Members of the Kv1 and Kv2 Voltage-Dependent K+ Channel Families Regulate Insulin Secretion
Patrick E. MacDonald,
Xiao Fang Ha,
Jing Wang,
Simon R. Smukler,
Anthony M. Sun,
Herbert Y. Gaisano,
Ann Marie F. Salapatek,
Peter H. Backx and
Michael B. Wheeler
Departments of Medicine (H.Y.G., P.H.B., M.B.W.) and Physiology
(P.E.M., X.F.H., J.W., S.R.S., A.M.S., A.M.F.S., P.H.B., M.B.W.),
University of Toronto, Toronto Ontario, Canada M5S 1A8
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ABSTRACT
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In pancreatic ß-cells, voltage-dependent K+ (Kv)
channels are potential mediators of repolarization, closure of
Ca2+ channels, and limitation of insulin secretion. The
specific Kv channels expressed in ß-cells and their contribution to
the delayed rectifier current and regulation of insulin secretion in
these cells are unclear. High-level protein expression and mRNA
transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and
insulinoma cells. Inhibition of these channels with tetraethylammonium
decreased IDR by approximately 85% and enhanced
glucose-stimulated insulin secretion by 2- to 4-fold.
Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit,
specifically eliminating Kv2 family currents, reduced delayed rectifier
currents in these cells by 6070% and enhanced glucose-stimulated
insulin secretion from rat islets by 60%. Expression of a C-terminal
truncated Kv1.4 subunit, abolishing Kv1 channel family currents,
reduced delayed rectifier currents by approximately 25% and enhanced
glucose-stimulated insulin secretion from rat islets by 40%. This
study establishes that Kv2 and 1 channel homologs mediate the majority
of repolarizing delayed rectifier current in rat ß-cells and
that antagonism of Kv2.1 may prove to be a novel glucose-dependent
therapeutic treatment for type 2 diabetes.
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INTRODUCTION
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THE ABILITY OF pancreatic islets of
Langerhans to secrete insulin in response to increased blood glucose
levels is essential for the maintenance of normoglycemia. Dysregulation
of islet insulin secretion is at least partly responsible for the
development of type 2 diabetes mellitus (1). In the
ß-cell, glucose stimulation is coupled to insulin secretion through
voltage-dependent and voltage-independent mechanisms (2, 3). Voltage-dependent mechanisms of stimulus-secretion coupling
are better characterized and are described in a number of reviews
(4, 5, 6, 7). Briefly, increased glucose metabolism in
pancreatic ß-cells, resulting from high postprandial blood glucose,
increases the intracellular ATP:ADP ratio. This leads to closure of
ATP-sensitive K+ (KATP)
channels and depolarization of the cell membrane (8), an
effect mimicked by the sulfonylurea drugs independent of blood glucose
(9, 10).
Depolarization of the ß-cell membrane results in the opening of
L-type Ca2+ channels, increasing the
intracellular Ca2+ concentration
([Ca2+]i) and
ultimately stimulating insulin secretion. ß-Cell repolarization is
mediated by a delayed rectifier current (IDR)
similar to those generated by voltage-dependent
K+ (Kv) or Ca2+-sensitive
voltage-dependent K+ (KCa)
channels (5, 11, 12, 13, 14). Accordingly, overexpression of a Kv
channel in transgenic mice was associated with hyperglycemia and
hypoinsulinemia, and in an insulinoma cell line this manipulation
attenuated [Ca2+]i
increases associated with glucose stimulation (15).
In addition, inhibitors of IDR are known to
enhance [Ca2+]i
oscillations (16) and insulin secretion (11, 13) in a glucose-dependent manner.
There are at least 11 currently known Kv channel families containing 26
homologs (17, 18, 19, 20, 21, 22), and of these, members of the Kv1, Kv2,
and Kv3 channel families mediate currents similar to those observed in
pancreatic ß-cells (5, 23, 24, 25). The task of identifying
the channel homologs responsible for repolarization of pancreatic
ß-cells is difficult because heterotetrameric Kv channels and
channels associated with regulatory ß-subunits often do not exhibit
the electrical and pharmacological properties of the constituent
pore-forming subunits (17, 26, 27, 28, 29).
Despite previous studies showing that insulin-secreting cells
express mRNA transcripts for a number of Kv and
KCa channels (5) and Kv2.1 protein
(11), no functional data exist for a role for specific
channels or channel families in ß-cell repolarization and the
regulation of insulin secretion. We have now characterized the mRNA and
protein expression of Kv1 and Kv2 channel family homologs in rat islets
and insulinoma cell lines. Pharmacological agents and
dominant-negative C-terminal truncated Kv1 (Kv1.4N) and Kv2
(Kv2.1N) channel subunit mutants were used to determine the role of
specific channels in mediating IDR and regulating
insulin secretion in the glucose-responsive HIT-T15 cell line and in
rat islets.
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RESULTS
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Effect of IDR Inhibition on Insulin Secretion
HIT-T15 cells or rat islets were incubated with the general Kv and
KCa channel antagonist tetraethylammonium (TEA)
at concentrations known to inhibit delayed rectifier currents while
having minimal effects on KATP channels
(12, 30, 31). In HIT-T15 cells, TEA (20 mM)
enhanced glucose-stimulated insulin secretion (GSIS) (from 0.51 ±
0.10 to 1.43 ± 0.14 ng/ml/2 h, n = 15;
P < 0.001), but most importantly, had no effect in the
absence of glucose (Fig. 1A
). Similarly,
GSIS from rat islets was enhanced by TEA (20 mM) (from
0.17 ± 0.03, n = 24 to 0.81 ± 0.18 ng/islet/h, n
= 13; P < 0.01), which had no effect in the absence of
stimulatory concentrations of glucose (control, n = 23; 20
mM TEA, n = 13) (Fig. 1B
). TEA enhanced GSIS
from rat islets in a dose-dependent manner (Fig. 1C
) with an
EC50 of 8.24 mM (n =
9). The effects of TEA were not related to cellular toxicity, since a
2-h exposure (20 mM) did not affect the survival
of HIT-T15 cells, as detected by propidium iodide fluorescence (not
shown).

