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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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).
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). 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 TEA’s
insulinotropic effect and that TEA enhances the insulinotropic effects
of agonists acting through the KATP and PKA
pathways.
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). 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.
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). 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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, 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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). 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
(31–33 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 TEA’s 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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). 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 20–30% and about
60–70% of the IDR in insulin-secreting cells,
respectively, potentially accounting for 80–100% 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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). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 TEA’s 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 Kv2.1’s 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 60–70% 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 TEA’s 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 Student’s 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 3–6
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 (22–24 C) and normalized to cell capacitance unless stated
otherwise. For experiments at 31–33 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 (24–48 h after
isolation), rat brain, and HIT-T15 cells using Trizol (Life Technologies, Inc.) as per the manufacturer’s 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
manufacturer’s 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 Lowry’s 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 supplier’s
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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