecular Probes; ref. 14);
meanwhile, 1
␮
M of ethidium homodimer-1 (5 min at RT;
Molecular Probes) stained the DNA containing nucleoplasm.
Fluorescence was visualized by using a Zeiss LSM 510 confocal
microscope (63⫻objective, 1.25 N.A.) and 1–2
␮
m optical slices.
Intensity values were obtained by calculating the average bright-
ness value on the corresponding ring-like staining of the nuclei
and measured on an arbitrary gray scale from 0 (blackest) to 255
(whitest).
Patch-Clamp Experiments. Single-channel currents were recorded
from nuclei positively labeled with ethidium homodimer-1 by
using standard patch-clamp recording procedures (16). The bath
solution contained 140 mM KCl, 1 mM MgCl
2
, 10 mM Hepes,
1 mM EGTA, pH 7.2, and the pipette solution contained 5 mM
KCl, 135 mM NaCl, 10 mM Hepes, 1.1 mM MgCl
2
, pH 7.4.
Pipette potential was held at 0 mV throughout the record.
Experiments were performed at RT. High resistance seals were
formed (1–5G⍀), indicating minimal contamination by the
endoplasmic reticulum (ER) membrane (13). Under these cir-
cumstances, it has been proposed that nuclear pores would be
occluded or nonconducting because of experimental conditions
or lack of cytosolic factors (13, 14, 17).
Ca
2ⴙ
Measurements in Isolated Nuclei. Single isolated nuclei were
loaded as described (11, 18). The membrane impermeant Ca
2⫹
probe Calcium Green-1 dextran (30
␮
g兾ml; 30 min at 4°C;
Molecular Probes) loaded the nucleoplasm while the membrane
permeant Ca
2⫹
probe Fluo-3兾AM (20
␮
M; 60 min at 4°C)
loaded the nuclear envelope. After loading, the nuclei were
washed twice with the intracellular medium and then were
equilibrated in the same medium supplemented with 1
␮
Mof
ATP and 300 nM Ca
2⫹
for a few minutes to load nuclei with Ca
2⫹
(11, 18). After that, they were washed twice again with the
intracellular buffer (without AT P and Ca
2⫹
). Experiments were
done at RT. No probe leakage was detected during the exper-
iment. Ca
2⫹
imaging in single isolated nuclei was performed by
using a Zeiss LSM 510 confocal microscope with a Zeiss 63X oil
immersion lens, N.A. 1.25. Images were collected at 3 s intervals,
and fluorescence was measured by using the Zeiss LSM software
package. Ca
2⫹
-induced fluorescence intensity ratio (F
t
兾F
0
) was
plotted as a function of time.
Nuclear Transmembrane Potential (⌬⌿
n
) Measurements. Isolated
nuclei were equilibrated in intracellular medium containing the
ER marker DiOC
6
(3) (200 nM, RT; ref. 19). After 5 min, the
nuclear envelope became stained. DiOC
6
(3) is a fluorescent
cationic lipophyllic dye whose incorporation into lumen is pro-
portional to ⌬⌿.
P-CREB Immunofluorescence. CREB phosphorylation was induced
in isolated nuclei by increasing Ca
2⫹
in an EGTA-buffered
intracellular medium from 65 nM to 1
␮
MCa
2⫹
(20) or by
addition of tolbutamide or AP
4
A for 2 min. After 10 min, nuclei
were fixed with 0.1% paraformaldehyde (wt兾vol) and perme-
abilized with 0.1% Triton X-100 for 10 min. Nuclei were
preincubated with blocking buffer; then, anti-CREB phospho-
specific rabbit antibodies were applied for 16 h at 4°C (1:200,
Calbiochem). P-CREB was visualized with fluorescein-
conjugated secondary antibodies (1 h, RT, 1:64; Sigma). High
Ca
2⫹
gave the maximum response.
Fluorescence
in Situ
Hybridization. Fragments (237 and 530 bp) of
c-myc and
␤
-actin cDNAs cloned in pBSSK (Stratagene) were
used as templates for digoxigenin-labeled double-stranded DNA
probes, synthesized by PCR using specific primers (21, 22).
