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Molecular Endocrinology 16(9):2135–2144
Copyright © 2002 by The Endocrine Society
doi: 10.1210/me.2002-0084
Glucagon-Like Peptide-1 Inhibits Pancreatic
ATP-Sensitive Potassium Channels via a Protein
Kinase A- and ADP-Dependent Mechanism
PETER E. LIGHT, JOCELYN E. MANNING FOX, MICHAEL J. RIEDEL,
AND
MICHAEL B. WHEELER
Department of Pharmacology (P.E.L., J.E.M.F., M.J.R.), Faculty of Medicine and Dentistry, University
of Alberta, Edmonton, Alberta, Canada T6G 2H7; and Department of Physiology (M.B.W.), University
of Toronto, Ontario, Canada M5S 1A8
Glucagon-like peptide-1 (GLP-1) elicits a glucosedependent insulin secretory effect via elevation of
cAMP and activation of protein kinase A (PKA).
GLP-1-mediated closure of ATP-sensitive potassium (KATP) channels is involved in this process,
although the mechanism of action of PKA on the
KATP channels is not fully understood. KATP channel currents and membrane potentials were measured from insulin-secreting INS-1 cells and
recombinant ␤-cell KATP channels. 20 nM GLP-1
depolarized INS-1 cells significantly by 6.68 ⴞ 1.29
mV. GLP-1 reduced recombinant KATP channel currents by 54.1 ⴞ 6.9% in mammalian cells coexpressing SUR1, Kir6.2, and GLP-1 receptor clones.
In the presence of 0.2 mM ATP, the catalytic subunit
of PKA (cPKA, 20 nM) had no effect on SUR1/Kir6.2
activity in inside-out patches. However, the stimulatory effects of 0.2 mM ADP on SUR1/Kir6.2 currents were reduced by 26.7 ⴞ 2.9% (P < 0.05) in the
presence of cPKA. cPKA increased SUR1/Kir6.2
currents by 201.2 ⴞ 20.8% (P < 0.05) with 0.5 mM
ADP present. The point mutation S1448A in the
ADP-sensing region of SUR1 removed the modulatory effects of cPKA. Our results indicate that
PKA-mediated phosphorylation of S1448 in the
SUR1 subunit leads to KATP channel closure via an
ADP-dependent mechanism. The marked alteration of the PKA-mediated effects at different ADP
levels may provide a cellular mechanism for the
glucose-sensitivity of GLP-1. (Molecular Endocrinology 16: 2135–2144, 2002)
A
properties of the channel complex (4, 6–8). SUR1 is
responsible for conferring the unique pharmacological
characteristics to the channel complex, including inhibition by antidiabetic sulphonylurea compounds
such as glibenclamide and activation by diazoxide.
SUR1 is predicted to contain 17 trans-membrane domains and possesses two nucleotide-binding folds
(NBFs), the second of which appears to act as the ADP
sensor (2, 7, 9). ADP antagonizes the inhibitory action
of ATP and fluctuations in intracellular ADP serve to
couple KATP channel activity to membrane excitability
and subsequent insulin secretion (4, 9).
KATP channels have recently been found to be cellular targets for receptor-mediated signal transduction
pathways that modulate insulin release including the
cAMP/protein kinase A (PKA) pathway (10, 11), protein
kinase C (12), and tyrosine kinase (13, 14).
Glucagon-like peptide-1 (GLP-1) is a gut hormone
released from intestinal L-cells in response to food
ingestion and stimulates insulin secretion from pancreatic ␤-cells in a glucose-dependent manner (15,
16). GLP-1-stimulated insulin release is mediated via
activation of the GLP-1 receptor (GLP-1R), G proteincoupled production of cAMP, and subsequent activation of PKA (17). The cellular targets for PKA-mediated
phosphorylation include the KATP channel, the L-type
calcium channel, intracellular calcium stores, and the
exocytotic machinery (10). It has been demonstrated
that GLP-1 facilitates closure of ␤-cell KATP channels,
TP-SENSITIVE POTASSIUM (KATP) channels
serve to couple cellular metabolism to electrical
excitability in many different tissues including pancreatic ␤-cells, heart, smooth muscle, skeletal muscle,
and brain (1–4). ␤-Cell KATP channels act as metabolic
sensors coupling glucose metabolism to insulin secretion. At low plasma glucose, KATP channels permit a
small efflux of potassium ions, keeping the ␤-cell in a
hyperpolarized inexcitable state. At elevated plasma
glucose levels, KATP channels are inhibited via increases in the intracellular ATP/ADP ratio upon
changes in glucose metabolism (4, 5) and the resultant
membrane depolarization leads to an increase in ␤-cell
excitability, calcium influx, and subsequent insulin
release.
