Members of the Kv1 and Kv2 Voltage-Dependent K+ Channel Families Regulate Insulin Secretion (original) (raw)

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1Departments of Medicine (H.Y.G., P.H.B., M.B.W.), Toronto Ontario, Canada M5S 1A8

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2Physiology (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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1Departments of Medicine (H.Y.G., P.H.B., M.B.W.), Toronto Ontario, Canada M5S 1A8

2Physiology (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

*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.

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1Departments of Medicine (H.Y.G., P.H.B., M.B.W.), Toronto Ontario, Canada M5S 1A8

2Physiology (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

*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.

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Received:

31 January 2001

Published:

01 August 2001

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Patrick E. MacDonald, Xiao Fang Ha, Jing Wang, Simon R. Smukler, Anthony M. Sun, Herbert Y. Gaisano, Ann Marie F. Salapatek, Peter H. Backx, Michael B. Wheeler, Members of the Kv1 and Kv2 Voltage-Dependent K+ Channel Families Regulate Insulin Secretion, Molecular Endocrinology, Volume 15, Issue 8, 1 August 2001, Pages 1423–1435, https://doi.org/10.1210/mend.15.8.0685
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Abstract

In pancreatic β-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 β-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 60–70% 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 β-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.

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 theβ -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 (4–7). Briefly, increased glucose metabolism in pancreatic β-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 β-cell membrane results in the opening of L-type Ca2+ channels, increasing the intracellular Ca2+ concentration ([Ca2+]i) and ultimately stimulating insulin secretion. β-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, 11–14). 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 (17–22), and of these, members of the Kv1, Kv2, and Kv3 channel families mediate currents similar to those observed in pancreatic β-cells (5, 23–25). The task of identifying the channel homologs responsible for repolarization of pancreaticβ -cells is difficult because heterotetrameric Kv channels and channels associated with regulatory β-subunits often do not exhibit the electrical and pharmacological properties of the constituent pore-forming subunits (17, 26–29).

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 β-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.

RESULTS

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).

Fig. 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.

To determine whether TEA’s 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 (2μ m) 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).

Fig. 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.

Similarly, islets were incubated in nonstimulatory concentrations of glucose (2.5 mm) with TEA (20 mm) and/or the sulfonylurea drug glyburide. Glyburide at 2μ m 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-β-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.

Fig. 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, 1μ m IBMX) pathways. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 compared with controls unless otherwise indicated.

Pancreatic Islet and β-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.

Table 1.

Identification of Kv Channel Homologs in HIT-T15 Cells and Rat Islets by RT-PCR of Total RNA

a

Identity as compared to human cDNA and protein sequences.

Table 1.

Identification of Kv Channel Homologs in HIT-T15 Cells and Rat Islets by RT-PCR of Total RNA

a

Identity as compared to human cDNA and protein sequences.

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 βTC-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 βTC-6f7 cells (Fig. 4). Detection of Kv2.1 was used as a positive control in all protein samples from islets andβ -cell lines.

Fig. 4.

Kv and KCa Channel Protein Expression HIT-T15 cell, βTC-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.

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 β-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).

Fig. 5.

β-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 (31–33 C, dashed line), sustained outward currents were moderately larger and also were largely blocked by 20 mm TEA. ***, P < 0.001 compared with controls.

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 (37–39). 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.

Table 2.

Effect of Kv1 and KCa Specific Antagonists on Glucose-Stimulated Insulin Secretion from HIT-T15 Cells and Rat Islets

a

P < 0.05 compared to control.

Table 2.

Effect of Kv1 and KCa Specific Antagonists on Glucose-Stimulated Insulin Secretion from HIT-T15 Cells and Rat Islets

a

P < 0.05 compared to control.

Because both large- and small-conductance KCa currents have been detected in insulin-secreting cells (43–48), 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 onβ -Cell IDR

To further investigate the role of the Kv1 and 2 family channels in mediating β-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).

Fig. 6.

Kv1.4N Expression Reduces β-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.

Fig. 7.

Kv2.1N Expression Reduces β-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.

Fig. 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.

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 30–50% 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.

Fig. 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.

