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Molecular Endocrinology 21 (3): 753-764
Copyright © 2007 by The Endocrine Society

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Pancreatic ß-Cells

Wasim El-Kholy, Patrick E. MacDonald, Jocelyn Manning Fox, Alpana Bhattacharjee, Tian Xue, Xiaodong Gao, Yi Zhang, Juliane Stieber, Ronald A. Li, Robert G. Tsushima and Michael B. Wheeler

Departments of Medicine and Physiology (W.E.-K., J.M.F., A.B., X.G., Y.Z., R.G.T., M.B.W.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Pharmacology (P.E.M.), University of Alberta, Edmonton, Alberta, Canada T6G 2E1; Department of Medicine (T.X., R.A.L.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and Institut fur Pharmakologie und Toxikologie (J.S.), Technischen Universitat Munchen, 80802 Munich, Germany

Address all correspondence and requests for reprints to: Michael B. Wheeler, Department of Physiology, 1 King’s College Circle, University of Toronto, Toronto, Canada M5S 1A8. E-mail: michael.wheeler{at}utoronto.ca; or Robert G. Tsushima, Department of Medicine, 1 King’s College Circle, University of Toronto, Toronto, Canada M5S 1A8. E-mail: r.tsushima{at}utoronto.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels mediate the pacemaker current (Ih or If) observed in electrically rhythmic cardiac and neuronal cells. Here we describe a hyperpolarization-activated time-dependent cationic current, ß-Ih, in pancreatic ß-cells. Transcripts for HCN1–4 were detected by RT-PCR and quantitative PCR in rat islets and MIN6 mouse insulinoma cells. ß-Ih in rat ß-cells and MIN6 cells displayed biophysical and pharmacological properties similar to those of HCN currents in cardiac and neuronal cells. Stimulation of cAMP production with forskolin/3-isobutyl-1-methylxanthine (50 µM) or dibutyryl-cAMP (1 mM) caused a significant rightward shift in the midpoint activation potential of ß-Ih, whereas expression of either specific small interfering (si)RNA against HCN2 (siHCN2b) or a dominant-negative HCN channel (HCN1-AAA) caused a near-complete inhibition of time-dependent ß-Ih. However, expression of siHCN2b in MIN6 cells had no affect on glucose-stimulated insulin secretion under normal or cAMP-stimulated conditions. Blocking ß-Ih in intact rat islets also did not affect membrane potential behavior at basal glucose concentrations. Taken together, our experiments provide the first evidence for functional expression of HCN channels in the pancreatic ß-cell.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN THE PANCREATIC ß-cell, stimulus secretion coupling involves a well characterized series of events (reviewed in Ref. 1). Briefly, entry of glucose via GLUT transporters into the cell is followed by oxidative metabolism in mitochondria, which increases the cytosolic ATP:ADP ratio. This increased ratio inhibits ATP-sensitive potassium (KATP) channels, which are primarily responsible for the hyperpolarizing currents under basal glucose conditions. Inhibition of KATP channels results in membrane depolarization, activation of voltage-dependent L-type calcium channels, and release of insulin. Voltage-dependent potassium (Kv) channels subsequently oppose the voltage-dependent L-type calcium channel current, thereby repolarizing the cell (2). Together, these channels give rise to the bursting action potential phenotype observed in pancreatic ß-cells under high-glucose conditions.

The hyperpolarization-activated cation current (Ih) is responsible for modulating the resting membrane potential and rhythmic electrical behavior of certain cardiac and neuronal cells, and the channel subunits responsible for this current have been cloned over the last decade (3, 4, 5, 6). To date, four mammalian hyperpolarization-activated cyclic-nucleotide-modulated channel isoforms (HCN1–4) have been cloned, each with a distinct pattern of expression throughout the body (7). HCN1 is the most abundant isoform in the brain and is also substantially expressed in the sino-atrial node of the heart (8). HCN2 and HCN4 are both expressed mainly in the central nervous system and heart, whereas HCN3 is present in the central nervous system but is absent in the heart (9). The four HCN subtypes can also combine to form functional hetero-tetrameric channels (10, 11). In these studies, expression of a dominant negative HCN1 or HCN2 channel was sufficient to block function of not only the same isoform, but also other HCN isoforms, indicating that the different channels form functional heteromers. HCN channels are activated at hyperpolarized membrane potentials, providing a depolarizing inward current that accelerates the rebound from these negative membrane potentials and consequently promotes activation of other voltage-gated channels, such as voltage-gated calcium and Kv channels (12, 13). HCN channels have been implicated in a wide range of physiological processes including cardiac function (12), synaptic plasticity (14), memory and motor learning (15, 16), vision (17), and taste perception (18).