The ␤-cell KATP channel complex consists of two
subunits, the sulphonylurea receptor (SUR1) and the
inwardly rectifying pore forming K⫹ channel subunit
Kir6.2. SUR1 and Kir6.2 are coassembled in a stoichiometry of (SUR1)4,(Kir6.2)4 to form the heterooctameric channel complex (1, 4, 6). The Kir6.2 subunit forms the potassium-conducting pore of the KATP
channel and is largely responsible for the ATP-sensing
Abbreviations: cPKA, Catalytic subunit of PKA; DMSO,
dimethylsulfoxide; GFP, green fluorescence protein; GLP-1,
glucagon-like peptide 1; GLP-1R, GLP-1 receptor; KATP,
ATP-sensitive potassium; NBF, nucleotide-binding fold;
NBF2, second NBF; PKA, protein kinase A; SUR1, sulphonylurea receptor; WT, wild-type.
2135
2136
Mol Endocrinol, September 2002, 16(9):2135–2144
leading to membrane depolarization and triggering of
the insulin secretory pathway (10, 11, 18). It is proposed that the PKA-induced closure of KATP channels
is the initial step in GLP-1-mediated insulin secretion
(10). The insulin stimulatory effects of GLP-1 are glucose dependent, such that GLP-1 only stimulates
insulin release when glucose levels are elevated. However, the underlying mechanism for this phenomenon
is still unclear.
Recent studies have shown that PKA may phosphorylate residues at positions 372 (19) or 224 (20) in
the pore-forming Kir6.2 subunit. However, both of
these studies observed activation of KATP channels
with PKA in the presence of ATP only. This situation is
somewhat unphysiological as in intact ␤-cells and cell
lines (11, 18, 21) intracellular nucleotides such as ATP
and ADP are preserved. Moreover, recent evidence
suggests that intracellular ADP levels fluctuate much
more than ATP levels at different glucose concentrations (22).
It was therefore the aim of this study to further
investigate the mechanisms by which GLP-1, acting
through PKA, modulates the ␤-cell KATP channel via
changes in nucleotide sensitivity.
RESULTS
The Effects GLP-1 on the Insulin-Secreting Cell
Line INS-1 Cell
The resting membrane potential of INS-1 cells after
more than 15 min exposure to 5 mM glucose was
⫺53.01 ⫾ 2.20 mV (n ⫽ 11 cells). In the presence of 5
mM glucose, application of 20 nM GLP-1 caused a
6.68 ⫾ 1.29 mV (n ⫽ 11 cells) depolarization of the
membrane potential (Fig. 1, A and B). This depolarization resulted in the initiation of action potential firing in
80% of the cells tested (Fig. 1A). To test whether the
effects of GLP-1 on membrane excitability are dependent on PKA activity, a 5-min pretreatment with
the selective PKA inhibitor H-89 (1 ␮M) was used.
H-89 prevented the GLP-1-induced depolarization
(⫺0.55 ⫾ 0.46 mV change, n ⫽ 5 cells, Fig. 1, B and C).
To confirm that these cells were receptive to KATP
channel inhibition, the sulphonylurea tolbutamide was
used. Application of 20 ␮M tolbutamide led to membrane depolarization and the firing of action potentials
(Fig. 1B). The addition of GLP-1 in the presence of the
GLP-1R antagonist exendin (9–39) (50 nM) caused a
slight membrane hyperpolarization (⫺4.9 ⫾ 0.8 mV,
n ⫽ 5 cells, data not shown).
In a parallel set of experiments, INS-1 cell cAMP
content was measured in response to the application
of GLP-1. In resting INS-1 cells at 5 mM glucose, the
cAMP concentration was 10.5 pmol/ml, upon 2-min
and 10-min exposure to 20 nM GLP-1, cAMP increased to 36 and 48 pmol/ml, respectively.
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
Fig. 1. The Effects of GLP-1 on Insulin-Secreting INS-1 Cells
The glucose concentration in all experiments was 5 mM. A
and B, Representative membrane potential measurements,
made using the perforated whole-cell patch-clamp technique
in current-clamp mode (see Materials and Methods for details). GLP-1 was superfused over cells via a fast-switching
perfusion device. The application of H-89 to cells was started
5 min before addition of GLP-1. Dashed lines denote initial
membrane potential. C, Grouped data from membrane experiments represented in A and B. Asterisk denotes a statistically significant difference (P ⬍ 0.05) compared with control
(no GLP-1).