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Figure 1. IDR Inhibition Enhances GSIS
The general Kv channel antagonist TEA (20 mM; black
bars) enhanced insulin secretion from HIT-T15 insulinoma cells
(A) and isolated rat islets (B) over 2 h compared to controls
(white bars). This effect occurred only in the presence
of stimulatory glucose. In rat islets, TEA dose-dependently enhanced
insulin secretion stimulated by 15 mM glucose in a
dose-dependent manner (C). The half-maximal effect of TEA was observed
at 8.24 mM. *, P < 0.05, **,
P < 0.01; and ***, P < 0.001
compared with controls.
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To determine whether TEAs insulinotropic activity was dependent
upon depolarization through KATP channel closure
and not glucose per se, we examined whether TEA could
enhance insulin secretion stimulated by KATP
channel inhibition in the absence of glucose. Micromolar concentrations
of the KATP channel antagonist
glyburide (Sigma, St. Louis, MO) have been
shown to stimulate insulin secretion from HIT-T15 cells in the absence
of glucose (32, 33). Glyburide (2
µM) simulated insulin secretion nearly 2-fold
from HIT-T15 cells (from 0.14 ± 0.01, n = 8 to 0.25 ±
0.01 ng/ml/2 h, n = 8; P < 0.001) in the absence
of glucose. Addition of TEA (20 mM) in the
presence of glyburide enhanced insulin secretion further
(to 0.56 ± 0.06 ng/ml/2 h, n = 8; P < 0.01
compared with glyburide alone) (Fig. 2
).

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Figure 2. IDR Inhibition Enhances
Glyburide-Stimulated Insulin Secretion from HIT-T15 Cells
In the absence of glucose, the KATP channel antagonist
glyburide (2 µM; hatched bar)
depolarizes HIT-T15 cells and stimulates insulin secretion compared
with control (white bar). The general Kv channel
antagonist TEA (20 mM) alone (gray bar) had
no effect on unstimulated insulin secretion but further enhanced
insulin secretion from HIT-T15 cells depolarized by
glyburide (black bar). **,
P < 0.01 and ***, P < 0.001
compared with control.
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Similarly, islets were incubated in nonstimulatory concentrations of
glucose (2.5 mM) with TEA (20 mM) and/or the
sulfonylurea drug glyburide. Glyburide at 2
µM elicited a large insulin response that was not
enhanced by 20 mM TEA (Fig. 3A
). Because the micromolar
concentrations of glyburide commonly used to stimulate
insulin secretion are approximately 4,000 times the published
EC50 in rodent islets (34),
nonspecific effects on ion channels or non-ß-cells are possible. With
10 nM glyburide, TEA (20 mM)
significantly enhanced insulin secretion compared with
glyburide alone in both the presence (n = 10;
P < 0.05) and absence (n = 12; P
< 0.05) of stimulatory glucose (Fig. 3B
). PKA pathway signaling
enhances GSIS, partly through actions on ion channels (35, 36). In the present study, TEA (20 mM)
enhanced the insulinotropic effect of the PKA pathway agonist
3-isobutyl-1-methylxanthine (IBMX) (1 µM) in
the presence of stimulatory glucose (Fig. 3D
). These results
demonstrate that membrane depolarization is sufficient to allow TEAs
insulinotropic effect and that TEA enhances the insulinotropic effects
of agonists acting through the KATP and PKA
pathways.

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Figure 3. IDR Inhibition Enhances the
Insulinotropic Effect of KATP and PKA Pathway Agonists
In the presence of 2.5 mM glucose (A and B) the
KATP channel antagonist glyburide
[hatched bars, at 2 µM (A) or 10
nM (B)] stimulates insulin secretion from isolated rat
islets compared with controls (white bars). The general
Kv channel antagonist TEA (20 mM; gray bars)
had no effect on unstimulated insulin secretion, but further enhanced
insulin secretion from isolated rat islets together (black
bars) with 10 nM glyburide (B). With
stimulatory glucose (15 mM, panels C and D), TEA (20
mM) enhanced insulin secretion and the effects of
secretagogues acting through the KATP (panel C, 10
nM glyburide) and PKA (panel D, 1
µM IBMX) pathways. *, P < 0.05; **,
P < 0.01; and ***, P < 0.001
compared with controls unless otherwise indicated.
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Pancreatic Islet and ß-cell Kv Channel
Expression
The above results demonstrate that the blockade of
IDR can enhance insulin secretion when glucose or
channel antagonists close KATP channels. To
determine which K+ channels mediate
IDR in insulin-secreting cells, HIT-T15 cell
and rat islet total RNA were examined for Kv gene transcripts via
RT-PCR (Table 1
). RT-PCR of HIT-T15 cell
RNA with Kv1.1, 1.3, and 1.4 specific primers resulted in amplification
products of the expected size. RT-PCR of rat islet RNA yielded cDNA
fragments of the correct size for Kv1.2, 1.3, 1.4, 1.6, and 2.1.
Sequencing confirmed that each fragment corresponded to the appropriate
channel with a high degree of nucleotide and predicted amino acid
identity with the respective human channel. All primer sets produced
PCR products of the appropriate size upon RT-PCR of rat brain or mouse
skeletal muscle (Kv1.7) total RNA as a positive control. No PCR product
was visible in the water blank controls.
Western blot studies confirmed the protein expression of Kv1.4,
1.6, 2.1, and 1.2 (at lower levels) in rat islets (Fig. 4
). Expression of Kv1.4 and 2.1 protein
was detected in the HIT-T15 and ßTC-6f7 insulinoma cell lines.
Despite failure to detect Kv2.1 mRNA in HIT-T15 cells by RT-PCR,
protein expression by this cell line is clearly abundant. It is
possible that species selectivity of our primers resulted in our
inability to detect the mRNA transcript in this hamster cell line.