The current study demonstrates the presence of HCN channels in pancreatic rat islets and the MIN6 mouse insulinoma cells using RT-PCR and quantitative PCR. We also provide electrophysiological, pharmacological, and genetic support for the expression of HCN channels in ß-cells. Hyperpolarizing steps in MIN6 insulinoma and ß-cells elicited a slow-activating inward current (ß-Ih) with kinetics and voltage sensitivity similar to those of previously characterized HCN isoforms. Several known pharmacological inhibitors of HCN channels blocked this current, and expression of a dominant negative HCN channel or siRNA directed against HCN2 significantly inhibited ß-Ih. However, genetic suppression of these channels using siRNA did not affect insulin release from MIN6 cells. Our findings indicate the presence of an HCN-encoded current in the pancreatic ß-cell that is sensitive to cAMP, but does not directly modulate acute insulin secretion.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HCN Channels Are Expressed in Pancreatic ß-Cells
Using RT-PCR, mRNA transcripts for HCN1–4 were discovered in rat islets, and in the insulin-secreting MIN6 ß-cell line (Fig. 1AGo). Rat brain and heart were used as positive controls, and a reverse transcriptase-lacking sample was used as a negative control. Transcripts were obtained in all cases using primers based on the rat and mouse HCN sequences. Sequencing of MIN6 transcripts indicated that they shared 100% identity with HCN2 and HCN3, and 99% for HCN4. In the case of MIN6 HCN4, there was a 99% identity with the corresponding mouse mBCNG-3 sequence and a 94% identity with the rat HCN4 sequence.


Figure 1
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  		Fig. 1. HCN Channels Are Expressed in the Pancreatic Islets and MIN6 Cells

A, RT-PCR analysis of HCN channel expression in rat islets and MIN6 cells. Reverse transcriptase negative lanes were used as negative control and rat brain and heart (right atria) as positive control for HCN expression. mRNA expression of HCN1–4 was found in rat islets and MIN6 cells using RT-PCR. B, Quantitative PCR measurements of HCN expression in rat brain and heart, MIN6 cells, and rat and mouse islets. Copy numbers were normalized to ß-actin levels (n = 10). KCNJ11 (KATP channel Kir6.2 subunit) message levels were measured also for comparison of relative HCN levels.

 
Quantitative PCR (qPCR) was performed to quantitatively assess the expression levels of HCN mRNAs in MIN6, mouse islets, and rat islets (Fig. 1BGo and Table 1Go). Transcripts for all HCN isoforms were present, with HCN2 being the most predominant isoform in MIN6 and mouse islets. In contrast, the expression profile differed in rat islets where HCN3 and HCN4 were the more abundant isoforms. In addition, the HCN family of channels is functionally expressed in brain and heart, and we also report our quantification of these levels compared with that in islet and MIN6 cells. As can be observed from Fig. 1BGo, the expression levels of HCN channel isoforms are less in islet and MIN6 cells in comparison with brain and heart. To better assess the significance of the expression levels observed in islet cells, we examined the expression level of the well-characterized ATP-sensitive potassium channel Kir6.2 subunit (KCNJ11) as a comparison. Surprisingly, HCN channel isoform levels are higher than Kir6.2 expression levels in mouse islets and MIN6 cells, but much less in rat islets. The large difference in normalized expression levels of KCNJ11 between mouse and rat islets is due to a combination of higher KNCJ11 copy number and lower ß-actin copy number per nanogram of cDNA used in rat vs. mouse samples (Table 1Go).


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  		Table 1. qPCR Analysis of HCN Channel Isoform, KCNJ11 and ß-Actin Copy Numbers in MIN6, Mouse Islet, Rat Islet, Rat Brain, and Rat Heart (n = 4)

 
Electrophysiological Characteristics of ß-Ih
We next attempted to measure HCN currents in isolated pancreatic ß-cells and MIN6. A hyperpolarization-activated cationic current in ß-cells, ß-Ih, was measured in both rat ß-cells and MIN6 cells using the whole-cell patch-clamp method at physiological (35–37 C) temperatures. The pulse protocol and representative current traces are displayed in Fig. 2AGo. Under our recording conditions the high (5 mM) ATP levels present in the intracellular solution inhibited KATP currents. In MIN6 and rat ß-cells, sequential hyperpolarizing steps from a holding potential of –40 mV elicited time-dependent inward currents that increased in amplitude with progressive hyperpolarization. Time-dependent ß-Ih current density at –130 mV with 30 mM external potassium was –36.4 ± 6.1 picoamperes (pA)/picofarads (pF) (n = 12) and –37.4 ± 4.6 pA/pF (n = 10) for MIN6 and rat ß-cells, respectively. Using a physiological external potassium concentration (5 mM) ß-Ih current density was –8.2 ± 1.4 pA/pF (n = 13) in MIN6 cells at –130mV (Fig. 2BGo). The steady-state activation midpoint (V50) and Boltzmann constant of ß-Ih in MIN6 cells at 37 C was –87.1 ± 1.7 mV and –6.1 ± 0.9 (n = 10), respectively. In rat ß-cells, the steady-state activation midpoint (V50) and Boltzmann constant of ß-Ih was –84.3 ± 3.9 mV and –8.2 ± 1.0 (n = 10), respectively. Activation kinetics of ß-Ih displayed a sigmoidal distribution typical of HCN-encoded channels when fitted to a single exponential function. The activation time constants of ß-Ih in MIN6 cells ranged from 244 ± 14 msec (n = 39) to 1.6 ± 0.3 sec (n = 15) at –140 mV and –90 mV, respectively. In rat ß-cells, the activation time constants were 251 ± 27 msec (n = 17) to 3.4 ± 0.8 sec (n = 8) at –140 and –90 mV, respectively (Fig. 2CGo).