Reconstitution of the GLP-1R/cAMP Pathway in a
Recombinant System
The GLP-1R, SUR1, and Kir6.2 clones were coexpressed in tsA201 cells to directly confirm the coupling
of GLP-1R activation, cAMP, PKA, and the ␤-cell KATP
channel isoform. Whole-cell perforated patch-clamp
experiments were performed on cells that exhibited a
barium-sensitive inward current (basal KATP current)
and fluoresced green (␭ ⫽ 520 nm) upon excitation
with 488 nm epifluorescent light, indicating expression
of the green fluorescent protein (GFP)-tagged GLP1R. In positively identified cells, application of 100 nM
GLP-1 caused a time-dependent decrease in wholecell KATP channel current (54.1 ⫾ 6.9% reduction (P ⬍
0.05), n ⫽ 6 cells, Fig. 2, A and C). In cells expressing
SUR1 and Kir6.2 only, 100 nM GLP-1 had no effect
(data not shown). To test the PKA dependence of the
observed reduction in current, cells expressing SUR1,
Kir6.2, and GFP-tagged GLP-1R were incubated for 5
min before recording with the selective PKA inhibitor
H-89 (1 ␮M). In these cells, application of 100 nM
GLP-1 did not cause any reduction in the observed
whole-cell KATP channel current [5.2 ⫾ 4.9%, n ⫽ 5
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
Mol Endocrinol, September 2002, 16(9):2135–2144
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Fig. 2. The Effects of GLP-1 on KATP Channels in a Recombinant Intact Cell System
A–C, Whole-cell KATP channel currents measured using the perforated patch-clamp technique in voltage-clamp mode. tsA201
cells were transiently transfected with GFP-tagged GLP-1R, SUR1, and Kir6.2 clones 48–72 h before recording. Currents were
recorded with 140 mM K⫹ in the bath solution and were held at 0 mV and stepped to ⫺100 mV every 10 sec. H-89 was applied
to cells 5 min before application of GLP-1. Barium chloride (Ba2⫹, 2 mM) was used as a fully washable potassium current blocker
to confirm the amount of recombinant inward KATP channel current present. Arrows in A and B denote zero current level. In Aii,
whole-cell current traces labeled a–d are derived from the respective time points labeled in Ai. C, Grouped data from the
experiments illustrated in A and B. D, Representative whole-cell recording of recombinant KATP channel current expressed with
the GLP-1R (i) and the lack of current in a nontransfected cell (ii). Cells were dialyzed with an ATP-free pipette solution to elicit
any ATP-sensitive current. E, Grouped data from cAMP assay experiments on tsA201 cells transfected with SUR1, Kir6.2, and
GLP-1R. *, Statistically significant difference (P ⬍ 0.05).
cells, (P ⬎ 0.95), Fig. 2, B and C]. In a parallel series of
experiments, cAMP content was measured in this recombinant cellular system. Nontransfected tsA201
cells had a resting cAMP content of 7.9 ⫾ 4.0 pmol/ml
and addition of 20 nM GLP-1 did not increase this
value (8.2 ⫾ 4.6 pmol/ml). In tsA201 cells expressing
SUR1, Kir6.2, and GLP-1R, the resting cAMP content
was 10.2 ⫾3.3 pmol/ml, a value not significantly different to that observed in nontransfected cells. However, in cells expressing SUR1, Kir6.2 and GLP-1R,
10-min exposure to 20 nM GLP-1, increased the cAMP
content significantly [60.3 ⫾ 7.1 pmol/ml (P ⬍ 0.01),
Fig. 2E]. Taken together, these data indicate that, in an
intact cellular system, activation the GLP-1 receptor
2138 Mol Endocrinol, September 2002, 16(9):2135–2144
leads to a significant cAMP/PKA dependent decrease
in recombinant KATP channel activity.
PKA Modulates Recombinant KATP Channels in
an ADP-Dependent Manner
To investigate the molecular mechanism by which
PKA inhibits ␤-cell KATP channels, the effects of the
catalytic subunit of PKA (cPKA) were tested on insideout membrane patches containing recombinant KATP
channels. Figure 3A shows a representative recording
of macroscopic Kir6.2/SUR1 currents. In all experiments, the measured inward current was maximally
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
active in the absence of internal ATP, inhibited by 1 mM
ATP and was partially restored upon exposure to intermediate levels of ATP such as 0.1 or 0.2 mM. Application of 20 nM cPKA had no significant effect on
macroscopic KATP channel current in the presence of
0.2 mM ATP alone (Irel ⫽ 109.8 ⫾ 11.0%, n ⫽ 8
patches, P ⬎ 0.05). In the presence of 0.2 mM ATP, the
addition of 0.2 mM ADP partially relieved the ATP
inhibition (0.2 mM ATP; Irel ⫽ 10.15 ⫾ 1.23%, n ⫽ 9
patches: 0.2 mM ATP and 0.2 mM ADP; Irel ⫽ 65.64 ⫾
9.6%, n ⫽ 9 patches, Fig. 3B). In the presence of both
ATP and ADP, application of 20 nM cPKA caused a
significant decrease in current (Irel ⫽ 73.34 ⫾ 2.9%,
n ⫽ 9 patches, P ⬍ 0.05, Fig. 3B). However, in the
presence of 0.2 mM ATP and elevated ADP (0.5 mM),
application of cPKA caused a significant increase in
KATP channel activity [Irel ⫽ 201.16 ⫾ 20.18%, n ⫽ 19
patches, (P ⬍ 0.05), Fig. 3C]. The grouped results from
this series of experiments are presented in Fig. 3D.