Levels of Kv2.1 protein detected in islets were roughly equivalent to
those in the rat brain control using the Kv2.1b antibody. However, the
levels of Kv2.1 detected differed markedly between the two antibodies,
possibly reflecting variable species affinity (Fig. 4
). Kv1.1 protein
was detectable at low levels in HIT-T15 cells with longer exposures
(not shown) but was not detected in rat islets. Specific protein bands
for the KCa channels BK, SK2, and SK3 were not
detected in insulin-secreting cells, with the exception of a light but
detectable band for SK2 in ßTC-6f7 cells (Fig. 4
). Detection of Kv2.1
was used as a positive control in all protein samples from islets and
ß-cell lines.

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Figure 4. Kv and KCa Channel Protein Expression
HIT-T15 cell, ßTC-6f7 cell, rat islet, and rat brain lysates (50 µg
protein) were probed for Kv1, Kv2, and KCa channel proteins
using specific antibodies (see Materials and Methods).
When available, control antigen (blocking peptide) was incubated with
the channel antibody before probing of membranes to demonstrate the
specificity of detection. Kv2.1 protein was detected with two separate
antibodies (anti-Kv2.1a and anti-Kv2.1b). Anti-Kv2.1b was found to be
more species specific for rat.
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Characterization of TEA-Sensitive IDR in
Insulin-Secreting Cells
As illustrated in Fig. 5
, IDR recorded from HIT-T15 cells or rat islet
cells were noninactivating over 500 msec. Despite similar kinetic
properties, current amplitudes (at the end of a 500-msec pulse to 70 mV
from a holding potential of -70 mV) in HIT-T15 cells were
approximately double those observed in rat islet cells (Fig. 5
).
Because of the inclusion of 1 mM EGTA and 5 mM
MgATP within the pipette solution, the outward currents are expected to
primarily reflect the opening of Kv channels, with minimal
contributions from KCa or
KATP channels. Since native ß-cells operate
over a range of membrane potentials, we studied outward currents in
islet cells from a range of holding potentials and found no differences
between currents elicited from -90, -70, or -50 mV. Steady-state
inactivation protocols (over 15 sec) showed sustained currents
displaying a half-maximal voltage sensitivity
(V1/2) of -32.47 ± 1.53 mV (n =
12).

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Figure 5. ß-Cell IDR Is Blocked by TEA
Outward K+ currents were recorded by depolarizing with a
series of 500-msec pulses from a holding potential of -70 mV in 20-mV
increments to a maximal depolarization to 70 mV. Data were normalized
to cell capacitance. Representative traces from a typical HIT-T15 cell
(open marks) and rat islet cell (black
marks) are shown under control conditions
(triangles) and in the presence of 20 mM TEA
(circles) in panel A. In panel B, the current-voltage
relationship of maximum sustained current was plotted for both HIT-T15
cells (open marks) and rat islet cells (black
marks). At more physiological temperatures (3133 C,
dashed line), sustained outward currents were moderately
larger and also were largely blocked by 20 mM TEA. ***,
P < 0.001 compared with controls.
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Consistent with its ability to inhibit Kv and KCa
channels far more potently than KATP channels
(12, 31), TEA (20 mM) inhibited outward
K+ currents from HIT-T15 and rat islet cells by
85.5 ± 2.7% (n = 9; P < 0.001) and
87.9 ± 1.2% (n = 11; P < 0.001),
respectively (Fig. 5
). The effect of TEA was reversible upon washing
after exposures of as long as 2 h, similar to the exposures
used for the above insulin secretion studies (data not shown). The
biophysical and pharmacological properties of these currents most
closely resembled those mediated by members of the Kv1, 2, and 3
families, but not the Kv4 family (37, 38, 39). Outward
currents from rat islet cells at more physiological temperatures
(3133 C) were somewhat larger at the end of a 500-msec depolarization
to 70 mV from -70 mV. However, current inhibition by 20
mM TEA (86.4 ± 1.2%, n = 9:
P < 0.001) was not significantly different compared
with room temperature.
Effect of Kv and KCa Channel Antagonists on Insulin
Secretion
To investigate whether specific Kv or KCa
channels contribute to the regulation of insulin secretion, experiments
were performed using selective channel antagonists. Margatoxin (100
nM), which inhibits Kv1.3 and 1.6 with an
IC50 of 30 pM and 5 nM,
respectively (40), did not effect insulin secretion from
either HIT-T15 cells or rat islets (Table 2
). Dendrotoxin (200 nM), an
inhibitor of both Kv1.1 and 1.2 channels with an
IC50 of 20 nM (41, 42),
did enhance GSIS from HIT-T15 cells (Table 2
) accompanied by a
26.3 ± 9.7% (n = 7; P < 0.001) reduction
in IDR, but did not enhance insulin secretion
from rat islets. This is consistent with our ability to detect mRNA
transcripts for Kv1.1 and variable but low Kv1.1 protein in HIT-T15
cells, but not rat islets. Specific antagonists are not available
against cloned Kv1.4 channels, the other Kv1 family member that was
detected. However, heterotetrameric channels formed from this subunit
are insensitive to TEA (41) and are therefore less likely
contributors to TEAs insulinotropic effect. Because no specific
antagonists to Kv2 family channels are commercially available, this
characterization was limited to antagonists of Kv1 channel family
members.
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Table 2. Effect of Kv1 and KCa Specific
Antagonists on Glucose-Stimulated Insulin Secretion from HIT-T15 Cells
and Rat Islets
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Because both large- and small-conductance KCa
currents have been detected in insulin-secreting cells
(43, 44, 45, 46, 47, 48), we investigated the effect of
KCa channel antagonists on GSIS from rat islets.
Neither the small conductance KCa antagonist
apamin (200 nM) nor the large- and medium-conductance
KCa antagonist iberiotoxin (100 nM)
had a significant effect on GSIS from rat islets compared with controls
(Table 2
). However, this does not rule out the possibility that an
apamin-insensitive small-conductance KCa current
may have a role in regulating insulin secretion (49, 50).