DISCUSSION

Repolarization of pancreatic β-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, 11–14). Accordingly, the general IDR inhibitor TEA enhances glucose-stimulated [Ca]i oscillations and insulin secretion (11–13, 16, 31). Consistent with an important role for these currents in β-cells, we found that 20 mm TEA reduced IDR (by 85–90% 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 β-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 β-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, 44–46, 48–50, 57–59). 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 β-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 α-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 (61–63), 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 βTC3-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 βTC-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 β-subunits, channel phosphorylation, and the channels oxidative state are known to significantly alter channel pharmacology and kinetics (27, 28, 64–67). 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 β-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.

MATERIALS AND METHODS

Cell Culture and Islet Isolation

HIT-T15 cells, a gift from R. P. Robertson (Pacific NW Research Institute, Seattle, WA), passage 80–95, 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, 250–350 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× 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 × 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 × 105 cells per dish. Islets or HIT-T15 cells were cultured in 0.5 ml of normal media with 1 × 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 ofβ -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) areβ -cells, and all electrophysiological experiments were confirmed in a clonal β-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 MΩ 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/2×SSC followed by a 30-min wash in 0.1% SDS/0.1× 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.

Acknowledgments

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.

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
  intracellularCa2+ concentration;
• EGFP
  enhanced green fluorescent protein;
• FT
• 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
• TEA

1

Dagogo-Jack

S

,

Santiago

JV

1997

Pathophysiology of type 2 diabetes and modes of action of therapeutic interventions.

Arch Intern Med

157

:

1802

–

1817

2

Straub

SG

,

James

RF

,

Dunne

MJ

,

Sharp

GW

1998

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets.

Diabetes

47

:

758

–

763

3

Aizawa

T

,

Komatsu

M

,

Asanuma

N

,

Sato

Y

,

Sharp

GW

1998

Glucose action ’beyond ionic events’ in the pancreatic β cell.

Trends Pharmacol Sci

19

:

496

–

499

4

Ammala

C

,

Kane

C

,

Cosgrove

KE

, et al.

1997

Characterization of ion channels in stimulus-secretion coupling in pancreatic islets.

Digestion

58

(Suppl 2):

81

–

85

5

Dukes

ID

,

Philipson

LH

1996

K+ channels: generating excitement in pancreatic β-cells.

Diabetes

45

:

845

–

853

6

Newgard

CB

,

McGarry

JD

1995

Metabolic coupling factors in pancreatic β-cell signal transduction.

Annu Rev Biochem

64

:

689

–

719

7

Rorsman

P

1997

The pancreatic β-cell as a fuel sensor: an electrophysiologist’s viewpoint.

Diabetologia

40

:

487

–

495

8

Tanabe

K

,

Tucker

SJ

,

Matsuo

M

, et al.

1999

Direct photoaffinity labeling of the Kir6.2 subunit of the ATP- sensitive K+ channel by 8-azido-ATP.

J Biol Chem

274

:

3931

–

3933

9

Boyd III

AE

1992

The role of ion channels in insulin secretion.

J Cell Biochem

48

:

235

–

241

10

Dunne

MJ

,

Cosgrove

KE

,

Shepherd

RM

,

Ammala

C

1999

Potassium channels, sulphonylurea receptors and control of insulin release.

Trends Endocrinol Metab

10

:

146

–

152

11

Roe

MW

, Worley

III

JF

,

Mittal

AA

, et al.

1996

Expression and function of pancreatic β-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

J Biol Chem

271

:

32241

–

32246

12

Fatherazi

S

,

Cook

DL

1991

Specificity of tetraethylammonium and quinine for three K channels in insulin-secreting cells.

J Membr Biol

120

:

105

–

114

13

Henquin

JC

1990

Role of voltage- and Ca2(+)-dependent K+ channels in the control of glucose-induced electrical activity in pancreatic B-cells.

Pflugers Arch

416

:

568

–

572

14

Smith

PA

,

Bokvist

K

,

Arkhammar

P

,

Berggren

PO

,

Rorsman

P

1990

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic β-cells.

J Gen Physiol

95

:

1041

–

1059

15

Philipson

LH

,

Rosenberg

MP

,

Kuznetsov

A

, et al.

1994

Delayed rectifier K+ channel overexpression in transgenic islets and β-cells associated with impaired glucose responsiveness.