A Single Residue Substitution in the SUR1
Subunit Abolishes the Effects of cPKA
Fig. 3. The Effects of Purified PKA on Recombinant KATP
Channels
A–C, Representative recordings of SUR1/Kir6.2 currents
from excised inside-out membrane patches from tsA201
cells. The purified cPKA was applied to the internal face of the
membrane patch at a concentration of 20 nM. The functional
expression of recombinant KATP channels was confirmed by
the presence of large macroscopic currents that were inhibited by 1 mM ATP. Steady-state currents were recorded at a
holding potential of ⫺60 mV under symmetrical K⫹ conditions (140 mM). Dotted lines denote the zero current level. B
and C, Arrows show the inhibitory (B) or stimulatory (C) effects of cPKA are dependent on internal ADP levels. D,
Grouped data from the representative experiments illustrated
in A–C. Asterisks denote a statistically significant difference
(P ⬍ 0.05) compared with control.
The results from the section above indicate that PKA
reduces the ADP-induced release of ATP inhibition
leading to a more pronounced channel closure in the
presence of ATP and 0.2 mM ADP. These findings
suggest that the molecular site of action of PKA may
reside in the ADP-sensing region of SUR1. The second
nucleotide binding fold (NBF2) of SUR1 has been identified as the intracellular binding site for ADP (2, 7, 9).
Consensus PKA phosphorylation motifs typically consist of two basic residues such as arginine or lysine, a
residue that is not critical followed by a phosphoacceptor serine or threonine (23). Sequence analysis
of NBF2 in the hamster SUR1 subunit revealed four
potential consensus PKA motif sequences that are
conserved in human and rat. One of these, a KKCS
motif, is located in the linker between the Walker A and
B motifs of NBF2 (Fig. 4). The putative serine phosphoacceptor residue at position 1448 (S1448) is in close
proximity to G1479, a residue found to be important in
the ADP-sensing properties of the KATP channel (9). In
the following set of experiments, we set out to determine if substitution of the serine at 1448 prevented the
effects of cPKA observed in wild-type (WT) KATP channels comprised of SUR1 and Kir6.2. We therefore generated a S1448A substitution mutant in the hamster
SUR1 sequence and coexpressed this mutant with
Kir6.2. Inside-out patch experiments revealed that the
S1448A mutant was functionally expressed and possessed similar ATP and ADP-sensing properties when
compared with WT SUR1/Kir6.2 channels (Fig. 5D).
Under the identical conditions that produced a cPKAinduced significant reduction in WT KATP channel current (0.2 mM ATP and 0.2 mM ADP), SUR1(S1448A)/
Kir6.2 current was not significantly changed in the
presence of 20 nM cPKA [95.40 ⫾ 2.8%, n ⫽ 8 patches
(P ⬎ 0.05), Fig. 5, A and C]. At higher levels of ADP
(0.5 mM), where WT KATP current is enhanced by
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
Mol Endocrinol, September 2002, 16(9):2135–2144
2139
Fig. 4. SUR1 Nucleotide Binding Fold 2 and PKA Phosphorylation Sites
A, Amino acid sequence alignment of the NBF2 of SUR1 from the hamster, human and rat clones; residues 1358-end (K1582)
are shown. The nucleotide binding Walker A and B motifs are underlined. The PKA consensus phosphorylation site at position
1448, G1479 and the human clone-specific PKA consensus site (S1571) are highlighted in reverse type. B, Schematic representation of the 17 trans-membrane domains of the SUR1 clone and the Kir6.2 subunits of the KATP channel. The nucleotide
binding folds 1 and 2 (NBF1, NBF2) and the Walker A and B motifs (A and B) are illustrated. The relative positions of residues
S1448, G1479, and S1571 in SUR1 and T224 and S372 in Kir6.2 are marked.
cPKA, the SUR1(S1448A)/Kir6.2 current was also unaffected by application of cPKA (95.31 ⫾ 5.94, n ⫽ 13
patches, Fig. 5B).
DISCUSSION
Glucose-mediated depolarization of the ␤-cell membrane potential is a key event that occurs before the
initiation of insulin secretion (10, 24, 25). Nucleotidemediated closure of KATP channels is thought to account for the observed depolarization via alterations in
the intracellular ATP and ADP levels (7–9) as glucose
levels increase. Therefore, receptor-linked pathways
such as GLP-1, that facilitate KATP channel closure in
␤-cells will promote insulin secretion. In accordance
with previous work, our current study confirms that
GLP-1 induces closure of ␤-cell KATP channels via a
cAMP/PKA-dependent process. More importantly, our
data suggest that the mechanism of KATP channel
inhibition by PKA is dependent on the presence of
intracellular ADP. Such a nucleotide-dependent
mechanism may contribute to the observed glucosedependent GLP-1-induced insulin secretory response.