Effect of Dominant-Negative Knockout of Kv1 and 2 Channels on
ß-Cell IDR
To further investigate the role of the Kv1 and 2 family channels
in mediating ß-cell IDR, we used a recombinant
adenovirus approach to express dominant-negative Kv1 (AdKv1.4N) and
2 (AdKv2.1N) channel subunits. Mutation or truncation involving all or
part of the pore-forming loop results in nonfunctional subunits that
can coassemble with and eliminate ion flow through endogenous channels
of the same family. Similar approaches have been used to study and
identify subunit assembly of native Kv channels (24, 51, 52).
Expression of the Kv1.4N subunit in HIT-T15 cells and rat islet cells
decreased IDR by 26.8 ± 5.9% (n = 14;
P < 0.05) and 22.3 ± 5.3% (n = 8;
P < 0.05), respectively, compared with controls (Fig. 6
). Expression of Kv2.1N reduced
IDR in HIT-T15 cells and rat islets cells to a
far greater extent (72.9 ± 2.9%; n = 24; P
< 0.001 and 61.6 ± 3.2%; n = 22; P <
0.001, respectively) compared with enhanced green fluorescent protein
(EGFP)-expressing controls (Fig. 7
). TEA
(20 mM) further reduced outward
K+ currents in cells expressing Kv2.1N,
eliminating a total of 94.3 ± 1.8% (n = 7;
P < 0.001) (HIT-T15) and 86.9 ± 1.8% (n =
11; P < 0.001) (rat islet cells) of
IDR compared with EGFP controls (Fig. 7
).
Remaining currents in Kv2.1N-expressing rat islet cells after the
addition of 20 mM TEA resembled A currents
mediated by cloned Kv1.4 and could be inactivated by holding at -50
mV, a protocol known to inactivate A currents (53) (Fig. 8
). These results suggest that the Kv1
and Kv2 channel families contribute approximately 2030% and about
6070% of the IDR in insulin-secreting cells,
respectively, potentially accounting for 80100% of total
IDR observed under the present conditions.
Steady-state inactivation of K+ currents recorded
from rat islet cells was unchanged by the expression of the Kv1.4N or
Kv2.1N constructs, showing no differences in voltage sensitivity of the
inactivating portion of the remaining currents with
V1/2 values of -33.6 ± 1.6 and -37.7
± 1.7 mV (n = 4 and 9).

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Figure 6. Kv1.4N Expression Reduces ß-Cell IDR
Current-voltage relationships were obtained from HIT-T15 cells (A) and
rat islet cells (B) expressing control EGFP (triangles)
or the dominant-negative Kv1.4N construct (circles).
Inset, Western blotting for the Kv1.4N construct showed
expression of the truncated protein in Kv1.4N-GW1H-transfected (2 ) and
AdKv1.4N-infected (3 ) HIT-T15 cells; only the full-length protein was
detected in Kv1.4-GW1H-transfected (4 ) or AdKv1.4-infected (5 ) cells.
Upon longer exposure, endogenous Kv1.4 would be detectable in control
lysates (1 ). *, P < 0.05 compared with controls.
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Figure 7. Kv2.1N Expression Reduces ß-Cell IDR
Current-voltage relationships were obtained from HIT-T15 cells (A) and
rat islet cells (B) expressing control EGFP (triangles)
or the dominant-negative Kv2.1N construct (circles).
Outward currents in cells expressing Kv2.1N could still be reduced by
addition of 20 mM TEA (open squares).
Inset, Northern blotting for the Kv2.1N transcript
showed expression in AdKv2.1N-infected (2 ) HIT-T15 cells (n = 2);
no transcript was detected in control-infected (1 ) cells (n = 2).
***, P < 0.001 compared with controls; and ###,
P < 0.001 compared with Kv2.1N-expressing cells.
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Figure 8. Outward K+ Currents in
Kv2.1N-Expressing Cells Exposed to TEA
Remaining outward currents in AdKv2.1N-infected rat islet cells exposed
to TEA (20 mM) were small and displayed an A current
component when depolarized to 30 mV from a holding potential of -90 mV
(triangle). Holding the cells at a more positive
potential (-50 mV; square) before depolarization did
not affect sustained currents (A), but dramatically reduced the
Kv1.4-like A current component (B). Each trace is an average of
recordings from eight AdKv2.1N-infected rat islet cells; the time
represented by the black bar in panel A is shown on an
expanded scale in panel B. The very fast component (within 5 msec of
depolarization) results from uncompensated capacitance transient,
and the small differences in initial holding current result from the
different holding potentials.
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Effect of Dominant-Negative Knockout of Kv1 and Kv2 Family Channels
on Insulin Secretion
Isolated islets were infected in vitro with AdKv1.4N,
AdKv2.1N, or AdEGFP (control). Coexpression of EGFP allowed
visualization of infected cells and estimation of infection efficiency.
Laser confocal microscopy (not shown) and our previous studies
(54) have shown that infection efficiencies of 3050%
are typical and cells within the islet core can be infected. Expression
of Kv1.4N in rat islets had no effect on basal insulin secretion but
significantly enhanced GSIS compared with control (0.031 ± 0.004
to 0.043 ± 0.007 ng/islet/h, n = 12; P <
0.05) (Fig. 9A
). Likewise, expression of
Kv2.1N in rat islets did not effect basal insulin secretion and caused
a much larger enhancement of GSIS compared with control (0.044 ±
0.009 to 0.070 ± 0.018 ng/islet/h, n = 9; P
< 0.001) (Fig. 9B
). These results appear to be in good agreement with
our electrophysiological observations, providing further evidence for a
link between enhanced insulin secretion and reduction of
IDR.

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Figure 9. Kv1.4N and Kv2.1N Expression Enhances GSIS from Rat
Islets
Insulin secretion from AdKv1.4N (panel A, black bars)
and AdKv2.1N (B, black bars)-infected rat islets was
enhanced compared with controls (white bars). These
dominant-negative subunits enhanced insulin secretion only in the
presence of stimulatory glucose, while no effect was observed under
nonstimulatory conditions. *, P < 0.05; and **,
P < 0.01 compared with controls.