Isolated islet cells were cultured at least for 24 h in a culture
medium containing 3 mM of glucose and then were placed in
different stimulating conditions in a Krebs–Ringer buffer for 10
min. After that, cells were equilibrated in 3 mM of glucose for
45 min. Only when AP
4
A was used as a stimulator, cells were
permeabilized as mentioned above. Then, cells were fixed in a
solution containing 4% (vol兾vol) formaldehyde, 5% (vol兾vol)
acetic acid, and 0.9% NaCl and permeabilized with 1:1,000
Triton X-100 for 10 min. Hybridization was performed under
standard conditions and was revealed by immunof luorescence
detection with f luorescein-conjugated antidigoxigenin (1:500;
Roche, Barcelona, Spain). Nonspecific binding was reduced with
a commercial blocking reagent (Roche, Barcelona, Spain). Cells
were counterstained with 1
␮
M ethidium homodimer for 5 min
before visualization under a Zeiss LSM 510 confocal microscope
(10⫻objective, 0.45 N.A.). The average intensity value from
each stained cell was calculated and expressed on an arbitrary
gray scale from 0 (blackest) to 255 (whitest). Cells whose
fluorescence intensity was above the range of values of unstimu-
lated control cells were scored as activated cells for gene
expression.
Statistical Analysis. Statistical analysis was performed by using
SIGMAPLOT (Jandel, San Rafael, CA). Values are mean ⫾SE.
Except where indicated, P⬍0.05 by Student’sttest.
Results
Nuclear Ca
2ⴙ
Changes. Ca
2⫹
changes were measured in the nuclear
space of isolated pancreatic beta cells by spot confocal micros-
copy (Fig. 1A; refs. 9 and 10). Cells equilibrated in an extracel-
lular medium containing 16.7 mM glucose exhibited a transient
increase of [Ca
2⫹
]
n
(Fig. 1B). Exposure to the sulfonylurea
tolbutamide (20
␮
M), which directly closes K
ATP
channels,
produced a similar increase (Fig. 1B). Both increases of [Ca
2⫹
]
n
were still produced in the absence of extracellular Ca
2⫹
(Fig. 1C).
Increased [Ca
2⫹
]
n
was probably due to direct Ca
2⫹
release from
the nuclear envelope to the nuclear space (11, 14, 23), because
tolbutamide induces [Ca
2⫹
]
n
changes not only in the absence of
extracellular Ca
2⫹
but in digitonin permeabilized cells (8) with
a buffered Ca
2⫹
concentration (Fig. 1D). The tolbutamide-
induced Ca
2⫹
increase was counteracted by the sulfonamide
Fig. 1. Ca2⫹changes in the nuclear space in intact cells revealed by spot
confocal microscopy. (A) Hybrid phase contrast image with a fluorescent spot
(see arrow) focused on an intact cell nucleus loaded with Calcium-Green-1兾
AM. (B) Intact cells were stimulated with different agents at the time indicated
by the line. Values are mean ⫾SE and were pooled from six cells [16.7 mM
glucose (G)] and seven cells [20
␮
M tolbutamide (T)]. (C) Beta cells were
stimulated with 16.7 mM G (n⫽5) and 20
␮
MT(n⫽6) in a Ca2⫹-buffered
medium containing 5 mM EGTA. (D) Permeabilized cells were stimulated with
100
␮
M tolbutamide or 100
␮
M tolbutamide plus 100
␮
M diazoxide (n⫽4)
inaCa
2⫹-buffered intracellular medium. G, glucose; T, tolbutamide; D,
diazoxide.
Quesada et al. PNAS
兩
July 9, 2002
兩
vol. 99
兩
no. 14
兩
9545
PHYSIOLOGY

diazoxide, a K
ATP
channel opener (Fig. 1D). These results
strongly suggest that intracellular K
ATP
channels responsible for
eliciting nuclear Ca
2⫹
signals might be present close to the beta
cell nucleus.