GLP-1 and ␤-Cell KATP Channels
Previous patch-clamp studies have suggested that
GLP-1 inhibits KATP channels from rodent ␤-cells in a
reversible manner (10, 11, 18). The effects of GLP-1
observed in this present study were measured at an
intermediate glucose concentration of 5 mM. This concentration of glucose was chosen as it has previously
been shown to be just subthreshold for the maintenance of sustained depolarization and action potential
firing in INS-1 cells (26), although occasional firing was
observed. In all experiments, a GLP-1-induced membrane depolarization preceded increases in action potential firing. Our data demonstrate that 1) GLP-1 significantly depolarizes INS-1 cells; 2) GLP-1 increases
cAMP content in INS-1 cells; and 3) the observed
effects are blocked by the PKA inhibitor H-89 (Fig. 1).
These findings are in accordance to the majority of
previous studies (Refs. 10, 11, and 18, but see Refs. 27
and 28) and further confirm that GLP-1 acts by a
cAMP/PKA-dependent membrane depolarization via
inhibition of KATP channels. Physiologically, the GLP1-mediated depolarization would likely have several
effects. Firstly, it may trigger glucose-unresponsive
cells to become excitable, therefore increasing the
percentage of ␤-cells secreting insulin. Secondly,
GLP-1 could further depolarize ␤-cells already in an
excitable state, leading to increased action potential
firing and augmented insulin secretion.
In addition to its effects on KATP channels, GLP-1
likely modulates membrane excitability via changes in
the activity of other ion channels. For example, GLP-1
has been shown to regulate nonspecific cation channels (29) and voltage gated L-type calcium channels
(28, 30). GLP-1 also directly modulates release of calcium from intracellular stores (31) and the exocytotic
release of insulin-containing granules directly (11). Be-
2140 Mol Endocrinol, September 2002, 16(9):2135–2144
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
ceptor (33). Results from this set of experiments demonstrate that stimulation of the GLP-1R increases
intracellular cAMP content and leads to a marked
PKA-dependent reduction in whole-cell KATP channel
current. The reconstitution of the signaling pathway
using recombinant overexpression of the respective
components of the signaling cascade has limitations,
for example, the limited availability of endogenous
signaling components such as adenylyl cyclase to
couple with overexpressed receptors. Nevertheless,
these results demonstrate directly that simulation of
GLP-1R can functionally modulate KATP channel activity via a cAMP/PKA-dependent pathway.
The Effects of PKA on the ␤-Cell KATP Channel
Fig. 5. The Effects of Purified PKA on Recombinant WT or
KATP Channels Expressing the SUR1 Substitution Mutant,
SUR1(S1448A)
A and B, Representative recordings of Kir6.2/SUR1(S1448A)
currents from excised inside-out membrane patches from
tsA201 cells in response to application of 20 nM cPKA in the
presence of either 0.2 mM ADP (A) or 0.5 mM ADP (B). Dotted line
denotes zero current level. C, Grouped data from WT (Kir6.2/
SUR1) or mutated (S1448A) KATP channels in response to
application of cPKA, either in the presence or absence of ADP
(0.2 mM) and ATP (0.2 mM) ATP continuously present. Asterisk
denotes a statistically significant difference (P ⬍ 0.05) compared with control (WT). D, Grouped data, from inside-out patch
experiments with WT and S1448A mutant KATP channels, showing a similar response to ATP alone (0.2 mM) and a release of this
ATP-induced inhibition by ADP (0.2 mM). There were no statistical differences in nucleotide sensitivities between WT and
S1448A KATP channels.
cause of the complexity of GLP-1 signaling in intact
␤-cells, we decided to reconstitute this pathway in an
intact cellular system. This approach has the advantage of isolating and amplifying the signal transduction
pathway, while maintaining the integrity of the intracellular environment. This was achieved by coexpression of the recombinant GLP-1 receptor and the KATP
channel subunits SUR1 and Kir6.2 in tsA201 cells.
GLP-1 was used at 100 nM, a concentration that has
previously been shown to maximally activate recombinant GLP-1R in mammalian cell lines (30, 32), while
having no effect on other related receptors such as
the glucose-dependent insulinotropic polypeptide re-
Evidence from this study clearly demonstrates a modulatory effect of PKA on recombinant KATP channels in
the presence of ATP and ADP. At lower levels of ADP,
PKA inhibits KATP channels, while at elevated ADP
levels PKA augments KATP channel activity. Previous
data suggest that intracellular ATP levels in insulin
secreting cells remain relatively unchanged upon increasing glucose, whereas the ADP levels fluctuate to
a greater extent (22). Regarding the nucleotide-dependent control of ␤-cell KATP channels, it is generally
accepted that intracellular ADP is a key component in
regulating channel activity and therefore ␤-cell excitability and insulin release (7–9). These findings, taken
together with the data presented in this study, suggest
the effects of PKA, mediated through GLP-1-R activation, may be dependent on the metabolic status of
the cell.