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DISCUSSION
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Repolarization of pancreatic ß-cells after a glucose-induced
depolarization is mediated by a voltage-dependent outward
K+ current, which assists in closure of
voltage-dependent Ca2+ channels, thereby
modulating insulin secretion (5, 11, 12, 13, 14). Accordingly, the
general IDR inhibitor TEA enhances
glucose-stimulated [Ca]i oscillations and
insulin secretion (11, 12, 13, 16, 31). Consistent with an
important role for these currents in ß-cells, we found that 20
mM TEA reduced IDR (by 8590% at
both room temperature and near-physiological temperature) and enhanced
glucose-stimulated insulin secretion (
2- to 4-fold) in both HIT-T15
cells and isolated rat islets. As expected, since ß-cell
IDR currents are postulated to activate only
after glucose induced depolarization, TEA had no insulinotropic effect
in the absence of stimulatory glucose. The ability of TEA to block
IDR and enhance glucose-dependent insulin
secretion suggests that repolarizing K+ channels
underlie IDR. However, the effects of TEA do not
resolve which K+ channels are responsible for
IDR in ß-cells.
For a number of reasons, it is unlikely that TEA exerts its
glucose-dependent insulinotropic effect by inhibiting
KATP channels. Unlike KATP
antagonists such as glyburide, TEA (20 mM) did
not enhance unstimulated insulin secretion (Figs. 2
and 3
) (9, 10). In fact, the combination of TEA and glyburide
enhanced insulin secretion to a greater degree than either alone,
suggesting separate targets. Moreover, the glucose-dependent
insulinotropic effect of TEA was observable at concentrations far lower
than the published EC50 for
KATP channels (Fig. 1C
). Finally, in the presence
of high glucose, the majority of KATP channels
are closed, owing to an increase in the ATP:ADP ratio (11, 55).
Glyburide enhances insulin secretion from rodent islets with an
EC50 of 0.5 nM (56),
while human islets bind glyburide with a dissociation
constant (Kd) of 1 nM
(34). Here, a glyburide concentration of 10
nM stimulated a 2-fold increase in insulin secretion from
isolated rat islets in the absence of stimulatory glucose. TEA enhanced
glyburide-stimulated insulin release, indicating that
membrane depolarization is sufficient to allow TEAs insulinotropic
effect. The inability of TEA to significantly enhance rat islet insulin
secretion stimulated by 2 µM glyburide (Fig. 3A
) may result from nonspecific effects of this high dose of
glyburide on other cell types within the islet, a problem
that would not be present in a homogenous insulinoma cell line.
Interestingly, in the presence of stimulatory glucose, the effects of
glyburide or the phosphodiesterase inhibitor IBMX were
enhanced by TEA (Fig. 3
, C and D), suggesting that TEA-like drugs may
be used in combination with KATP or PKA pathway
agonists for a greater insulinotropic effect.
It is conceivable that Ca2+-sensitive
K+ currents mediate the effects of TEA in our
studies. Indeed KCa currents have been detected
in insulin-secreting cells; however, reports regarding the
pharmacological identification of these currents and their contribution
to glucose-induced electrical activity are conflicting (12, 30, 44, 45, 46, 48, 49, 50, 57, 58, 59). There is little functional evidence
supporting a major role for KCa channels in
regulating insulin secretion, and we were unable to detect
KCa protein or an insulinotropic effect of
general KCa channel antagonists (100
nM iberiotoxin and 200 nM apamin) in rat islets
(Table 2
). It is possible, nevertheless, that an apamin-
insensitive small-conductance KCa current,
possibly mediated by SK1 (60), can modulate insulin
secretion (45, 49, 50).
Although it seems clear that Kv channels are mediators of ß-cell
membrane repolarization, a role for specific channels in mediating
IDR has not been established. Since Kv channels
consist of homo- or heterotetrameric proteins from the same family
(17, 23, 25, 29), we chose to express truncated subunits
lacking the pore-forming region to selectively knock out functional
channels in a family-specific manner. Similar approaches have been used
to study and identify
-subunit assembly of native Kv channels
(24, 51, 52). In our study, the dominant-negative Kv1.4N
and Kv2.1N constructs inhibited outward K+
currents when coexpressed with wild-type channels of the same family in
HIT-T15 cells, but did not inhibit currents resulting from different
channel families (members of the Kv1, 2, 3, and 4 channel families were
tested; data not shown).
Expression of Kv2.1N in HIT-T15 cells or rat islet cells had a
dramatic effect on IDR, reducing it by
approximately 70 and 60%, respectively. This correlated with an
approximately 60% increase in GSIS from Kv2.1N infected islets
compared with EGFP-expressing controls. Supported by the fact that the
EC50 for the insulinotropic effect of TEA is
within the range reported for Kv2.1s IC50 for
block by TEA (61, 62, 63), our data suggest an important role
for the Kv2 family in insulin secretion. Kv2.1 protein was detected at
levels comparable to the rat brain control in both the insulinoma cell
lines and rat islets. This is consistent with previous studies showing
high-level protein expression of Kv2.1 in ßTC3-neo insulinoma cells
and Kv2.1 mRNA in insulin-secreting cells (5, 11).
Transcripts for Kv2.2, the only other Kv2 family member that forms
functional channel pores, were not detected. Kv2.1N expression did not
enhance insulin secretion to the same degree as seen with TEA and may
be explained in a number of ways. The insulinotropic effect of TEA was
measured in response to an acute application of the drug, whereas the
effect of Kv2.1N expression was measured after a more chronic
expression protocol (2 days) that may have led to changes in the
machinery controlling insulin secretion. In addition, our adenoviral
expression of the Kv2.1N construct was limited to approximately 50% of
the cells. Infection of rat islets with control EGFP virus decreased
basal insulin secretion and reduced insulin secretion induced by
glucose. Although the degree of insulin secretion enhancement by Kv2.1N
expression was compared with EGFP controls, it is conceivable that
Kv2.1N might contribute additional effects on insulin secretion
independent of IDR reduction. To minimize the
possible effects of differential expression efficiency between control
and experimental groups, islets were infected with equal numbers of
viral particles and inspected for qualitatively similar levels of EGFP
expression. Finally, it is still uncertain whether the relationship
between IDR reduction and enhancement of GSIS is
linear, meaning that a reduction in IDR greater
than 6070% may be required for a 2- to 4-fold increase in insulin
secretion to occur.