J Biol Chem

269

:

27787

–

27790

16

Eberhardson

M

,

Tengholm

A

,

Grapengiesser

E

1996

The role of plasma membrane K+ and Ca2+ permeabilities for glucose induction of slow Ca2+ oscillations in pancreatic β-cells.

Biochim Biophys Acta

12

:

8367

–

8372

17

Christie

MJ

1995

Molecular and functional diversity of K+ channels.

Clin Exp Pharmacol Physiol

22

:

944

–

951

18

Salinas

M

, de

Weille

J

,

Guillemare

E

,

Lazdunski

M

,

Hugnot

JP

1997

Modes of regulation of shab K+ channel activity by the Kv8.1 subunit.

J Biol Chem

272

:

8774

–

8780

19

Hugnot

JP

,

Salinas

M

,

Lesage

F

, et al.

1996

Kv8.1, a new neuronal potassium channel subunit with specific inhibitory properties towards Shab and Shaw channels.

EMBO J

15

:

3322

–

3331

20

Stocker

M

,

Kerschensteiner

D

1998

Cloning and tissue distribution of two new potassium channel α-subunits from rat brain.

Biochem Biophys Res Commun

248

:

927

–

934

21

Salinas

M

,

Duprat

F

,

Heurteaux

C

,

Hugnot

JP

,

Lazdunski

M

1997

New modulatory α subunits for mammalian Shab K+ channels.

J Biol Chem

272

:

24371

–

24379

22

Dilks

D

,

Ling

HP

,

Cockett

M

,

Sokol

P

,

Numann

R

1999

Cloning and expression of the human kv4.3 potassium channel.

J Neurophysiol

81

:

1974

–

1977

23

Xu

J

,

Yu

W

,

Jan

YN

,

Jan

LY

,

Li

M

1995

Assembly of voltage-gated potassium channels. Conserved hydrophilic motifs determine subfamily-specific interactions between the α-subunits.

J Biol Chem

270

:

24761

–

24768

24

Blaine

JT

,

Ribera

AB

1998

Heteromultimeric potassium channels formed by members of the Kv2 subfamily.

J Neurosci

18

:

9585

–

9593

25

Koch

RO

,

Wanner

SG

,

Koschak

A

, et al.

1997

Complex subunit assembly of neuronal voltage-gated K+ channels. Basis for high-affinity toxin interactions and pharmacology.

J Biol Chem

272

:

27577

–

27581

26

Standen

NB

,

Quayle

JM

1998

K+ channel modulation in arterial smooth muscle.

Acta Physiol Scand

164

:

549

–

557

27

Rettig

J

,

Heinemann

SH

,

Wunder

F

, et al.

1994

Inactivation properties of voltage-gated K+ channels altered by presence of β-subunit.

Nature

369

:

289

–

294

28

Heinemann

SH

,

Rettig

J

,

Graack

HR

,

Pongs

O

1996

Functional characterization of Kv channel β-subunits from rat brain.

J Physiol (Lond)

493

:

625

–

633

29

Po

S

,

Roberds

S

,

Snyders

DJ

,

Tamkun

MM

,

Bennett

PB

1993

Heteromultimeric assembly of human potassium channels. Molecular basis of a transient outward current?

Circ Res

72

:

1326

–

1336

30

Bokvist

K

,

Rorsman

P

,

Smith

PA

1990

Block of ATP-regulated and Ca2(+)-activated K+ channels in mouse pancreaticβ -cells by external tetraethylammonium and quinine.

J Physiol (Lond)

423

:

327

–

342

31

Bokvist

K

,

Rorsman

P

,

Smith

PA

1990

Effects of external tetraethylammonium ions and quinine on delayed rectifying K+ channels in mouse pancreatic β-cells.

J Physiol (Lond)

423

:

311

–

325

32

Kulkarni

RN

,

Wang

ZL

,

Wang

RM

,

Smith

DM

,

Ghatei

MZ

,

Bloom

SR

2000

Glibenclamide but not other sulphonylureas stimulates release of neuropeptide Y from perifused rat islets and hamster insulinoma cells.