It should be mentioned that, while our findings are in
accordance with the majority of previous studies, it
has been suggested by Suga et al. (27) that the effects
of GLP-1 on the ␤-cell KATP channel are cAMP/PKA
independent. In our study, we used the cell-permeant
specific PKA inhibitor H-89 at a concentration of 1 ␮M
(IC50 48 nM). In the study by Suga et al. (27), the cAMP
analog Rp-cAMPs (100 ␮M) was used to inhibit the
cAMP pathway; however, Rp-cAMPs possesses limited membrane-permeability and was used at 100 ␮M
(only 9-fold the IC50 of 11 ␮M). Suga et al. (27) also
showed that forskolin significantly stimulated insulin
secretion despite the presence of Rp-cAMPs. Therefore, incomplete inhibition of the cAMP/PKA pathway
may account for the apparent discrepancy. Mechanistically, Suga et al. also observed that GLP-1 directly
increases ATP-sensitivity of the KATP channel reducing
the IC50 for ATP-inhibition from to 12 to 6 ␮M, with no
change in the Hill coefficient. Physiologically, this is
likely to have negligible effects on KATP channel activity, as intracellular ATP is in the millimolar range and KATP
channel open probability, in the presence or absence of
GLP-1, will be virtually identical at these physiological
ATP levels. In contrast, data from our own study link
PKA-induced modulation of KATP channel activity to the
more metabolically variable ADP concentration under
more physiological conditions.
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
The molecular site of PKA action on the KATP channel is of considerable importance. Although the possibility exists that PKA acts indirectly upon the KATP
channel via phosphorylation of an accessory protein,
the observed effects of the purified catalytic subunit of
PKA on the recombinant KATP channel in excised
patches argues against this notion (Fig. 3 and Refs. 19
and 20). The location of the phosphorylation site on
the KATP channel complex has been the subject of
recent research in several laboratories. In the study by
Béguin et al. (19), it is suggested that S372 in the Kir6.2
subunit and the S1571 residue (found only in the human SUR1 subunit) are the targets for PKA. In another
study by Lin et al. (20), T224 in Kir6.2 was identified as
another putative molecular target for PKA. In both of
these studies, inside-out excised patch experiments
were performed in the presence of ATP only, and a
PKA-induced increase in KATP channel activity was
observed. In direct contrast to their findings, our own
observations in intact cells and in excised inside-out
patches, in the presence of ATP and ADP, demonstrate that PKA modulates KATP channel activity under
more physiological conditions in an ADP-dependent
fashion. Moreover, the inhibitory effects of GLP-1 and
PKA have been previously observed in several species
(10, 11, 18) suggesting the human specific S1571 residue found by Béguin et al. (19) is unlikely to play a
significant physiological role in any common GLP-1mediated cross-species mechanism.
Data from our study now suggest that a serine located at position 1448 in SUR1 also contributes to the
PKA-mediated regulation of KATP channel activity, as
substitution of the serine with an alanine, prevented
ADP-dependent PKA-mediated KATP channel modulation (Fig. 5). S1448 is found in the linker between the
Walker A and B motifs in NBF2 and is highly conserved
among different species (Fig. 4). The importance of
NBF2 in the ADP-sensing properties of the KATP channel is well established (2, 7, 9). For example, mutations
in the human SUR1 gene in NBF2 lead to persistent
hyperinsulinemic hypoglycemia of infancy characterized by very low plasma glucose resulting from uncontrolled insulin release (9, 34–36). Taken together, the
ADP dependence and the involvement of S1448 in the
action of PKA on the KATP channel suggest a novel
molecular mechanism for the regulation of ␤-cell KATP
channel activity.
It is worth noting that mutagenic substitution of residues T224 and S372 to alanines in Kir6.2 resulted in
PKA-induced inhibition of SUR1/Kir6.2 currents in the
presence of ATP alone (20). This finding and the data
from our study suggest that PKA-mediated control of
KATP channel activity is probably dependent on the
phosphorylation of one or more phospho-acceptor
residues on either SUR1 or Kir6.2. It is likely that,
depending on the metabolic status of the cell (ATP:
ADP ratio), phosphorylation of respective residues
may lead to markedly different modulation of KATP
channel activity.
Mol Endocrinol, September 2002, 16(9):2135–2144
2141
Physiological Consequences
Understanding the effects of GLP-1 on ␤-cell ionic
currents such as KATP channels has direct relevance to
insulin secretion in vivo. GLP-1 is known to promote
insulin secretion in a glucose-dependent manner (18)
(for reviews see Refs. 10 and 15). The mechanisms of
GLP-1 action seem to be predominately mediated via
elevation of cAMP and activation of PKA (10, 15). It has
also been suggested that calmodulin may play a role in
mediating the effects of GLP-1 in ␤-cells (37).