Figure 2
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  		Fig. 2. Biophysical Properties of ß-Ih

A, Pulse protocol used (top) and current traces of ß-Ih in MIN6 and rat ß-cells measured at 32–35 C (bottom). Dashed line indicates zero current. B, Current-voltage relationship of ß-Ih in MIN6 cells and dispersed rat ß-cells with 30 mM extracellular KCl, and with 5 mM external KCl in MIN6 cells. Data points represent the mean ± SEM from n ≥ 10 recordings per condition. C, Activation kinetics of ß-Ih in both MIN6 cells and rat ß-cells from –140 mV to –90 mV fit to a monoexponential function (n = 8–39).

 
cAMP Modulation of ß-Ih
A hallmark property of HCN channels is their direct sensitivity to cAMP (19). This second messenger causes a rightward shift in the voltage dependence of activation of different HCN isoforms to varying degrees, with HCN4>HCN2>>HCN1 in sensitivity (7). Figure 3AGo shows representative current traces for the effect of cAMP on ß-Ih at –70 mV. The V50 of ß-Ih in MIN6 cells was significantly shifted in the depolarizing direction upon addition of 50 µM forskolin/3-isobutyl-1-methylxanthine (IBMX) for 10 min to increase intracellular cAMP levels (–88.0 ± 2.2 mV control vs. –74.0 ± 3.7 mV forskolin/IBMX; n = 7–8, P < 0.01; Fig. 3BGo). Consistent with these results, the cell-permeable cAMP analog dibutyryl-cAMP (1 mM) also caused a rightward shift of ß-Ih V50 in MIN6 cells (–87.2 ± 1.7 mV control vs. –79.6 ± 1.4 mV dibutyryl-cAMP (db-cAMP), n = 9–10, P < 0.05; Fig. 3CGo). These studies were performed in different populations of control and treated cells due to the presence of a rapid hyperpolarizing shift in the activation curve observed in both whole-cell and perforated-patch recordings (data not shown). This hyperpolarizing shift confounded measuring the effects of cAMP on the current and has been reported previously (20).


Figure 3
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  		Fig. 3. cAMP Causes a Rightward Shift in the Voltage Sensitivity of ß-Ih from MIN6 Cells

A, Representative current traces from control conditions and cells treated with 50 µM forskolin/IBMX or 1 mM db-cAMP at –70 mV. Line denotes zero current. B, Voltage dependence of activation relationship of ß-Ih under control conditions V50 = –88 ± 2.2 mV and in the presence of 50 µM forskolin/IBMX V50 = –74 ± 3.7 mV (P < 0.01) or C) under control conditions V50 = –87.2 ± 1.7 mV and in the presence of 1 mM db-cAMP V50 = –79.6 ± 1.4 mV mV (P < 0.05) (n = 7–10). DMSO, Dimethylsulfoxide; Forsk, forskolin.

 
This evidence for a hyperpolarization-activated, cAMP-sensitive inward current, which shares the biophysical properties of Ih previously described in cardiac and neuronal tissues, provides further support for the functional expression of HCN channels in ß-cells. ß-Ih, therefore, most closely shares the biophysical properties of the HCN2 isoform, with respect to activation kinetics and magnitude of response to cAMP.

ß-Ih Is Blocked by Known Inhibitors of HCN Channels
Cesium, ZD7288, cilobradine (DK-AH 269), and zatebradine (UL-FS49) are established inhibitors of HCN channels (6, 21, 22). Cesium (5 mM) and ZD 7288 (50 µM) blocked time-dependent ß-Ih in rat ß-cells at –130 mV by 78.0 ± 3.6% (n = 7) and 51.9 ± 5.9% (n = 10), respectively (P < 0.01) (Fig. 4Go). Cesium (2 mM), ZD 7288 (50 µM), cilobradine (5 µM), and zatebradine (5 µM) blocked time-dependent ß-Ih in MIN6 cells at –130 mV by 82.2 ± 5.4% (n = 10), 57.3 ± 7.9% (n = 6), 84.6 ± 7.8% (n = 4), and 72.4 ± 14.8% (n = 4), respectively (P < 0.01 for all compounds vs. control) (Fig. 4BGo). The concentrations used in this study were similar to those used in previous studies of Ih, and these findings provide pharmacological evidence that ß-Ih is HCN encoded.


Figure 4
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  		Fig. 4. ß-Ih Currents in Rat ß-cells and MIN6 Cells Are Blocked by Cesium, ZD7288, Cilobradine, and Zatebradine

A, Current traces of ß-Ih at –130 mV under control conditions and in the presence of several HCN inhibitors. B, Percent inhibition of ß-Ih in MIN6 cells and rat ß-cells using these four HCN antagonists (P < 0.01 vs. same cell control for all compounds, n = 4–10). Cilo, Cilobradine; Zate, zatebradine; ms, millisecond.

 
Effects of ZD7288 and Cilobradine on Insulin Secretion
To determine whether HCN channels play a role in functionally regulating the pancreatic ß-Ih cell, glucose-stimulated insulin secretion (GSIS) assays were performed on MIN6 and rat ß-cells using ZD7288 and cilobradine. Figure 5AGo shows that 50 µM ZD7288 inhibited GSIS from MIN6 cells by 59.3 ± 5.1% (n = 3; P < 0.01). Figure 5BGo shows that 50 µM ZD7288 also reduced GSIS from rat islets by 34.9 ± 8.3% (n = 4; P < 0.05), indicating this effect was not restricted to insulinoma cells. In contrast to the striking effects of ZD2788 on insulin secretion, 5 µM cilobradine did not produce a significant alteration in GSIS when used to treat isolated rat islets (Fig. 5CGo). Because both compounds blocked ß-Ih to a similar extent at the concentrations used in these GSIS studies, the lack of agreement in these results suggests that one, or both, compounds behave in a nonselective manner.


