Department of Medicine, University of Chicago, Chicago, Illinois
Submitted 8 February 2005 ; accepted in final form 5 May 2005
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ABSTRACT |
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delayed-rectified potassium conductance; hanatoxin; tetraethylammonium; intracellular calcium
Pancreatic islets display characteristic fast spikes and bursts in electrical activity and [Ca2+]i oscillations, which are important in generating pulsatile insulin secretion (11). Periodic depolarization and repolarization of the Vm is critical to the regulation of Ca2+ influx through voltage-dependent Ca2+ channels and oscillatory [Ca2+]i responses of the -cell. Alterations of the balance between depolarizing and repolarizing currents in the
-cell can lead to severe perturbations of insulin secretion (13). Repolarization of the Vm can, in principle, be mediated by several K+-selective ion channel proteins such as KATP, voltage-gated channels (KV), and Ca2+-activated K+ channels (KCa), which include small-conductance and large-conductance channels (12, 23, 28, 31). The identification and characterization of specific K+ channels that regulate this process in normal
-cells remains incomplete.
Voltage-operated K+ channels regulate the Vm of electrically excitable cells (5) and are important components of the -cell plasma membrane. Patch-clamp studies and whole cell microelectrode studies on
-cell outward K+ currents have demonstrated that they are predominantly (80%) of the delayed-rectifier type and are sensitive to external tetraethylammonium (TEA) with Kd values of 15 mM (4, 25, 26, 30). The KV currents in human cells are similar to those in mouse and rat and are all sensitive to the blocker TEA (Kd in the 57 mM range; see Refs. 2 and 16). It has been suggested that the fast spike repolarization of rodent
-cell membrane results from the opening of delayed-rectifier K+ channels (26). TEA blockade of K+ channels has been shown to dramatically increase glucose-dependent electrical activity and insulin secretion in mouse islets (16). Several groups have made an effort to define the KV channel gene or genes encoding this current. Some of the genes expressed in insulin-secreting cell types include members of the KV1.x, KV 2.x, and KV 3.x gene families (3, 19, 22, 24, 35). Dominant-negative mouse transgenes for KV1.x channels strongly suggest that this class of K channels does not play a significant role in regulation of Vm repolarization (23). On the other hand, KV2.1 channels contribute significantly to the
-cell delayed-rectifier current (9, 19, 20, 24). It has also been shown that increased action potential burst frequency and increased insulin secretion result from the block of the KV2.1 channels in mouse
-cells and insulinoma cell lines by pharmacological agents or by coexpression of dominant-negative KV2.1 subunits (19, 20).
Here we have examined the role of KV2.1 channels in glucose-dependent [Ca2+]i oscillations. Western blot analysis indicated that KV2.1 protein is expressed in mouse and human islets. In these islets, we found that hanatoxin (HaTx), the most specific KV2.1 channel inhibitor known, profoundly affects oscillatory [Ca2+]i responses. Although HaTx also blocks KV4 channels, these are not present in -cells (4, 9, 35). HaTx also inhibits the
-cell delayed-rectifier K+ current by inducing a right shift in the current-voltage (I-V) relationship, just as described for its effect on KV2.1 expressed in Xenopus oocytes (30). However, its action on [Ca2+]i is not identical to that of TEA, and here we show using a modeling approach that the different responses are most likely because of the specific nature of the HaTx blockade of KV2.1. These new observations provide evidence that the critical delayed-rectifier channel in mouse and human islet
-cells is comprised of KV2.1 subunits.
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MATERIALS AND METHODS |
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Human islets. Human islets were provided by Dr. B. Herring (University of Minnesota). Human islets were cultured in the same conditions as mouse islets.