The cellular sites of action of PKA in the pancreatic
␤-cell include classes of ion channels such as KATP
channels, nonspecific cation channels, and voltagegated calcium and potassium channels (for review see
Ref. 10). PKA is also known to facilitate the exocytotic
release of insulin-containing vesicles (11). To date, the
cellular mechanism for the observed glucose-sensitivity of GLP-1 is poorly understood, although it has been
suggested that glucose-induced increases in intracellular ATP prime insulin-containing vesicles for release
(38). However, recent data suggest that, in the ␤HC9
insulin-secreting cell line, intracellular ATP does not
fluctuate greatly (⬃2 mM) in response to elevated glucose, whereas intracellular free ADP decreases exponentially as glucose levels increase (22). Glucosedependent increases in the ATP/ADP ratio have also
been observed in INS-1 cells (39). Data from our study
demonstrate that PKA inhibits KATP channels in an
ADP-dependent manner, and we suggest that at low
glucose concentrations, when intracellular ADP is elevated, the effect of GLP-1 (via PKA) on KATP channel
is negligible or even stimulatory. However, as intracellular ADP drops in response to increased glucose,
GLP-1 elicits a more pronounced closure of KATP
channels. The physiological consequence of such a
pathway would be a GLP-1-facilitated membrane depolarization and subsequent initiation of ␤-cell excitability only when glucose levels are elevated. We
therefore propose this sequence of events as a possible mechanism for the glucose-dependent action of
GLP-1. Our data also indicate that GLP-1-induced
membrane depolarization, via closure of KATP channels, may be the initial trigger for a chain of interrelated events that promote insulin release (for review
see Ref. 10).
KATP channels are also found in other tissues, and
PKA has been shown to activate these channels in
smooth muscle (40) and neurons (41). These documented tissue-specific effects may be accounted
for by a combination of the following three observations: 1) the existence of multiple phosphorylation
sites in both the SUR and Kir6.x subunits; 2) KATP
channel isoform-specific PKA phosphorylation
sites, for example the equivalent consensus PKA
site at S1448 is not found in the SUR2A or SUR2B
isoforms (42); and 3) the different metabolism and
glucose/substrate sensitivity of the cell-type in
question. Further studies are required to elucidate
the molecular mechanisms underlying PKA-mediated
2142
Mol Endocrinol, September 2002, 16(9):2135–2144
regulation of KATP channel activity in a variety of other
cell types.
MATERIALS AND METHODS
Cell Culture and Transfection
The insulin-secreting ␤-cell line INS-1 was maintained in culture with Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 11 mM glucose, 2 mM L-glutamine,
10% fetal calf serum, and 0.1% penicillin/streptomycin.
tsA201 cells (a SV40-transformed variant of the HEK293 human embryonic kidney cell line) were maintained in DMEM
supplemented with 25 mM glucose, 2 mM L-glutamine, 10%
fetal calf serum, and 0.1% penicillin/streptomycin. INS-1 and
tsA201 cells were kept at 37 C with 5% CO2. The KATP
channel Kir6.2 subunit clone from mouse was generously
provided by Dr. S. Seino (43). The SUR1 subunit clone from
hamster was generously provided by Drs. L. Aguilar Bryan
and J. Bryan (44). Clones were inserted into the mammalian
expression vector pCDNA3. The green fluorescent protein
tagged GLP-1 receptor clone was created by us (45). tsA201
cells were plated at 50–70% confluency on 35-mm culture
dishes 4 h before transfection. Clones were then transfected
into tsA201 cells using the calcium phosphate precipitation
technique. Transfected cells were identified using fluorescence optics in combination with either coexpression of the
green fluorescent protein plasmid (pGreenLantern, Life Technologies, Inc., Gaithersburg, MD) or the GFP-tagged GLP-1
receptor. Recordings were made from cells 48–72 h after
transfection. In contrast to cells expressing SUR1/Kir6.2 and
GLP-1R, nontransfected cells did not exhibit any inward barium-sensitive potassium current when dialyzed with pipette
solution containing no ATP (see Fig. 2D).
Molecular Biology
The S1448A point mutation was introduced into the hamster
SUR1 clone, using the Unique Site Elimination (U.S.E.) Mutagenesis Kit as per the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ) and confirmed by
sequence analysis.
Patch-Clamp Experiments
The perforated patch technique (46) was used to measure
whole-cell currents and membrane potentials from INS-1
cells whilst maintaining the intracellular signaling environment. Amphotericin (Sigma) was dissolved in dimethylsulfoxide (DMSO) (40 mg/ml) and diluted into the pipette solution
immediately before use to give a final concentration of 80
␮g/ml. Pipettes were then back-filled with this solution containing Amphotericin. The pipette solution used for all wholecell recordings contained the following (in mM): KCl 10, K
Aspartate 130, HEPES 10, MgCl2 1.4, EGTA 1, glucose 10.