Figure 5
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  		Fig. 5. Pharmacological HCN Inhibitors Have Divergent Effects on Insulin Secretion from MIN6 Cells and Rat Islets

ZD7288 (50 µM) significantly inhibits glucose-stimulated insulin secretion from A) MIN6 cells (n = 3) and B) rat islets (n = 4). C, Cilobradine (5 µM) has no effect on basal or glucose-stimulated insulin secretion from rat islets (n = 4). LG, Low glucose (0 mM glucose); HG, high glucose (11.1 mM glucose); NS, not significantly different.

 
ZD7288 Directly Inhibits Depolarization-Induced Exocytosis from Pancreatic ß-Cells
It has been previously demonstrated that ZD7288 blocks exocytosis independent of its effects on HCN channels (23). More recently, ZD7288 has been shown to inhibit prolactin release from pituitary lactotrophs downstream of calcium influx and independently of HCN function (24). Direct recordings of cell capacitance have been used to determine the amount of exocytotic activity occurring in pancreatic ß-cells (25). These recordings are commonly performed using a train of depolarizing pulses while measuring the concomitant effect on whole-cell capacitance (Fig. 6AGo). Treatment of primary rat ß-cells with 50 µM ZD7288 caused a significant reduction in capacitive increases vs. control cells, suggesting that ZD7288 exerts an inhibitory effect at a distal step of the ß-cell stimulus-secretion process (n = 7; P < 0.05 for first depolarization; P < 0.01 for second depolarization) (Fig. 6BGo). These results are not likely to have occurred via an effect on HCN channels because the exocytotic events proceed downstream of HCN channel activity. Furthermore, the same experiment using 5 µM cilobradine did not significantly alter the exocytotic response compared with control cells [total change = 505.22 ± 133.57 femtofarads (fF) control (n = 5) vs. 442.72 ± 100.89 fF cilobradine (n = 9)]. These results are the first to demonstrate that ZD7288 directly blocks exocytosis, in addition to inhibiting HCN channels, likely via a direct interaction with the exocytotic machinery.


Figure 6
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  		Fig. 6. Nonselective Pharmacological Properties of HCN Inhibitors

A, Pulse protocol used (above) and changes in single-cell capacitance (below) from single rat ß-cells in the absence or presence of 50 µM ZD7288. ZD7288 blocks exocytosis directly. B, Increases in cell capacitance per pulse in control ß-cells and ß-cells exposed to 50 µM ZD7288. ZD7288 significantly reduced the capacitive increases due to the first two depolarizing step pulses (n = 7). C, Current traces of MIN6 Kv control currents (above) and in the presence of 50 µM cilobradine (below). D, Current-voltage relationship from MIN6 cells in the absence and presence of cilobradine. Cilobradine significantly inhibits sustained outward Kv currents between –10 and +70 mV (*, P < 0.05; **, P < 0.01; n = 4).

 
Cilobradine Blocks Kv Currents from MIN6 Insulinoma Cells
Voltage-gated potassium (Kv) channels are important regulators of ß-cell function (2). Inhibition of Kv2.1 causes a glucose-dependent enhancement of insulin secretion (26). Previous reports have shown that cilobradine and zatebradine inhibit potassium conductances at concentrations of 100 µM and 10 µM, respectively (27, 28). Given the apparent lack of selectivity of these agents on HCN channel inhibition, we tested whether cilobradine could also block ß-cell Kv currents.

At 5 µM, cilobradine significantly inhibited sustained Kv current density at +70 mV from 258 ± 38 pA/pF to 138± 13 pA/pF (n = 5; P < 0.05), whereas peak currents were slightly reduced from 300 ± 51 pA/pF to 204 ± 22 pA/pF (n = 5; P = 0.12). Figure 6Go, B and D, show that at 50 µM cilobradine dramatically reduced the sustained Kv current density from 225 ± 44 pA/pF to 76 ± 12 pA/pF (n = 4; P < 0.05).

There was no difference in Kv channel function at +70 mV from MIN6 cells under control conditions (current density = 273 ± 11 pA/pF) or after treatment with up to 100 µM ZD7288 (current density = 264 ± 15 pA/pF), indicating that ZD7288 does not appear to affect Kv channel function (n = 9).

Our findings suggest that the HCN channel blocker cilobradine is also a potent Kv channel inhibitor, making it an inappropriate compound for studying insulin secretion due to the important role Kv currents play in regulating insulin secretion (2). We initially hypothesized that inhibition of a depolarizing HCN current by cilobradine would have attenuated insulin release by potentially blocking the transition from the resting membrane potential at basal glucose levels to the bursting pattern typically observed at high glucose levels. However, the concurrent block of Kv channels by this compound, which we have previously shown would augment insulin secretion, makes it difficult to interpret cilobradine’s effect on insulin release (29).