Expression of Kv1.4N in HIT-T15 or rat islet cells reduced
IDR by approximately 30 and 20%, respectively,
and increased GSIS from rat islets by about 40% compared with EGFP
controls. Of the Kv1 channel family, Kv1.6 protein was detected at high
levels in rat islets, while Kv1.4 protein was detected at high levels
in rat islets and the insulinoma cell lines HIT-T15 and ßTC-6f7.
Kv1.2 protein was detected at low levels in rat islets, and Kv1.1
protein was detected variably at low levels in HIT-T15 cells. We did
not examine the protein expression of Kv1.5 or 1.7, as neither was
detectable in insulin-secreting cells by RT-PCR, and both are known to
be insensitive to TEA. Variable detection of Kv1.1 in HIT-T15 cells is
consistent with the ability of Dendrotoxin to reduce
IDR and enhance insulin secretion in these cells.
Our results suggest a minimal contribution of homotetrameric Kv1.6 or
Kv1.4 channels to the insulinotropic effect of TEA since the former is
sensitive to Margatoxin and the latter is insensitive to TEA. However,
heterotetrameric channels containing these subunits cannot be ruled out
since heterotetrameric channels do not necessarily possess the
pharmacological sensitivities of their constituent subunits
(29). Also, the presence of regulatory ß-subunits,
channel phosphorylation, and the channels oxidative state are known to
significantly alter channel pharmacology and kinetics (27, 28, 64, 65, 66, 67). We did observe a small A current component in
Kv2.1N-expressing rat islet cells in the presence of 20 mM
TEA that was inactivated by holding the cell at -50 mV. This provides
confirmatory evidence for the presence of Kv1.4-containing channels but
suggests a limited role for them under normal conditions.
Current type 2 diabetes treatments aimed at enhancing insulin secretion
are limited to the sulfonylurea drugs, which act in a
glucose-independent manner. This is because their mechanism involves
inhibition of Kir6.2 through an interaction with
the associated SUR1, depolarizing the cell, and triggering influx of
Ca2+ and ultimately insulin secretion. Because
TEA acts in a glucose-dependent fashion, enhancing ß-cell
depolarization rather than initiating it, drugs acting at TEAs
specific target may be considered useful therapies that could also be
expected to enhance the insulinotropic effect of
KATP or PKA pathway agonists. In this study we
identified high-level expression of Kv1.4, 1.6, and 2.1 in rat islets
and have used an adenoviral approach to functionally knock out
these channels in isolated islets. Dominant-negative knockout of Kv2.1
enhanced insulin secretion by 60% in a glucose-dependent manner, while
knockout of the Kv1 channel family members had a similar, but lesser,
effect. It seems clear, however, that Kv2.1, and potentially members of
the Kv1 channel family, may represent novel targets for the treatment
of type 2 diabetes.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Islet Isolation
HIT-T15 cells, a gift from R. P. Robertson (Pacific NW
Research Institute, Seattle, WA), passage 8095, were cultured in
Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with
10% FBS, 1% L-glutamine, and 1%
penicillin-streptomycin. Islets of Langerhans were isolated from
male Wistar rats, 250350 g, by perfusion of the pancreas through the
common bile duct with 10 ml of a collagenase solution (10 mg/100 g body
wt) and incubation of the excised pancreas with shaking at 37 C. The
digestion was washed, filtered through 355 µm mesh, and separated on
a density gradient created by resuspending the pellet in
histopaque-1077 (Sigma, St. Louis, MO) and layering on
serum-free media [low-glucose (LG)-RPMI 1640 described below without
serum). Islets were collected from the interphase and further purified
from contaminating single cell types by sedimentation. Isolated islets
were cultured in LG-RPMI 1640 (7.5% FBS, 1% penicillin/streptomycin,
0.25% HEPES, and 2.5 mM glucose) at 37 C and 5%
CO2.
Insulin Secretion Studies
Twenty islets per well were plated in 24-well plates with
LG-RPMI 1640 for insulin secretion studies. Twenty-four to 48 h
after isolation, islets were washed and LG-RPMI 1640 was replaced by 2
ml of experimental media. Experimental media consisted of either
LG-RPMI 1640 or high glucose (HG)-RPMI 1640 (15 mM glucose)
with or without various experimental agents (see figures).
For HIT-T15 cell studies, cells were plated in 12-well plates at 5
x 105 cells per well. Forty-eight hours after
plating, HIT-T15 cells were washed with, and preincubated for 2x 30 min
in, Krebs Ringer bicarbonate (KRB) buffer (115 mM NaCl, 5
mM KCl, 24 mM NaHCO3, 2.5
mM CaCl2, 1 mM
MgCl2, 10 mM HEPES, and 0.1% BSA).
After preincubation, cells were washed with KRB buffer and then
incubated in 1 ml of KRB buffer alone or with 10 mM glucose
with and without experimental agents (see figures).
All secretion studies were performed for 2 h at 37 C and 5%
CO2, after which media samples were taken and
centrifuged at 700 x g. RIAs were performed using a
Rat Insulin RIA Kit (Linco Research, Inc., St. Charles,
MO). Each experiment was performed with an n value of at least 8 in at
least three separate experiments, and data were normalized to an
unstimulated control to account for variation between preparations and
are expressed as nanograms/islet/h or nanograms/ml/2 h. Data were
analyzed with Students t test or Wilcoxon matched pairs
test as appropriate. Dose-response curves and
EC50 values for insulin secretion studies were
generated using PRISM software (GraphPad Software, Inc.,
San Diego, CA).