Immunoblots. Anti-KV2.1 rabbit polyclonal IgG (raised against amino acids 837853 of rat KV2.1, well conserved among mammalian KV2.1 proteins) was obtained from Upstate Biotechnology (32). For immunoblots, 520 mg membrane protein or whole cell lysates were denatured in reducing sample buffer, fractionated on 9% polyacrylamide-SDS gels adjacent to marker proteins, and transferred to nylon membranes. These were blocked, incubated with affinity-purified rabbit IgG followed by goat anti-rabbit horseradish peroxidase, and detected using an enhanced chemiluminescence technique (Amersham).
Electrophysiology.
Dissociated -cells were prepared by mild trypsin digestion of isolated mouse islets. Bath solution contained (in mM) 135 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, and 10 HEPES, pH 7.3. Pipette solution contained (in mM) 140 KCl, 2 MgCl2, 2 CaCl2, 11 EGTA, 5 MgATP, and 10 HEPES, pH 7.3. In some experiments, the bath solution was supplemented with up to 1 µM HaTx (generously provided by Dr. K. Swartz, National Institutes of Health). For whole cell current recordings, the cells were held at 70 mV, and voltage steps were applied in the interval from 80 to 60 mV in 10 mM increments. Experiments were performed at room temperature using an Axopatch 200B amplifier and DigiData 1500A interface (Axon Instruments). pClamp software (Axon Instruments) was used for both data acquisition and data analysis (modified from Ref. 24).
Measurement of [Ca2+]i.
Cells were loaded with fura 2 for 25 min at 37°C in Krebs-Ringer bicarbonate buffer [containing (in mM): 119 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 25 NaHCO3] supplemented with 5 µM fura 2-AM (Molecular Probes, Eugene, OR). [Ca2+]i was estimated as described elsewhere (22). Dual-wavelength digitized video fluorescent microscopy with fura 2 in islets and -cells was performed using an intensified charge-coupled device (Hammatsu C2400) and Metafluor imaging software (Universal Imaging).
Statistical analysis. Statistical significance was determined by t-test, and the results were reported as averages ± SE.
Mathematical simulation and analysis.
To analyze the experimental results, we used our previously described detailed mathematical model of ionic flux in -cells that includes the critical channels and pumps in the plasma membrane: delayed-rectifying K+ channels, voltage-independent small-conductance KCa channels, KATP channels, and others. This model is coupled to the equations describing cytoplasmic Ca2+, inositol 1,4,5-trisphosphate, ATP, and sodium homeostasis (10). This model is available for direct simulation on the website "Virtual Cell" (http://www.nrcam.uchc.edu) in "MathModel Database" on the "math workspace" in the library "Fridlyand" under the name "Chicago.1."
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RESULTS |
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To evaluate the effect of HaTx on the outward K+ current in mouse -cells, the I-V relationship was determined in the absence and presence of 1 µM HaTx (Fig. 2), a concentration well above the Kd of 42 nM (29). In the control cells, activation of the voltage-dependent outward current is seen at 20 ± 5 mV (n = 10), whereas in cells exposed to HaTx it was +20 ± 5 mV (n = 10). Because no current is left at 20 mV, these results clearly show that essentially all the KV current in mouse
-cells is sensitive to HaTx.
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Mouse islets cultured at 1114 mM glucose demonstrate regular [Ca2+]i oscillations of the slow type (31). HaTx (0.2 µM, Fig. 4A and 1 µM, data not shown) had an effect on the waveform of [Ca2+]i oscillations, in that each peak had a shorter duration (decreased by 39.8 ± 4.2%, P < 0.01 in 0.2 µM HaTx), with fast oscillations at the plateau of each peak (2.03 ± 0.2 oscillations/min in 0.2 µM HaTx). This pattern remained similar after the addition of TEA (20 mM) except for an increase in the amplitude of oscillations by 29.4 ± 2.4% (n = 9) and 32.4 ± 4.2% (n = 4) in experiments with 0.2 and 1 µM HaTx correspondingly (the difference between the two concentrations is not significant, P > 0.05).