Verification of K
ATP
Channels on Nuclei. To verify the existence of a
K
ATP
channel on the nuclear envelope from mouse beta cells, we
used a high-specificity, high-affinity (K
i
s⫽4 nM) binding assay
of glibenclamide-BODIPY-FL to the sulfonylurea receptor
(SUR1; ref. 14). SUR1 is the molecular complement of the
potassium inward rectifier (K
ir
; refs. 24 and 25). The association
of SUR1 and K
ir
6.2 forms the K
ATP
channel in beta cells. Fig. 2
A–Cshows a confocal optical section of pancreatic beta cells with
an intracellular ring-like labeling, suggesting the binding of
glibenclamide-BODIPY to the nuclear envelope. Similar results
were observed in isolated nuclei (Fig. 2 D–F). The gliben-
clamide-BODIPY binding site colocalized with rhodamine B
hexyl ester, a marker of the nuclear membrane (ref. 14; Fig. 2E).
The binding was specific because it was displaced by the non-
fluorescent sulfonylurea tolbutamide in a dose-dependent man-
ner (Fig. 2G). This evidence agrees with previous observations
reporting an intracellular location of K
ATP
channels including
perinuclear sites (26–31) in addition to plasma membrane K
ATP
channels.
Excised patch-clamp recordings (16) from the nuclear enve-
lope of isolated nuclei in a 140 mM K
⫹
兾5mMK
⫹
solution
exhibited K
⫹
-channel activity with conductance of approxi-
mately 25 pS (ref. 25; Fig. 2I). This channel was activated by ADP
and inhibited by ATP in a concentration-dependent manner
(Fig. 2I). Nuclear K
ATP
(nK
ATP
) channels display kinetic prop-
erties similar to channels found on the beta cell plasma mem-
brane, with openings grouped in bursts separated by long closing
periods (data not shown). The pharmacological profile of the
nK
ATP
channel also resembles that of the plasma membrane
K
ATP
channel (25). At concentrations similar to those acting on
the plasma membrane K
ATP
channel, tolbutamide (100
␮
M)
blocks the nK
ATP
channel whereas diazoxide (200
␮
M) opens it
(Fig. 2Iiv) in the presence of ATP. Therefore, this nK
ATP
channel seems remarkably similar to the plasma membrane K
ATP
channel (25).
Blockade of nK
ATP
Channels Elicits Ca
2ⴙ
Signals in Isolated Nuclei.
Depending upon their lipophylicity, the Ca
2⫹
-sensitive fluores-
cence dyes Fluo-3兾AM or Calcium Green-1 dextran localized
preferably in the nuclear envelope or the nucleoplasm (11, 14,
18), respectively (Fig. 3 Aand B). In isolated nuclei, confocal
microscopy revealed that tolbutamide induced opposite Ca
2⫹
changes in the nuclear envelope and the nucleoplasm (Fig. 3 A
and B). Either 100
␮
Mor500
␮
M of tolbutamide generated
Ca
2⫹
increases in the nucleoplasm, which paralleled Ca
2⫹
de-
creases observed in the nuclear envelope (Fig. 3 Aand B).
Tolbutamide did not produce any Ca
2⫹
transient in the presence
of the K
ATP
channel opener diazoxide (data not shown). More-
over, the diadenosine polyphosphate AP
4
A (100
␮
M), which
blocks the K
ATP
channel (2), induced a similar Ca
2⫹
release (Fig.
3C). Because AP
4
A is membrane-impermeant, this experiment
also suggests that the regulatory site of this K
⫹
channel does not
face the perinuclear space. Thus, the nuclear envelope may act
asaCa
2⫹
reservoir that is mobilized as a result of glucose
metabolism.