Dominant-Negative Kv Channel Constructs and Adenoviral
Vectors
E1-deleted recombinant adenovirus shuttle vectors expressing a
C-terminal truncated Kv1.4 subunit (AdKv1.4N) or enhanced green
fluorescent protein (AdEGFP-RSV) alone under the control of the rous
sarcoma virus promoter was provided by Dr. Roger J. Hajjar
(Cardiovascular Research Center and Heart Failure Transplantation
Center, Massachusetts General Hospital, Harvard Medical School, Boston,
MA). Recombinant adenoviruses expressing a C-terminal truncated Kv2.1
subunit (AdKv2.1N) or EGFP alone (AdEGFP-CMV) under the control of the
cytomegalovirus promoter were prepared by CRE-lox recombination
(68). All of these adenovirus constructs coexpress EGFP
with the gene of interest to facilitate the identification of infected
cells. Adenoviruses were amplified by passage in HEK 293 cells or CRE-8
cells (for viruses constructed by CRE-lox recombination). Infected
cells were resuspended and lysed in 10 mM Tris, 1
mM MgCl2, pH 8.0 [1 mM
freeze-thaw media (FT)] and purified by centrifuging the lysate on a
gradient created by layering 3 ml each of 1.20 g/ml, 1.33 g/ml, and
1.45 g/ml CsCl in 1 mM FT at 27,000 rpm for 2 h in a
SW41-T1 rotor (Beckman Coulter, Inc., Fullerton, CA).
Resultant bands were removed and dialyzed overnight against 1
mM FT and 10% glycerol and stored at -70 C until use.
Infection of isolated rat islets was performed in 24-well plates with
either 20 (insulin secretion studies) or 50 (electrophysiological
studies) islets per well on the day of isolation. Infection of HIT-T15
cells for electrophysiological studies (AdKv2.1N only) was performed in
35-mm dishes seeded 24 h previously with 5 x
105 cells per dish. Islets or HIT-T15 cells were
cultured in 0.5 ml of normal media with 1 x
1010 virus particles/ml for 2 h at 37 C and
5% CO2 after which 1.5 ml of LG-RPMI 1640 were
added. Forty-eight hours later, islets or HIT-T15 cells were examined
under UV light to detect the expression of EGFP. Insulin secretion
studies, electrophysiological studies, RNA isolation, or protein
isolation was carried out 48 h post infection.
For HIT-T15 cell electrophysiological studies, a wild-type Kv1.4 or a
Kv1.4N construct (in the GW1H plasmid; provided by Dr. Hajjar) was
expressed by transfection with Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) as per instructions of the manufacturer.
This plasmid was cotransfected with the pEGFP plasmid (CLONTECH Laboratories, Inc. Palo Alto, CA) that expresses EGFP as a
marker for transfection. Control cells were transfected with pEGFP
alone.
Electrophysiological Studies
Islets were washed in and incubated with PBS and 0.2
mM EDTA with 1.5% trypsin for 11 min, followed by
mechanical dispersion and plating of single-islet cells overnight in
LG-RPMI 1640 in 35-mm culture dishes. Cells were voltage clamped in the
whole-cell configuration using an EPC-9 amplifier and Pulse software
(Heka Electronik, Lambrecht, Germany). Electrical identification of
ß-cells using a current clamp was not possible due to the
intracellular solution required to measure IDR
currents; however, the majority of islet cells (
70% or more) are
ß-cells, and all electrophysiological experiments were confirmed in a
clonal ß-cell line (HIT-T15). HIT-T15 cells were trypsinized and
replated in 35-mm dishes 24 h before electrophysiological studies.
Patch pipettes were prepared from 1.5-mm thin-walled borosilicate glass
tubes using a two-stage micropipette puller (Narishige, Tokyo, Japan).
Pipettes were heat polished and typically had a tip resistance of 36
M
when filled with intracellular solution containing (in
mM): KCl, 140; MgCl2·6
H2O, 1; EGTA, 1; HEPES, 10; MgATP 5 (pH 7.25)
with KOH. The bath solution contained (in mM): NaCl, 140;
CaCl2, 2; KCl, 4; MgCl2 ·
6 H2O, 1; HEPES, 10 (pH 7.3) with NaOH. All
electrophysiological measurements reported were made at room
temperature (2224 C) and normalized to cell capacitance unless stated
otherwise. For experiments at 3133 C, temperature was maintained with
an Olympus America Inc. temperature control unit (Melville, NY) and
continuous perfusion with warmed solutions. Outward currents were
elicited with a 500-msec depolarization in steps of 20 mV to +70 mV
from a holding potential of -70 mV. Outward currents were also
compared from holding potentials of -90, -70, and -50 mV using
500-msec depolarizing pulses to 30 mV. To minimize variation, maximum
sustained current was determined from a third degree polynomial
function fit to the final 25 msec of the 500-msec depolarizing
pulse.
The voltage dependence of steady state inactivation was investigated by
holding the cells at potentials from -80 to 30 mV for 15 sec followed
by a 5-msec prepulse to -70 and a 500-msec depolarization to 30 mV to
elicit outward currents. Steady state inactivation curves were fit with
a Boltzman function: I/Imax = 1/[1 +
exp([V - V1/2]/s)] where
V1/2 is the voltage at which half the channels
are inactivated, and s is the slope of the curve. For pharmacological
studies, the drug was applied by perfusion for at least 5 min before
recording. Outward currents at the end of the 500-msec depolarizing
pulse were compared using the t test.
RNA Analysis
Total RNA was obtained from rat islets (2448 h after
isolation), rat brain, and HIT-T15 cells using Trizol (Life Technologies, Inc.) as per the manufacturers instructions.