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The effects of 1 µM HaTx on glucose-induced elevation of [Ca2+]i in human islets are illustrated on Fig. 5. The addition of HaTx induced an initial decrease in [Ca2+]i similar to one observed in mouse islets (Figs. 3B and 4A). In several independent experiments, among eight islets that responded to 20 mM glucose with an increase of [Ca2+]i, six displayed oscillations after the application of HaTx. The frequency of HaTx-induced oscillations was 0.54 ± 0.11/min. In other experiments using freshly isolated human islets, the response to TEA was similar to that observed with HaTx (data not shown).
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To simulate elevated glucose concentration conditions, we adjusted the rate coefficient for ATP production (Fig. 6, left). In "high glucose conditions," a constant [Ca2+]i level and constant spikes in the Vm could be seen in the model trace. In these conditions, complete block of KV channels results in the expected plasma membrane depolarization and a sustained increase in [Ca2+]i (data not shown).
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To differentiate separate processes and parameters more precisely, we also performed an analysis of model simulation at higher time resolution, where single spikes can be represented (Fig. 7). The oscillatory behaviors of Vm, [Ca2+]i, KV channel gating variable (n), and delayed-rectifying K+ current (IKDr) are shown. The detailed solution from Fig. 6, left (before application of KV "blocker"), is shown in Fig. 7A. However, a similar analysis at decreased KV conductance (as in Fig. 6, right, after KV "block") is hindered by the influence of slow [Ca2+]i oscillations, since spikes in the plasma Vm are absent in the "rest" period (low [Ca2+]i ) and spikes change during the "active" period (high [Ca2+]i; Fig. 6). Our general model (10) indicates the importance of ATP, Na+, and inositol 1,4,5-trisphosphate for generation of slow oscillations, but not for spiking. For this reason, for the analysis only, we fixed intracellular ATP, intracellular Na+, and inositol 1,4,5-trisphosphate concentrations at levels equivalent to their values established in Fig. 6, left, at "high glucose" simulation before simulation of KV channel block. No slow [Ca2+]i oscillations occur in this case; therefore, changes in spike activity introduced by KV inhibition became clearly apparent (Fig. 7B). The corresponding spike frequency and amplitude were also increased in the active period of slow Ca+ oscillations in Fig. 6 in the same manner as in Fig. 7B (data not shown).
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DISCUSSION |
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In addition, we employed an inhibitor of KV2.1, HaTx (29), as a specific pharmacological probe for its role in the glucose-induced Ca2+ responses in -cells within intact normal islets. We first found that HaTx caused a shift in the I-V relation for the
-cell delayed-rectifier, resulting in a 40-mV rightward shift in threshold of activation (Fig. 2). We compared HaTx effects on [Ca2+]i with those of the less-specific K+ channel blocker TEA. The effect of HaTx on [Ca2+]i oscillations was similar to that of TEA at 14 mM glucose. However, at higher glucose only HaTx, but not TEA, was able to stimulate slow [Ca2+]i oscillations.
To analyze these results, we used a detailed mathematical model of ionic flux in -cells based on Fridlyand et al. (10). In our model, the following equation (Eq. 16 from Ref. 10) was used for KV channels: IKDr = g'mKDr x n(Vm VK), where g'mKDr is the maximal whole cell conductance, n is a voltage- and time-depending general gating variable, and VK is K+ equilibrium potential. In this equation, "n" increases with membrane depolarization in a similar fashion to the current in Fig. 2, and the term Vm VK determines the dependence of outward K+ flux through the open channels on Vm.
According to our mathematical model, slow [Ca2+]i oscillations can be created by stimulation of a -cell at an intermediate glucose level (514 mM) by closing KATP channels followed by depolarization of the plasma membrane. These simulated slow [Ca2+]i oscillations (see Fig. 3 from Ref. 10) closely resemble the experimental [Ca2+]i oscillations in the left part of Fig. 4, A and B.