ß-Ih Is Inhibited by Genetic Suppression of HCN Channels
We have demonstrated previously that the HCN1-AAA mutant channel, the permeation determinants of which in the channel pore, GYG349–352, have been substituted by alanines, specifically inhibits HCN1- and HCN2-encoded channels in a dominant-negative manner by coassembling with functional HCN subunits, thereby rendering the channel complex nonconducting (10). In fact, a similarly designed construct, HCN2-GYG402–404->AYA, has also been shown to perform similar functions by suppressing native cardiac If in rat neonatal ventricular cardiomyocytes as well as the associated spontaneous electrical activity (11). To further verify the molecular identity of ß-Ih, we studied the effect of HCN1-AAA on ß-Ih in MIN6 cells. Figure 7BGo illustrates that HCN1-AAA overexpression significantly reduced ß-Ih (P < 0.01), providing additional support that this pancreatic current is HCN encoded. HCN1-AAA did not affect outward Kv currents in MIN6 cells (data not shown).


Figure 7
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  		Fig. 7. Effect of Genetic Knockdown of HCN Channels on ß-Ih

A, ß-Ih current traces in MIN6 cells transfected with green fluorescent protein (GFP) (control; upper traces) or the dominant-negative HCN1-AAA construct (lower traces). B, Current-voltage relationship for ß-Ih in MIN6 cells under control conditions (solid squares, n = 15) or overexpressing HCN1-AAA (open circles, n = 8). HCN1-AAA significantly reduced ß-Ih between –70 and –130 mV (P < 0.01). C, Current trace of ß-Ih from MIN6 cells transfected with a scrambled siRNA (upper traces) or siRNA directed against HCN2 (siHCN2b; lower traces). D, Current-voltage relationship of siRNA-mediated knockdown of time-dependent ß-Ih in MIN6 cells (n = 5–12; **, P < 0.01 vs. scrambled by ANOVA). ms, Millisecond.

 
Based on our PCR data and the biophysical similarities between ß-Ih and recombinant HCN2 with respect to activation kinetics and cAMP sensitivity, this isoform appeared to be the most consistently expressed HCN isoform in the ß-cell (30). Using small interfering (si)RNA against HCN2 (siHCN2a-c) we examined whether inhibition of this isoform could induce a significant inhibition of ß-Ih. Figure 7DGo shows that transfection of three different small interfering (si)HCN2 constructs caused a significant inhibition of ß-Ih from MIN6 cells 48–72 h posttransfection. At –130 mV, ß-Ih from scrambled siRNA-transfected control cells was –34 ± 5 pA/pF (n = 10), whereas from siHCN2a-, siHCN2b-, and siHCN2c-transfected cells it was –7 ± 2 pA/pF (n = 11), –4 ± 2 pA/pF (n = 5), and –8 ± 3 pA/pF (n = 5), respectively (all P < 0.01 vs. scrambled by ANOVA). Additional measurements indicated that these siRNAs did not affect Kv currents. These results demonstrate that HCN2 is a key isoform in MIN6 cells responsible for generating ß-Ih.

Genetic Suppression of ß-Ih Does Not Affect Insulin Secretion from MIN6 Cells
To assess the effect of ß-Ih on glucose-stimulated insulin secretion, MIN6 cells were transfected with scrambled siRNA or a sequence directed against HCN2 (siHCN2b) shown to be highly effective at inhibiting ß-Ih (see Fig. 7Go). Figure 8Go shows that glucose stimulation in the presence of vehicle increased insulin secretion from 1.89 ± 0.13 to 4.20 ± 0.29 ng/µg DNA/30 min in scrambled siRNA cells (P < 0.01), and from 1.99 ± 0.21 to 3.92 ± 0.30 ng/µg DNA/30 min (P < 0.01). In the presence of 25 µM forskolin/IBMX to increase cAMP levels, secretion in scrambled cells increased from 2.55 ± 0.32 to 6.36 ± 0.37 ng/µg DNA/30 min in siHCN2b cells (P < 0.001), and from 2.28 ± 0.30 to 5.85 ± 0.34 ng/µg DNA/30 min in siHCN2b cells (P < 0.001). There was no significant difference between the scrambled siRNA and corresponding siHCN2b groups under any treatment condition, suggesting that HCN channels in ß-cells do not participate in the acute stimulus-secretion coupling pathway under normal or cAMP-enhanced conditions. HCN2 mRNA levels were reduced by 49.7 ± 10.8% in siHCN2b- vs. scrambled-transfected cells as measured by qPCR (P < 0.05; n = 3), but had no significant effect on the mRNA levels of the other HCN isoforms (data not shown).


Figure 8
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  		Fig. 8. Transfection of MIN6 Cells with siRNA Does Not Affect GSIS

Transfection of MIN6 cells with siHCN2b that blocks approximately 90% of ß-Ih does not affect basal or cAMP-augmented glucose-stimulated insulin secretion. Cells were incubated with either vehicle [dimethylsulfoxide (DMSO)] or 25 µM forskolin/IBMX (F + I) in 0 mM (LG) and then 10 mM (HG) glucose for 30 min each, 72 h after transfection with either scrambled or siHCN2b siRNA (**, P < 0.01; ***, P < 0.001; n = 4 for each group).