All of these results suggested that this Ca
2⫹
pool was sensitive
to changes in the nuclear membrane K
⫹
permeability but also
raised the possibility that Ca
2⫹
release may be elicited by means
of a voltage-dependent mechanism. To explore the hypothesis
that blockade of nK
ATP
channels provokes a Ca
2⫹
release from
the nuclear envelope by voltage variations, we changed the
concentrations of extraluminal K
⫹
. Reduction of the K
⫹
con-
centration (by N-methyl-D-glucamine replacement) and the en-
Fig. 2. KATP channels on the nuclear envelope. (A) Intact islet cells
incubated with glibenclamide-BODIPY-FL showed a strong ring-like label-
ing around the nucleus. (B) Cell in Aidentified as beta cell by immunoflu-
orescence against insulin. (C) Colocalization of images Aand B. Optical
sections range between 1–2
␮
m. (Bar ⫽10
␮
m.) Similar results were
obtained in 19 cells from four coverslips from three different cultures. (D)
Glibenclamide-BODIPY-FL ring-like staining of an isolated nucleus. (E)
Nucleus in Dwith rhodamine B hexyl ester staining the nuclear envelope.
(F) Colocalization of images Dand E(n⫽21). (Bar ⫽5
␮
m.) Confocal images
were taken with 1
␮
m optical slices. (G) Glibenclamide-BODIPY competi-
tion with nonfluorescent unlabeled tolbutamide reflected a displacement
of the binding site at the nuclear envelope. Glibenclamide labeling is
expressed as the percentage of fluorescence intensity with respect to the
control condition (0
␮
M tolbutamide). Results (mean ⫾SE) were pooled
from 94 nuclei for 0
␮
M tolbutamide, 29 for 1
␮
M, 20 for 10
␮
M, and 24 for
1.000
␮
M from 12 coverslips of 6 different nuclei preparations. (H) Image
showing a patch pipette on an isolated nucleus previously identified using
ethidium homodimer-1. (I) Single-channel records (filtered at 1 KHz) were
obtained from a nucleus membrane excised patch (n⫽5). The pipette
potential was held at 0 mV. Upward currents represent currents going into
the pipette. Single-channel current gives an estimate of 25 pS. A dashed
line represents changes in the external solution. (i) ADP (200
␮
M) increased
the burst length. The change from 200
␮
M ADP to 2 mM ATP rapidly and
completely closed K⫹channels. (ii) The K⫹channel activity absent in 2 mM
ATP was rapidly restored when ATP was removed. (iii) The change from 40
␮
Mto20
␮
M ATP increased channel activity. (iv) Diazoxide (200
␮
M)
activates the channel in the presence of ATP, whereas tolbutamide (100
␮
M) blocks it.
9546
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.142039299 Quesada et al.

suing change in the K
⫹
electrochemical gradient led to a
corresponding Ca
2⫹
discharge (Fig. 3D). Tolbutamide failed to
produce a Ca
2⫹
transient in nuclei pretreated with 10
␮
Mofthe
K
⫹
ionophore valinomycin, further suggesting that the collapse
of K
⫹
electrochemical gradient can suppress Ca
2⫹
release (data
not shown). We monitored changes of nuclear transmembrane
potential (⌬⌿
n
) by using DiOC
6
(3), which has been validated as
a potentiometric probe in mitochondria (ref. 19; see Materials
and Methods). DiOC
6
(3) accumulated in the nuclear envelope of
beta cells. Tolbutamide elicited a consistent increase in
DiOC
6
(3) fluorescence, indicating that the perinuclear lumen
became more negative (Fig. 3E). These results pointed to the
existence of a ⌬⌿
n
, which may change as a result of a decrease
in K
⫹
permeability. In beta cells, a [Ca
2⫹
]
n
pathway sensitive to
this ⌬⌿
n
may exist.
Several intracellular channels found in the endo-sarcoplasmic
reticulum (ER兾SR) and nucleus, including inositol-1,4,5-
trisphosphate receptors (InsP
3
-R) and ryanodine receptors
(RyR; refs. 11, 14, and 32) among others, have been shown to be
voltage-sensitive. Ca
2⫹
release induced by tolbutamide de-
creased 81.3 ⫾9.3% in isolated nuclei exposed to 10
␮
M
ruthenium red, a RyR blocker (Fig. 3F). Higher ruthenium red
concentrations (100
␮
M) completely blocked the Ca
2⫹
release.