A computer simulation of the effect of high glucose concentration (20 mM) is shown in Fig. 6. In this case, slow [Ca2+]i oscillations are absent, replaced by a constant elevation of [Ca2+]i (Fig. 6 1, left) which, as evident at higher time resolution, is actually a row of continuous fast small spikes (shown in Fig. 7A). This is in agreement with the [Ca2+]i data shown in Fig. 3A. The electrical activity, illustrated in Fig. 62 (before channel block), also consists of continuous spiking (Fig. 7, A-2). A decrease in IKDr conductance (consistent with a block of delayed-rectifier K+ channels) stimulates slow [Ca2+]i oscillations and a burst behavior in Vm (Fig. 6, right). These simulated [Ca2+]i oscillations closely resemble the HaTx-generated oscillation in Fig. 3B.
Spike activity can be generated by the alternate activation of delayed-rectifying K+ channels and voltage-gated Ca2+ channels (2, 6, 26). However, KV channels have a dual function. These channels serve to create the Vm spikes, since their gating variable ("n") has oscillatory properties, and second, they dampen down depolarization at the spike peaks and help to terminate the depolarization. Our modeling of single spikes (Fig. 7) illustrates this mechanism. As can be seen in Fig. 7A, the acceleration of Vm increases n. This in turn increases IKDr (see equation above), which serves to drive membrane repolarization and limits the increase in Vm.
A partial decrease in KV conductance in the model diminishes the dampening effect on spike amplitude and increases both the amplitude and the frequency of spikes (Fig. 7B). This leads to increased amplitude and frequency of outward KV currents (IKDr) during Vm spikes (Fig. 7.4), as well as, accordingly, total IKDr per unit of time (data not shown). The spike frequency and IKDr were also increased in the active period of slow Ca+ oscillations in Fig. 6 (data not shown). This shows that our analysis is also applicable to the simulation of slow Ca+ oscillations.
Our model reproduces the stimulation of Ca2+ oscillations by HaTx (Figs. 3B and 6). The intracellular [Na+] is a dynamic variable that governs slow [Ca2+]i oscillations in our model (10) and is likely to play a role in the stimulation of Ca2+ oscillations by the block of KV channels. A decrease in KV conductance in Fig. 6 leads to increased [Ca2+] flux through voltage-gated Ca2+ channels because the dwell time in the depolarized state increases with increasing amplitude and frequency of Vm spikes (Fig. 7.2, A and B). The increased [Ca2+]i activates the inward Na+ flux through Na+/Ca2+ exchangers (Fig. 6.4). The resulting increase in intracellular [Na+] leads to a slow rise of outward current through electrogenic Na+-K+ pumps. The resulting plasma membrane repolarization promotes a transition to the silent phase. During the silent phase, the declining intracellular [Na+] leads to the decreased outward current through electrogenic Na+-K+ pumps and then in turn to plasma membrane depolarization (Fig. 6, also see Ref. 10). This explains the experimental stimulation of [Ca2+]i oscillations by HaTx (Fig. 3B). We were able to identify the decrease in [Ca2+]i and the subsequent oscillations after addition of HaTx. The brief increase in [Ca2+]i, predicted by the model (Fig. 6), was not seen in the experiments because of the relatively slow rate of image acquisition.
Quite different conditions are created by application of TEA, which blocks KCa and KATP channels in addition to KV. The block of KCa and KATP channels in the model leads to decreased current through these channels and to membrane depolarization. The inability of TEA to stimulate slow [Ca2+]i oscillations (Fig. 3C) can be explained by the opposite effects of various K+ channel block on channel currents.
We also have modeled the effect of KV channel inhibition at intermediate glucose concentrations. In this case, the decrease in KV channel conductance (similar to that in Fig. 6) only leads to a small additional decrease in K+ outward current per unit time, with a corresponding small increase in membrane depolarization (as opposed to membrane repolarization at high glucose level) and in the amplitude of simulated slow [Ca2+]i oscillations (data not shown), which is in accordance with experimental data in Fig. 4, A and B.