RT-PCR was performed on 1 µg of total RNA using a GeneAmp RNA PCR kit
(Perkin-Elmer Corp., Branchburg, NJ) according to the
manufacturers instructions. PCR primers used were designed to
conserved sequences of rat Kv1.1 [Forward (F):
5'-AAGGATCCGTCATTGTGTCC-3'; Reverse (R): 5'-AAAGGCCTAAACATCGGTCAG-3'],
Kv1.2 (F: 5'-GTAAAGCACACTTCTCAAGCCCC-3'; R:
5'-CCTCCCGAAACATCTCAATTGC-3'); Kv1.3 (F:
5'-GAGATCCGCTTTTACCAGCTGGG-3'; R: 5'-CATGATATTTCTGGAGAAGG-3'); Kv1.4
(F: 5'-GATAGCCATTGTGTCCGTCCTGG-3'; R:
5'-GGCACACAGGGACCCGACAATC-3'); Kv1.5 (F:
5'-CTGAGAGGGAGAGAGGCAGGG-3'; R:
5'-GCAGCTCCTGAGGCATAGGG-3'); Kv1.6 (F: 5'-GTTGGTGATCAACATCTCCGGG-3'; R:
5'-GGCCGCCTTGCTGGGACAGG-3'); Kv1.7 (mouse) (F:
5'-TCTCCGTACTCGTCATCCGG-3'; R: 5'-AAATGGGTGTCCACCCGGTC-3'); Kv2.1 (F:
5'-CGAGGAGCTGAAGCGGGAGG-3'; R: 5'-GGAAGATGGTGACGTAGTAGGG-3'); and
Kv2.2 (F: 5'-GGATGCCTTTGCTAGAAGTATGG-3'; R:
5'-CGCTGGCACTGTCAGGTTGC-3'). PCR was also performed on water
blank controls containing no cDNA template and rat brain cDNA as a
positive control. PCR was performed with 35 cycles of 94 C for 30 sec,
60 C for 35 sec, and 72 C for 45 sec followed by a 10-min extension at
72 C. PCR products of the expected size were excised from an 1.2% low
melt agarose gel and ligated into the pCR2.1 vector and sequenced using
the universal M13 reverse primer. Resulting sequences were subjected to
analysis by NCBI Blast (NCBI, Bethesda, MD) and nucleotide and amino
acid identity analysis with MacDNASIS (Hitachi Software, San Francisco,
CA).
Northern analysis was used to detect expression of mRNA transcripts for
Kv2.1N in total RNA (7.5 µg) from AdKv2.1N- or AdEGFP-infected HIT
cells as described previously (69). Probes were generated
by random priming (Random Primers DNA Labeling System, Life Technologies, Inc.) of Kv2.1N cDNA and incorporation of
P32-dCTP. Blots were washed twice by shaking in
room temperature 0.1% SDS/2xSSC followed by a 30-min wash in 0.1%
SDS/0.1x SSC at 55 C. Blots were exposed overnight to X-OMAT AR film
(Eastman Kodak Co., Rochester, NY).
Protein Analysis
Immunoblotting of Kv channel proteins was performed as
previously described (70, 71). Briefly, the islets were
washed in ice-cold PBS, solubilized in 2% SDS loading buffer, boiled
for 10 min, and passed through a 23G needle. Fifty micrograms of the
protein from each sample, determined by Lowrys method, were loaded
and separated on a 10% polyacrylamide gel. The protein was transferred
to PVDF-Plus (Fisher Scientific Ltd., Nepean, Ontario,
Canada) membrane and immunodecorated with primary antibody or
antibody-antigen solutions (diluted according to the suppliers
instructions) for 1.5 h at room temperature. Primary antibodies
were from Alomone Labs (Jerusalem, Israel) (Kv1.2, 1.3, 1.4, 1.6, 2.1)
and Upstate Biotechnology, Inc. (Lake Placid, NY) (Kv1.1,
2.1). Primary antibodies were detected with appropriate secondary
antibodies (sheep antimouse, 1:10,000; donkey antirabbit, 1:7,500;
Amersham Pharmacia Biotech Ltd., Buckinghamshire, U.K.)
for 1 h, and then visualized by chemiluminescence (ECL-Plus,
Amersham Pharmacia Biotech Ltd.) and exposure of the
filters to Kodak film (Eastman Kodak Co.,
Rochester, NY) for 5 sec to 10 min. At least three blots were performed
for each protein investigated.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Robert Hajjar (Harvard Medical School) for
providing Kv1.4N plasmid and adenovirus vectors. Additionally, we thank
Dr. Robert Tsushima (University of Toronto) for helpful discussion, the
use of equipment, and critical reading of the manuscript; and Dr.
Sabine Sewing (Eli Lilly) for helpful discussion.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Michael B. Wheeler, Ph.D., or Peter H. Backx, D.V.M., Ph.D., University of Toronto, Department of Physiology, 1 Kings College Circle, Toronto, Ontario, Canada, M5S 1A8. E-mail: michael.wheeler@utoronto.ca or p.backx{at}utoronto.ca
This research was supported by research grants to M.B.W. and P.H.B.
from the Banting and Best Diabetes Centre (BBDC) and Eli Lilly & Co.
(Indianapolis, IN). P.H.B. holds a Career Investigator Award from the
Heart and Stroke Foundation of Ontario. P.E.M. was supported by
studentships from the Department of Physiology, University of Toronto,
and the BBDC/Novo Nordisk. S.R.S. was supported by an Institute of
Medical Science Summer Studentship.
Abbreviations: [Ca2+]i, intracellular
Ca2+ concentration; EGFP, enhanced green fluorescent
protein; FT, freeze-thaw media; GSIS, glucose-stimulated insulin
secretion; IBMX, 3-isobutyl-1-methylxanthine; HG-RPMI, high-glucose
Roswell Park Memorial Institute medium; IDR , delayed
rectifier current; KCa, Ca2+-sensitive
voltage-dependent K+ channel; KRB, Krebs Ringer
bicarbonate; Kv, voltage-dependent K+ channel;
LG-RPMI, low-glucose RPMI; TEA, tetraethylammonium.
Received for publication January 31, 2001.
Accepted for publication May 8, 2001.
 |
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