J Endocrinol

165

:

509

–

518

33

Seri

K

,

Sanai

K

,

Kurachima

K

,

Imamura

Y

,

Akita

H

2000

(R)-ACX is a novel sulfonylurea compound with potent, quick and short-lasting hypoglycemic activity.

Eur J Pharmacol

389

:

253

–

256

34

Giannaccini

G

,

Lupi

R

,

Trincavelli

ML

, et al.

1998

Characterization of sulfonylurea receptors in isolated human pancreatic islets.

J Cell Biochem

71

:

182

–

188

35

Gromada

J

,

Holst

JJ

,

Rorsman

P

1998

Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1.

Pflugers Arch

435

:

583

–

594

36

Kanno

T

,

Suga

S

,

Wu

J

,

Kimura

M

,

Wakui

M

1998

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet β-cells.

Pflugers Arch

435

:

578

–

580

37

Perney

TM

,

Kaczmarek

LK

1991

The molecular biology of K+ channels.

Curr Opin Cell Biol

3

:

663

–

670

38

Philipson

LH

,

Miller

RJ

1992

A small K+ channel looms large.

Trends Pharmacol Sci

13

:

8

–

11

39

Rorsman

P

,

Trube

G

1986

Calcium and delayed potassium currents in mouse pancreatic β-cells under voltage-clamp conditions.

J Physiol (Lond)

374

:

531

–

550

40

Garcia-Calvo

M

,

Leonard

RJ

,

Novick

J

, et al.

1993

Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels.

J Biol Chem

268

:

18866

–

18874

41

Grissmer

S

,

Nguyen

AN

,

Aiyar

J

, et al.

1994

Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines.

Mol Pharmacol

45

:

1227

–

1234

42

Hurst

RS

,

Busch

AE

,

Kavanaugh

MP

,

Osborne

PB

,

North

RA

,

Adelman

JP

1991

Identification of amino acid residues involved in dendrotoxin block of rat voltage-dependent potassium channels.

Mol Pharmacol

40

:

572

–

576

43

Satin

LS

,

Hopkins

WF

,

Fatherazi

S

,

Cook

DL

1989

Expression of a rapid, low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering.

J Membr Biol

112

:

213

–

222

44

Findlay

I

,

Dunne

MJ

,

Petersen

OH

1985

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium.

J Membr Biol

83

:

169

–

175

45

Kozak

JA

,

Misler

S

,

Logothetis

DE

1998

Characterization of a Ca2+-activated K+ current in insulin-secreting murine βTC-3 cells.

J Physiol (Lond)

509

:

355

–

370

46

Findlay

I

,

Dunne

MJ

,

Ullrich

S

,

Wollheim

CB

,

Petersen

OH

1985

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin- secreting cells.

FEBS Lett

185

:

4

–

8

47

Smith

PA

,

Bokvist

K

,

Arkhammar

P

,

Berggren

PO

,

Rorsman

P

1990

Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic β-cells.

J Gen Physiol

95

:

1041

–

1059

48

Tabcharani

JA

,

Misler

S

1989

Ca2+-activated K+ channel in rat pancreatic islet B cells: permeation, gating and blockade by cations.

Biochim Biophys Acta

982

:

62

–

72

49

Lebrun

P

,

Atwater

I

,

Claret

M

,

Malaisse

WJ

,

Herchuelz

A

1983

Resistance to apamin of the Ca2+-activated K+ permeability in pancreatic β-cells.

FEBS Lett

161

:

41

–

44

50

Gopel

SO

,

Kanno

T

,

Barg

S

,

Eliasson

L

,

Galvanovskis

J

,

Renstrom

E

,

Rorsman

P

1999

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreaticβ cells.

J Gen Physiol

114

:

759

–

770

51

Ribera

AB

,

Pacioretty

LM

,

Taylor

RS

1996

Probing molecular identity of native single potassium channels by overexpression of dominant negative subunits.

Neuropharmacology

35

:

1007

–

1016

52

Johns

DC

,

Marban

E

,

Nuss

HB

1999

Virus-mediated modification of cellular excitability.

Ann NY Acad Sci

868

:

418

–

422

53

Hille

B

2001

Potassium channels and chloride channels

. In:

Hille

B

, ed.

Ionic channels of excitable membranes

.