Despite the overall similarity in the effects of TEA and HaTx at intermediate glucose concentrations (Fig. 4, A and B), there are also interesting differences. The ability of TEA to induce oscillations is more robust than that of HaTx, suggesting that TEA is capable of additional depolarization of the Vm by inhibiting additional K+ channels compared with HaTx. This suggestion was confirmed by model simulations, where an additional decrease in the conductance of every K+ channel leads to increased amplitude and frequency of slow [Ca2+]i oscillations at intermediate glucose concentrations (data not shown).
This can also help to explain the similarity in TEA action in Figs. 3B and 4B. HaTx action on the -cell at high glucose creates the same oscillatory pattern as the one that occurs at intermediate glucose levels without HaTx (Figs 3B and 4B). Therefore, a subsequent addition of TEA in both cases leads to a similar effect, i.e., existing [Ca2+]i oscillations became more robust.
Modeling was able to closely reproduce the effect of HaTx on [Ca2+]i oscillations in islets by using partial block (1020%) of aggregated KV channels. In electrophysiological experiments performed on single mouse -cells, the delayed-rectifier current at potentials more negative than 0 mV was totally blocked (Fig. 2). Similar HaTx concentrations were employed when monitoring [Ca2+]i.
One way to resolve this disparity is to assume that HaTx did not block all the KV channels in islets, as it did in single dissociated -cells. HaTx is a relatively large protein molecule compared with TEA, and its ability to penetrate the islet may be low, since even glucose diffusion is limited within cultured islets (15). In addition, in a recent report studying HaTx-related toxins, it was suggested that there is an important role of membrane binding and partitioning in the successful encounter of the toxin with the channel (17). It is likely that these processes are less efficient in islets as opposed to single cells.
It is also possible that, since our model (10) was developed for a single cell, it does not take into account the multicellularity of a mouse islet, in particular the electrical coupling by gap-junctions (14). Neighboring cells could contribute to repolarization in the intact islet.
Our results extend recent data (19, 20), which emphasized the possible importance of KV2.1 in mouse islet physiology. A dominant-negative KV2.1 mutant construct delivered to islet cells by adenoviral vector was used to inhibit endogenous KV2.1 assembly (18). These islets were shown to secrete more insulin than nontransfected controls, which indicates that KV2.1 represents some of the repolarizing current in pancreatic -cells. In a more recent study by the same group, a KV 2.1 inhibitor termed C-1 (structure not given) was shown to increase insulin secretion, electrical activity, and [Ca2+]i response in
-cells (20).
Our experimental data and computer simulations confirm these results and show increased electrical activity and [Ca2+]i concentration after KV block at intermediate glucose concentration. In our model, further decrease in KV conductance below the level used for simulation in Fig. 6 leads to termination of the slow [Ca2+]i oscillations and to generation of stable elevated level of [Ca2+]i (several µM, data not shown) due to increased membrane depolarization. Simulation with increased [Ca2+]i is in good agreement with the experimental data from Ref. 20. The results of our simulations show that the effect of KV inhibition depends on the relative activity of channels and the glucose concentration.
Regulation of the electrical response of -cells to glucose is clearly a complex process. Although KATP channels are critical for maintaining the resting Vm, the mechanisms for establishing bursting and spiking behavior and their modulation are less well understood. We found that there is the glucose dependence to the effect of KV channel blockade by both a general and a more specific KV channel inhibitor. Our model simulations have helped to resolve these differences by showing that, although moderate changes in KV modulate oscillatory behavior, a further decrease in K+ conductance leads to extremely high levels of [Ca2+]i. Our model also demonstrates that the effects of different K+ channel block can be offsetting. These results show explicitly how the effect of KV inhibitors on
-cell depends on both their relative specificity and the glucose concentration.
Multiple drugs and molecular genetic approaches have been shown to modulate KV2.1 conductance and assembly (21). These approaches have been extremely useful to further understand the complex interrelationship of ion channels in the regulation of insulin secretion and may lead to further understanding of -cell functional defects in type 2 diabetes and approaches to amelioration of these defects.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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