 
Inhibiting ß-Ih with ZD7288 or Cilobradine Does Not Affect Membrane Potential in Rat Islets
Because we could not determine an effect of genetic inhibition of ß-Ih on insulin release from ß-cells at low or high glucose conditions, we examined whether ZD7288 or cilobradine could affect the membrane potential behavior of ß-cells from within intact rat islets at basal glucose levels, where the membrane potential is at its most negative in a ß-cell and therefore most likely to be affected by HCN activity. ß-Cells were identified by their bursting pattern at high (11.1 mM) glucose levels, followed by a transition to a hyperpolarized resting membrane potential upon exposure to low (2.8 mM) glucose (Fig. 9Go). Treatment with ZD7288 (Vm = –62.5 ± 0.5 mV control vs. –62.5 ± 2.5 mV; n = 2) or cilobradine (Vm = –53.0 ± 9.4 mV control vs. –57.4 ± 7.7 mV; n = 6) in this state did not cause a hyperpolarization, which would be expected if a significant inward current from HCN channels were contributing to the resting membrane potential at low glucose levels. These results suggest that HCN channels do not play a role in setting ß-cell membrane potential at basal glucose levels.


Figure 9
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  		Fig. 9. Membrane Potential of ß-Cells from Intact Rat Islets Is Not Affected by HCN Inhibitors

A, Control cell showing oscillations and bursting at high (11.1 mM) glucose, followed by a repolarization to basal membrane potentials upon switching the external solution to low (2.8 mM) glucose. Membrane potential was not affected in ß-cells treated with B) ZD7288 (50 µM) or C) cilobradine (5 µM) at basal glucose levels. s, Seconds; ZD, ZD7288.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we have demonstrated the expression and function of HCN channels in pancreatic ß-cells. Previous efforts aimed at detecting HCN channel expression in whole pancreatic extracts by Northern blot analysis have been unsuccessful, possibly because these studies did not use isolated endocrine tissue, in which the channels appear to be specifically located but which constitute only a small fraction of total pancreatic mass (30). RT-PCR analyses showed expression of HCN1–HCN4 in rat islets and MIN6 cells. Quantitative PCR demonstrated that HCN1–HCN4 channels were expressed in varying abundance in MIN6, mouse islets, and rat islets, with HCN2 gene expression being predominant in mouse cells and HCN3 and HCN4 being the most abundant in rat islets. We demonstrate that the expression level of HCN genes in islet cells is comparable to that in rat brain and heart, where they are known to be functionally important (7). In addition, in MIN6 cells and mouse islets, the gene expression level of each HCN isoform is greater than that of KCNJ11, the Kir6.2 subunit of KATP channels that is known to be functionally important for insulin secretion from these cells (31, 32).

Electrophysiological measurements identified a current we refer to as ß-Ih, which has similar biophysical properties to HCN channels observed in neuronal and cardiac cells (13). In both rat islet ß-cells and MIN6 cells, we consistently measured inward currents activated by hyperpolarizing step pulses between –60 and –130 mV. The currents displayed a voltage-dependent acceleration of current activation at more negative potentials, as has been shown previously for HCN currents (5, 6). A significant depolarizing shift in the midpoint of voltage-dependent activation, from –88 mV to –74 mV, was observed after elevating intracellular cAMP with forskolin and IBMX, whereas a shift of approximately 8 mV was measured using the cell-permeable cAMP analog db-cAMP. Because sensitivity to cAMP is one of the key properties of HCN channels, these results strongly support the notion that ß-Ih is HCN encoded in pancreatic ß-cells. Furthermore, because cAMP is a key factor in ß-cell glucose responsiveness, it is possible that ß-Ih plays a role in this cAMP-related enhancement pathway (33).

Cesium, ZD7288, cilobradine, and zatebradine, which are all established HCN blockers, significantly inhibited ß-Ih in rat ß-cells and MIN6 cells in the concentration ranges commonly used in past studies (21, 22). However, we found that these compounds could not be used for insulin secretion experiments due to their nonselective effects, in particular, their direct inhibition of exocytosis (ZD7288) and Kv currents (cilobradine, zatebradine, and cesium) (23, 24, 34). Cesium cannot be employed in insulin secretion studies because it also has nonselective effects on ß-cell potassium currents (34). It has been demonstrated that inhibiting Kv currents in the ß-cell enhances glucose-stimulated insulin secretion (29). Therefore, our results are difficult to interpret because cilobradine, which blocked both ß-Ih and Kv currents, had no effect on insulin secretion. Given the fact that siRNA against HCN2 caused a dramatic reduction in ß-Ih, but did not affect insulin release, it appears that inhibiting ß-Ih selectively does not affect insulin secretion.

A genetic knockdown strategy was employed using a dominant-negative HCN1 channel (HCN1-AAA) and siRNAs designed against the HCN2 isoform to selectively inhibit ß-Ih. The choice of HCN2 was based on 1) our findings that HCN2 mRNA is consistently and highly expressed in MIN6 cells as assessed by RT-PCR and qPCR, and 2) biophysical data indicating that ß-Ih shares the cAMP sensitivity of both HCN2 and HCN4, but that its activation kinetics are more in common with HCN1 and HCN2 as reported previously (30). Both approaches provided more selective inhibition of ß-Ih compared with pharmacological methods, which we found to be quite nonselective. Electrophysiological measurements indicated that both the HCN1-AAA channel and siRNA constructs were capable of potently inhibiting ß-Ih 48 h post transfection. Transfection efficiency of the HCN1-AAA construct did not exceed 30% in MIN6 cells, making them inappropriate for use in a GSIS assay. Because the transfection efficiency of siRNAs exceeded 75% in MIN6 cells, as determined by fluorescein (FAM) labeling, we used them to examine the role of HCN channels on regulation of insulin secretion. After 72 h, the same length of time needed to see significant inhibition of ß-Ih in our electrophysiological measurements, we found no change in secretion between the scrambled negative control and siHCN2b groups. Interestingly, under conditions of elevated cAMP levels using the adenylate cyclase activator forskolin, in conjunction with the phosphodiesterase inhibitor IBMX, there was still no effect of HCN knockdown on ß-cell secretion. This was surprising considering our results showing that HCN currents are not active under baseline conditions in ß-cells at physiological membrane potentials, but that they are active when cAMP levels are enhanced, providing a depolarizing current which could potentially increase ß-cell electrical excitability (see Fig. 3Go). These results indicate that ß-Ih is not required for acute insulin secretion from MIN6 cells. Due to the low transfection efficiency achievable in primary tissues, this experiment with siRNAs could not be replicated in isolated rat islets.