The pH of the solution was adjusted to 7.4 with KOH. Patch
pipettes were pulled using borosilicate glass (G85150T,
Warner Instrument Corp., Hamden, CT) to yield pipettes with
a resistance of 2–6 M⍀ when filled with pipette solution. Once
a G⍀ seal was formed, series resistance was monitored and
a perforation access of less than 20 M⍀ was deemed acceptable. All membrane potential recordings were made in
current-clamp mode. Cells were not used in which the resting
membrane potential was more positive than –40 mV. In order
not to overestimate the GLP-1-induced depolarization, the
interburst membrane potential was measured during the period of action potential firing. Cells were superfused with
control and test solutions containing (in mM) NaCl 140, KCl 5,
HEPES 10, CaCl2 1.0, and MgCl2 1.4.
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
Whole-cell currents were recorded in voltage-clamp mode
under symmetrical [K⫹] conditions (140 mM). The holding
potential was 0 mV and inward currents were elicited using
200 msec long voltage steps to ⫺100 mV every 10 sec. The
amplitude of potassium current was estimated as the current
blocked by 2 mM barium chloride. In several instances, 20 ␮m
tolbutamide (Sigma) was also used to estimate the KATP
channel current present. An Axopatch 200B patch-clamp
amplifier and Clampex 8.0 software (Axon Instruments, Foster City, CA) were used for data acquisition and analysis.
Cells were superfused with control and test solutions containing (in mM) NaCl 140, KCl 5, HEPES 10, CaCl2 1.0, and
MgCl2 1.4.
Standard patch-clamp techniques were used to record
macroscopic single-channel currents in the inside-out patch
configuration. Single channel currents were recorded at fixed
holding potentials, amplified, digitized, and acquired using
pClamp 8.0 software. Data were sampled at 1000 Hz and
filtered at 400 Hz except where otherwise stated. The pipette
solution used for all inside-out patch recordings contained
the following (in mM): KCl 140, HEPES 10, MgCl2 1.4, EGTA
1, and glucose 10. The pH of the solution was adjusted to 7.4
with KOH. This solution was also used in the recording chamber to superfuse the cells/patches for experiments using
symmetrical [K⫹].
Cells or membrane patches were directly exposed to test
solutions via a multi-input perfusion pipette (time to change
solution at the tip of the recording pipette was less than 2
sec). All patch clamp experiments were performed at room
temperature (20–22 C).
Experimental Compounds
MgATP or K2ADP (Sigma, St. Louis, MO) was added as
required from a 10 mM stock, which was prepared immediately before use. Tolbutamide (Sigma) was stored as a 100
mM stock in DMSO at 4 C. H-89 (Calbiochem, La Jolla, CA)
was stored as a 1 mM stock in DMSO at 4 C. The catalytic
subunit of PKA (cPKA) was generously provided by Dr. Michael P. Walsh (University of Calgary, Calgary, Alberta, Canada) (47) and was used at a final concentration of 20 nM.
GLP-1 (7–36) and Exendin (9–39) (Sigma) were stored frozen
as 100 ␮M stock solutions in Tris-HCl buffered solution (pH
7.4) and added to the experimental solution immediately
before use.
cAMP Assay
Intracellular cAMP levels were determined in tsA201 cells that
had been cultured in 35-mm plates. Cells were transfected
with SUR1, Kir6.2, and the GFP-tagged GLP-1R. Transfection efficiency was 70–80% in all experiments as denoted by
the number of cells fluorescing green. GLP-1 (20 nM) was
added to tsA201 cells for 10 min before the assay. Cells were
then washed three times in ice-cold PBS, cAMP was then
extracted with 5% trichloroacetic acid and measured using
an enhanced immunoassay kit as per the manufacturer’s
instructions (Biomedical Technologies Inc., Stoughton, MA).
Analysis and Statistics
Recombinant single-channel current data were normalized to
yield Irel where Irel is the current under test conditions relative
to the maximal control current observed and was expressed
as a percentage i.e. I(test)/I(control) ⫻ 100. Statistical significance was evaluated by Student’s paired t test. Differences
with values of probability P ⬍ 0.05 were considered to be
significant. All values in the text are given as mean ⫾ SEM.
Light et al. • GLP-1 Inhibits ATP-Sensitive Potassium Channels
Acknowledgments
We would like to thank Lynn Eisner and Diana Steckley for
their expert technical assistance.
Received February 26, 2002. Accepted June 3, 2002.
Address all correspondence and requests for reprints to:
Peter E. Light, Department of Pharmacology, Faculty of
Medicine and Dentistry, University of Alberta, 9-58 Medical
Science Building, Alberta, Canada T6G 2H7. E-mail:
[email protected].
This study was supported by grants from the Canadian
Diabetes Association in honor of Violet D. Mulcahy (to P.E.L.)
and the Canadian Institutes of Health Research (to
M.B.W. M.O.P. 12898). P.E.L. received salary support as an
Alberta Heritage Foundation for Medical Research (AHFMR)
Scholar and Canadian Institutes of Health Research (CIHR)
New Investigator. M.B.W. is a CIHR Investigator. J.E.M.F. is
an AHFMR Postdoctoral Fellow.
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