Similar results were observed when isolated nuclei were prein-
cubated with anti-RyR antibodies (1:100, 30 min; data not
shown). Conversely, heparin, a blocker of the InsP
3
-R (15) did
not produce a significant effect (data not shown).
Nuclear Function of nK
ATP
Channels. Several roles have been sug-
gested for nuclear channels, yet very few studies relate these
channels to nuclear functions. As has recently been proposed,
nuclear Ca
2⫹
signals may target the CREB protein, whose
phosphorylation may activate transcription (20, 33). By using
immunofluorescence in isolated functional nuclei (20), we have
observed CREB phosphorylation (Fig. 4) induced by tolbut-
amide and AP
4
A, both K
ATP
blockers that provoked an increase
of [Ca
2⫹
]
n
(Fig. 3). As shown in Fig. 1D, the K
ATP
channel opener
diazoxide counteracted the tolbutamide effects (Fig. 4D). These
observations further suggested that the closure of nK
ATP
chan-
nels initiate the transduction of nuclear signals.
In addition to transcription factors, other specific intranuclear
Ca
2⫹
targets may exist that regulate specific nuclear events, such
as gene induction. These targets might include nuclear kinases,
polymerases and chromatin remodeling, among others (34). To
evaluate the relationship between these nuclear Ca
2⫹
signals and
gene expression, we conducted f luorescence in situ hybridization
experiments in intact beta cells. C-myc expression was chosen as
a model because this immediate early gene is activated by
glucose in a Ca
2⫹
-dependent manner in both cultured cell lines
(21; E.R., unpublished work) and in islet cells (35). Fig. 5 shows
c-myc expression in islet cells under different stimuli. Remark-
ably, tolbutamide and AP
4
A activated gene expression (30 and
20%, respectively) even in the absence of extracellular Ca
2⫹
.In
this condition, both K
ATP
blockers induced Ca
2⫹
changes within
the nuclear space (Figs. 1Cand 3 A–C). Diazoxide abolished
these effects when present (data not shown).
Discussion
Exposure of beta cells to glucose results in a Ca
2⫹
-mediated
activation of a broad range of functions (3–6). However, the link
between glucose-induced Ca
2⫹
signals and specific organelles
remains obscure. Here, we focused on the mechanism of glucose-
regulated [Ca
2⫹
]
n
fluctuations and the effect of these Ca
2⫹
signals on nuclear function. Exposure of intact cells to glucose
and tolbutamide led to a [Ca
2⫹
]
n
rise both in cells equilibrated
in normal Krebs–Ringer containing 2.5 mM Ca
2⫹
and in Ca
2⫹
-
free Krebs–Ringer solution (Fig. 1 Band C). These results
suggest that both secretagogues must be acting on intracellular
Ca
2⫹
compartments and are in agreement with previous obser-
vations in beta cells (36) showing that tolbutamide can increase
cytosolic [Ca
2⫹
] in the absence of extracellular Ca
2⫹
. Mitochon-
Fig. 3. Nuclear Ca2⫹signals induced by KATP channel blockade. (A) Nuclei
were loaded with the membrane permeant probe Fluo-3兾AM, which was
preferentially accumulated in the nuclear envelope (ref. 11; see image).
[Ca2⫹]nwas measured by using confocal microscopy. Tolbutamide applied to
Fluo-3-loaded nuclei produced a Ca2⫹decrease (n⫽3). (B) Calcium Green-1
dextran, a nonpermeant probe, was distributed uniformly in the nucleoplasm
(see image). Tolbutamide provoked a transient Ca2⫹increase in the nucleo-
plasm (n⫽6). Spot confocal methods also were used to observe this tolbut-
amide-induced nuclear Ca2⫹release (n⫽3; data not shown). (Bar ⫽5
␮
m.) (C)
Same experiment described in Bshowing a Ca2⫹release induced by 100
␮
M
AP4A(n⫽3). (D) Replacement of 100 mM K⫹by NMG⫹(N-methyl-D-
glucamine) led to a similar Ca2⫹transient (n⫽4) in the nucleoplasm. (E)
Nuclear envelope was loaded with DiOC6(3), a voltage-sensitive probe (see
Materials and Methods). Fluorescence was increased upon addition of tolbu-
tamide (n⫽4). (F) Ruthenium red (RR) blocked almost 90% of the tolbut-
amide-induced Ca2⫹release (n⫽4). Two consecutive Ca2⫹transients were
induced in this experiment. Before the second discharge, nuclei were loaded
with Ca2⫹(see Materials and Methods) and then treated with ruthenium red.