Sunderland, MA

:

Sinauer Associates Inc.

;

99

–

116

54

Chan

CB

,

MacDonald

PE

,

Saleh

MC

,

Johns

DC

,

Marban

E

,

Wheeler

MB

1999

Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.

Diabetes

48

:

1482

–

1486

55

Dukes

ID

,

McIntyre

MS

,

Mertz

RJ

, et al.

1994

Dependence on NADH produced during glycolysis for β-cell glucose signaling.

J Biol Chem

269

:

10979

–

10982

56

Panten

U

,

Burgfeld

J

,

Goerke

F

, et al.

1989

Control of insulin secretion by sulfonylureas, meglitinide and diazoxide in relation to their binding to the sulfonylurea receptor in pancreatic islets.

Biochem Pharmacol

38

:

1217

–

1229

57

Light

DB

, Van

Eenenaam

DP

,

Sorenson

RL

,

Levitt

DG

1987

Potassium-selective ion channels in a transformed insulin-secreting cell line.

J Membr Biol

95

:

63

–

72

58

Atwater

I

,

Rosario

L

,

Rojas

E

1983

Properties of the Ca-activated K+ channel in pancreatic β-cells.

Cell Calcium

4

:

451

–

461

59

Lebrun

P

,

Malaisse

WJ

,

Herchuelz

A

1983

Activation, but not inhibition, by glucose of Ca2+-dependent K+ permeability in the rat pancreatic B-cell.

Biochim Biophys Acta

731

:

145

–

150

60

Bond

CT

,

Maylie

J

,

Adelman

JP

1999

Small-conductance calcium-activated potassium channels.

Ann NY Acad Sci

868

:

370

–

378

61

Frech

GC

,

VanDongen

AM

,

Schuster

G

,

Brown

AM

,

Joho

RH

1989

A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning.

Nature

340

:

642

–

645

62

Taglialatela

M

,

VanDongen

AM

,

Drewe

JA

,

Joho

RH

,

Brown

AM

,

Kirsch

GE

1991

Patterns of internal and external tetraethylammonium block in four homologous K+ channels.

Mol Pharmacol

40

:

299

–

307

63

Ikeda

SR

,

Soler

F

,

Zuhlke

RD

,

Joho

RH

,

Lewis

DL

1992

Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA.

Pflugers Arch

422

:

201

–

203

64

Jonas

JC

,

Gilon

P

,

Henquin

JC

1998

Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic β-cells.

Diabetes

47

:

1266

–

1273

65

Ruppersberg

JP

,

Stocker

M

,

Pongs

O

,

Heinemann

SH

,

Frank

R

,

Koenen

M

1991

Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation.

Nature

352

:

711

–

714

66

Stephens

GJ

,

Owen

DG

,

Robertson

B

1996

Cysteine-modifying reagents alter the gating of the rat cloned potassium channel Kv1.4.

Pflugers Arch

431

:

435

–

442

67

Walaas

SI

,

Greengard

P

1991

Protein phosphorylation and neuronal function.

Pharmacol Rev

43

:

299

–

349

68

Hardy

S

,

Kitamura

M

,

Harris-Stansil

T

,

Dai

Y

,

Phipps

ML

1997

Construction of adenovirus vectors through Cre-lox recombination.

J Virol

71

:

1842

–

1849

69

Salapatek

AM

,

MacDonald

PE

,

Gaisano

HY

,

Wheeler

MB

1999

Mutations to the third cytoplasmic domain of the glucagon-like peptide 1 (GLP-1) receptor can functionally uncouple GLP-1-stimulated insulin secretion in HIT-T15 cells.

Mol Endocrinol

13

:

1305

–

1317

70

Huang

X

,

Wheeler

MB

,

Kang

YH

, et al.

1998

Truncated SNAP-25 (1–197), like botulinum neurotoxin A, can inhibit insulin secretion from HIT-T15 insulinoma cells.

Mol Endocrinol

12

:

1060

–

1070

71

Wheeler

MB

,

Sheu

L

,

Ghai

M

, et al.

1996

Characterization of SNARE protein expression in β cell lines and pancreatic islets.

Endocrinology

137

:

1340

–

1348

Copyright © 2001 by The Endocrine Society

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