After this, we measured the membrane potential response to HCN inhibition on rat ß-cells from intact islets. We hypothesized that if ß-Ih were active in some capacity, it would be only at the most hyperpolarized membrane potentials, i.e. at basal glucose levels. Inhibition of this depolarizing HCN current would be expected to cause a measurable hyperpolarization of the membrane potential. Using either ZD7288 or cilobradine failed to yield such a response, suggesting that ß-Ih is not active under basal glucose conditions. It is therefore difficult to predict, based on our observations, what role this current has in the ß-cell.

The ß-cell translates a complex metabolic cascade induced by glucose entry into electrical activity that triggers insulin release (reviewed in Ref. 35). We initially hypothesized that HCN channels play different roles in ß-cell function, depending on the level of blood glucose. Briefly, under basal glucose conditions, when KATP channels are open and are responsible for setting the membrane potential, inward HCN currents will counteract the outward KATP currents, thereby contributing to the resting membrane potential of ß-cells. In support of this it has been reported that, under basal glucose conditions, the ß-cell membrane potential (~ –60 to –70 mV) is significantly more positive than the equilibrium potential for potassium, which would be the predicted resting membrane potential of the ß-cell if the KATP current was the sole ionic conductance present. Upon glucose stimulation, the ß-cell membrane potential depolarizes to the activation threshold for voltage-gated L-type Ca2+ channels, thereby causing calcium entry and insulin secretion. At these stimulated membrane potentials, however, it is unlikely that ß-Ih plays a significant role because the cell is too depolarized for it to be active based on our results.

The present study provides clear evidence for HCN channel expression in pancreatic ß-cells and therefore extends our knowledge of the electrophysiological regulation of the pancreatic ß-cell. Despite consistently measuring ß-Ih in MIN6 and rat ß-cells, we were unable to demonstrate an effect of this current and HCN channels in the regulation of acute insulin secretion or membrane potential behavior at basal glucose levels. Previous studies have shown that the ß-cell contains other currents including sodium, chloride and zinc, the functions of which are also unclear with regard to their physiological role in insulin release (36, 37, 38, 39). ß-Ih can now be added to the list of these currents. It now remains to be seen what long-term effects HCN channel inhibition may have on ß-cell function. Recent reports have shown that KATP channel activation using diazoxide or other selective openers induces ß-cell rest and protection against the effects of high-fat diet (40, 41, 42). Chronic studies where HCN channel function or expression is manipulated are required to determine the potential roles of these channels on ß-cell function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
RT-PCR and Quantitative PCR
Total RNA (5–20 µg) was isolated using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. A subsequent DNase I treatment was performed to remove any residual DNA contamination (QIAGEN, Chatsworth, CA). Isolated RNA (1 µg) was reverse transcribed and amplified using a One-Step RT-PCR kit (Invitrogen). Intron spanning primers for rat HCN1–4 isoforms were designed using Primerquest (Integrated DNA Technologies, Inc., Coralville, IA) (Table 2Go). The amplification protocol consisted of a pre-PCR cycle of 52–55 C for 30 min, followed by a 95 C step for 15 min. The PCR cycle was a 94 C step for 40 sec, a 53–59 C gradient segment for 30 sec, and a 72 C step for 40 sec, repeated for 30 cycles. The PCR was terminated with a 10-min segment at 72 C. Sequences were cloned and determined as described previously (29).


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  		Table 2. RT-PCR Primers for HCN Channel Isoforms

 
qPCR was performed as described previously (43). Briefly, total RNA was isolated and used to prepare cDNA as described above. Intraexon oligonucleotide primers were designed using Primerquest (Table 3Go). A master mix was aliquoted to a 384-well plates (Applied Biosystems, Foster City, CA) with each well containing: 2.85 µl of water, 1 µl of 10x PCR buffer, 0.2 µl of primer mix (or 0.1 µl of forward, 0.1 µl of reverse), 1.2 µl of 25 mM MgCl2, 0.2 µl of deoxynucleotide triphosphate mix (10 mM each), 0.025 µl of Platinum Taq polymerase (Invitrogen), 0.3 µl of SYBR green stock (Molecular Probes, Eugene, OR). The real-time PCR protocol employed was as follows: heat activation of polymerase at 95 C for 3 min, followed by 40 cycles of: 95 C for 10 sec, 65 C for 15 sec and 72 C for 20 sec. Readings were carried out on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) and compared against a standard curve created from genomic DNA.