When the blocker was not present, the two transients had the same ampli-
tude. Thus, the first transient was used as a control (C).
Fig. 4. CREB phosphorylation in isolated nuclei. Immunofluorescence de-
tection of P-CREB in nuclei treated with different stimuli. (A)Ca
2⫹(1
␮
M). (B)
Tolbutamide (100
␮
M). (C)Ca
2⫹(65 nM). Immunodetection is shown in green,
and ethidium homodimer-1 staining is shown in red. (D) Percentage of labeled
nuclei relative to the maximal response 1
␮
MCa
2⫹(HC; n⫽145) measured in
the following conditions: 100
␮
M tolbutamide (T; n⫽112); 100
␮
M tolbut-
amide plus 200
␮
M diazoxide (T⫹D; n⫽92); 100
␮
MAP
4A(n⫽56); and 65 nM
Ca2⫹(LC; n⫽134).
Quesada et al. PNAS
兩
July 9, 2002
兩
vol. 99
兩
no. 14
兩
9547
PHYSIOLOGY

dria have been suggested as an intracellular target of sulfonyl-
ureas in beta cells (36). However, the intracellular source of this
sulfonylurea-induced Ca
2⫹
release was not univocally identified.
Although Ca
2⫹
discharge from an organelle could diffuse to the
nuclear space, our observations that tolbutamide can increase
[Ca
2⫹
]
n
as well in permeabilized cells equilibrated in Ca
2⫹
-
buffered intracellular medium (Fig. 1D), indicate that in this case
the source of Ca
2⫹
may be located very close to the nucleus. This
tolbutamide effect was readily blocked by diazoxide, a K
ATP
channel opener (Fig. 1D). This observation indicates that K
ATP
channels may be present near the nucleus and their closure may
control [Ca
2⫹
]
n
in beta cells. Excised patches of nuclear mem-
brane confirmed the presence of a nuclear nK
ATP
channel that
exhibits kinetics and pharmacological properties very similar to
the K
ATP
channels found in the plasma membrane (ref. 25; Fig.
2I). Patch scission can provoke membrane remodeling, making
it difficult to know the channel orientation by using the patch-
clamp approach. However, the effect of the membrane-
impermeant K
ATP
channel blocker AP
4
A (2) on Ca
2⫹
release in
isolated nuclei (Fig. 3D) suggests that the regulatory site of this
channel is not facing the perinuclear space.
In addition to this functional characterization we have dem-
onstrated here a specific binding site for sulfonylureas at the beta
cell nuclear envelope (Fig. 2) that was revealed by glibenclamide-
BODIPY-FL binding. This method has been successfully used to
detect the presence of K
ATP
channels in different kinds of cells
including beta cells, neurons, and monocytes (15). Our results
are in agreement with previous work in cells transfected with
green fluorescent protein-tagged constructs linked to SUR1 or
Kir6.2 that showed fluorescence patterns not only in plasma but
in perinuclear membranes, as well as in ER and Golgi (30, 31).
Thus, three independent lines of evidence point to the presence
ofaK
ATP
channel in the nuclear envelope, broadening the idea
that together with the well characterized plasma membrane
K
ATP
channels (24, 25), sulfonylureas might have multiple sites
of action (26), including mitochondria (27),secretory granules
(28, 29), and, as shown here, the nuclear envelope.
Our results show that nK
ATP
channels are involved in beta cell
nuclear Ca
2⫹
signaling because specific K
ATP
blockers such as
tolbutamide (25) and AP
4
A (2) induced Ca
2⫹
release from the
nuclear envelope to the nucleoplasm in isolated nuclei (Fig. 3
A–C). Several lines of evidence suggest that the nuclear Ca
2⫹
pathway revealed here depends on a change in K
⫹
permeability
and ⌬⌿
n
in beta cell nuclei. First, valinomycin abolished tolbu-
tamide-induced Ca
2⫹
transients. Second, a change in K
⫹
elec-
trochemical gradient by NMG
⫹
replacement in the extraluminal
medium produced a similar Ca
2⫹
discharge (Fig. 3D). Third,
tolbutamide provoked a transient increase in ⌬⌿
n
(Fig. 3E)as
revealed the voltage-sensor DiOC
6
(3) (19, 37). It is plausible that
a potential difference between perinuclear space and cytosol
exists because the two membranes of the nuclear envelope
surround a lumen that separates different subcellular compart-
ments and contains a diversity of channels with different per-
meabilities (23, 34). The nuclear envelope is a structural exten-
sion of the ER兾SR network. Thus, similar properties and
molecular composition are shared among these organelles. The
existence of a low resting transmembrane potential described in
the SR (38) is compatible with transient voltage increases during
stimulation similar to those reported here.
Although the understanding of the molecular components of
the nuclear envelope and their regulation have undergone
important progress, their involvement in nuclear Ca
2⫹
signaling
still remains a challenge for exploration (23, 34). A variety of
ER兾SR and nuclear channels that may contribute to regulate
[Ca
2⫹
]
n
and other ion species have been reported (13, 32,
39–44). An important feature is that several ER兾SR and nuclear
channels are voltage-sensitive. Those include Cl
⫺
(13), K
⫹
(42),
RyR (32), InsP
3
-R (14), and InsP
3
-insensitive Ca
2⫹
channels (43,
44). Our results are consistent with RyR channels at the nuclear
envelope—as observed in exocrine cells (11) and oocytes (45)—
that may control the nuclear Ca
2⫹
release reported here, because
their blockade by ruthenium red or anti-RyR antibodies sup-
pressed the tolbutamide-induced nuclear Ca
2⫹
transients (Fig.
3F). It is noteworthy that RyR channels undergo an active state
of elevated open probability upon changes of voltage (32). Thus,
it is very likely that RyR channels may be affected by tolbut-
amide-induced nuclear voltage changes (Fig. 3E). Their activa-
tion may lead to Ca
2⫹
-release, which may be amplified by a
Ca
2⫹
-induced Ca
2⫹
-release process, as has been observed in ER
of pancreatic beta cells (46).
Our results show that this tolbutamide-induced [Ca
2⫹
]
n
con-
ductance seems to be associated with a change in ⌬⌿
n
as a result
of a decrease in K
⫹
permeability (Fig. 3 Dand E). This process
is not exclusive to beta cells. In fact, there is a body of evidence
that establishes a link between K
⫹
and Ca
2⫹
conductances in the
ER兾SR. For instance, Ca
2⫹
release from SR has been induced
not only by K
⫹
channel general blockers (47) but also by the
specific K
ATP
channel blocker glibenclamide (48), suggesting the
presence of this K
⫹
channel type and its role in intracellular Ca
2⫹
release. Moreover, lowering the extraluminal K
⫹
produced a
Ca
2⫹
discharge from SR microsomes of myocytes, suggesting a
voltage change that is not explained well by current ideas about
E–C coupling in muscle cells (49). Furthermore, a functional
K
ATP
channel has been proposed to control the K
⫹
permeability
and the granular membrane potential in glucagon-containing
secretory granules (29). The presence of nuclear Ca
2⫹
channels
sensitive to changes in transmembrane potential explains
tolbutamide-induced Ca
2⫹
release in beta cell nuclei. None-
theless, we do not exclude the involvement of other voltage-
sensitive pathways that may direct or indirectly trigger the
nuclear Ca
2⫹
release reported here. These possible pathways
may include ion exchange fluxes coupled to Ca
2⫹
(50, 51) or
other mechanisms such as the ER Ca
2⫹
leak pathway (52). In
fact, a remarkable characteristic is that the ER leak pathway is
increased with high concentrations of AT P, a blocker of the
Fig. 5. Gene expression induced by KATP channel blockers in the absence of
extracellular Ca2⫹. Fluorescence in situ hybridization and transmitted light
images of islet cells stimulated in different conditions. (A) Glucose (16 mM). (B)
Tolbutamide (100
␮
M) in the absence of extracellular Ca2⫹(buffered with
EGTA). (C) Glucose (3 mM) as a control condition. The green color shows c-myc
expression of beta cells counterstained with ethidium homodimer (red).
(Bar ⫽10
␮
m.) (D) Percentage of activated cells relative to unstimulated
control cells (3 mM glucose; n⫽360) in different conditions: 40 mM potassium
(K⫹;n⫽290); 16 mM glucose (G; n⫽369); 100
␮
M tolbutamide (T; n⫽329);
100
␮
M tolbutamide in the absence of extracellular Ca2⫹[T(0);n⫽466]; 100
␮
M
AP4A in the absence of extracellular Ca2⫹(n⫽105); and absence of extracel-
lular Ca2⫹[Ca2⫹(0);n⫽211]. Results were pooled from three independent
experiments.
␤
-actin constitutive expression was used as an invariable control
under the same conditions.
9548
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.142039299 Quesada et al.

K
ATP
channel (52). In summary, we suggest that blockade of
K
ATP
channels at the nuclear envelope may elicit a transient
transmembrane potential rise as a result of K
⫹
permeability
decrease, which may trigger a voltage-sensitive Ca
2⫹
discharge,
mainly through RyR channels.
By using immunofluorescence, we have proved that the nu-
clear Ca
2⫹
transients reported here increased the phosphoryla-
tion rate of the transcription factor CREB (Fig. 4) in isolated
nuclei, in agreement with similar results described in hippocam-
pal neuron nuclei (20). Our working model only allows Ca
2⫹
mobilization from the nucleus and unambiguously involves
nK
ATP
channels in pancreatic beta cell nuclear function.
In a whole-cell model, we evaluated the possibility of other
nuclear processes such as gene expression being affected. Tol-
butamide and AP
4
A, which induced Ca
2⫹
release in isolated
nuclei (Fig. 3), triggered c-myc expression in the absence of
extracellular Ca
2⫹
(Fig. 5). In the whole cell, we cannot rule out
the possibility that other Ca
2⫹
release in the cytosol may drive
gene expression in our conditions. However, as shown here, the
nucleus probably accounts for an important part of these intra-
cellular Ca
2⫹
signals (Figs. 1 and 3). Be that as it may, we have
demonstrated that these nuclear Ca
2⫹
signals are sufficient to
drive CREB phosphorylation in isolated nuclei in these cells.
Hence, Ca
2⫹
signals generated at the nuclear envelope have an
important role for nuclear function in beta cells.
We propose that signals generated by glucose metabolism not
only inhibit the plasma membrane K
ATP
channel, initiating
insulin release, but also interact with nK
ATP
channels, triggering
nuclear Ca
2⫹
signals that modulate nuclear functions such as
phosphorylation of the transcription factor CREB and likely
gene expression. Our data shows a new signal-transduction
pathway linking nuclear K
⫹
ion channels to fluctuations of
[Ca
2⫹
]
n
and nuclear function and also contributes to an under-
standing of the mechanism of action of sulfonylureas, drugs that
are broadly used in the clinic for the treatment of the diabetic
patient.
We thank P. Verdugo and D. Willows for the critical review of the
manuscript. This work was supported in part by Grant PM99-0142 from
Secretarı´a de Estado de Universidades e Investigacio´n, Fundacio´ Marato´
TV3 Grant 99-1210, Fundacio´n Salud 2000, Juvenile Diabetes Foun-
dation Grant 1-2000-575, and Generalitat Valenciana Grant GV99-
139-1-04.
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Quesada et al. PNAS
兩
July 9, 2002
兩
vol. 99
兩
no. 14
兩
9549
PHYSIOLOGY