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  		Table 3. qPCR Primers for Mouse and Rat HCN Channel Isoforms, Kir6.2 (KCNJ11) and ß-Actin

 
Electrophysiological Recordings
Rat islets were isolated as described previously (29), and ß-cells were identified as being more than 4 pF in size in a dispersed preparation of islet cells according to the criteria of Gopel et al. (44). Animal procedures were performed in accordance with the University of Toronto’s Animal Care Committee’s ethical guidelines. HCN currents were measured in the voltage-clamped whole-cell configuration using an EPC-9 amplifier and PULSE software from HEKA Electronik (Lambrecht, Germany). Patch pipettes were prepared from 1.5-mm thin-walled borosilicate glass tubes using a two-stage Narishige (Tokyo, Japan) micropipette puller and had typical resistances of 4–8 M{Omega} when heat polished and filled with intracellular solution. Intracellular solutions contained (in mM concentration): 130 KCl; 10 NaCl; 0.5 MgCl2; 1 EGTA; 5 HEPES; 5 MgATP (pH 7.3) with KOH. Extracellular solutions contained (in mM concentration): 110 NaCl; 30 KCl; 0.5 MgCl2; 1.8 CaCl2; 5 HEPES; 10 glucose (pH 7.4) with NaOH. For current-voltage relationships, cells were held at –40 mV, and inward currents were elicited by a 500-msec depolarizing pulse to 0 mV followed by 3-sec hyperpolarizing pulses from –130 mV to –40 mV, in 10-mV increments. Sustained inward current was taken as the average current over the final 150 msec of the 3-sec pulse. For steady-state activation, the pulse protocol was identical, except that a 500-msec pulse followed each hyperpolarizing sweep to –130 mV, and tail current measurements were taken as the average current over the first 10 msec of the hyperpolarizing pulses to –130 mV. The voltage ramp protocol consisted of a 1-sec hyperpolarizing pulse to –130 mV followed by a ramp from –130 mV to –30 mV in 1 sec. The resulting values were fitted using the Boltzmann equation y = [(A1 + A2)/(1 + exp(V – V1/2/k)] – A2, where A1 is the amplitude, A2 is the offset, V is the membrane potential, V1/2 is the activation midpoint voltage, and k is the Boltzmann constant. Islet membrane potential recording was performed as described previously (45).

Pharmacological compounds were applied via perfusion to the bath solution for at least 2 min before a final current recording was taken. All HCN channel recordings were performed at 35–37 C and normalized to cell capacitance, unless otherwise indicated. ZD7288 was obtained from Tocris (Ellisville, MO), and forskolin and IBMX were purchased from Sigma-Aldrich. Cilobradine and zatebradine were supplied by Dr. Juliane Stieber.

For capacitance measurements, the pipette solution contained (in mM concentration): 125 Cs-glutamate; 10 CsCl; 10 NaCl; 1 MgCl; 0.05 EGTA; 3 MgATP; 5 HEPES (pH 7.1 using CsOH). The extracellular solution consisted of (in mM concentration): 118 NaCl; 20 tetraethylammonium chloride; 5.6 KCl; 1.2 MgCl2; 2.6 CaCl2; 5 D-glucose; 5 HEPES (pH 7.4 with NaOH). The resistance of heat-polished patch pipettes was between 2 and 4 M{Omega}. Cells were held at –70 mV in the whole-cell configuration, and exocytosis was elicited with a train of ten 500-msec membrane depolarizations to 0 mV (1 Hz stimulation frequency). Capacitance was measured using the Sine+DC lock-in feature of an EPC-9 patch-clamp amplifier with Pulse software (HEKA Electronik).

Insulin Secretion Studies
For MIN6 secretion studies, cells were cultured in DMEM supplemented with 25 mM glucose, 10% fetal bovine serum, 0.004% ß-mercaptoethanol, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (penicillin-streptomycin; Invitrogen), and plated in 12-well plates at 5 x 105 cells per well. After culture for 48 h at 37 C and 5% CO2, the cells were washed with, and preincubated in Krebs-Ringer bicarbonate-HEPES (KRBH) buffer containing (in mM) 128.8 NaCl; 4.8 KCl; 1.2 KH2PO4; 1.2 MgSO4; 2.5 CaCl2; 5 NaHCO3; 10 HEPES; and 1% BSA at pH 7.4 without glucose for 30 min. After this, cells were washed with KRBH buffer with 0 mM glucose with or without drug for 60 min, and then with 11.1 mM glucose with or without drug for 60 min. All secretion studies were performed at 37 C and 5% CO2, after which media samples were collected and centrifuged at 700 x g to remove any residual cells. For primary tissue secretion studies, islets were isolated and maintained in the RPMI media containing 11.1 mM glucose overnight, followed by a 1-h incubation in KRBH buffer with either 2.8 mM or 11.1 mM glucose, in the presence or absence of drug treatments.

RIAs were performed using a rat insulin RIA Kit (Linco Research, St. Charles, MO). Experiments were performed in triplicate in at least three separate experiments and expressed as ng/µg DNA–1 · 30 min–1.

HCN1-AAA and siRNA Design and Transfection
Rabbit HCN1 was subcloned into the pCI expression vector (kind gift of Drs. Ishii and Ohmori, Kyoto University) as previously described (46). Site-directed mutagenesis was performed using PCR with overlapping mutagenic primers as previously described (47). Sequencing was performed to ensure that the desired mutations were present. HCN channel constructs were transfected into MIN6 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Plasmid DNA encoding the appropriate channel (1 µg/60-mm dish) was added to the cells with Lipofectamine 2000, followed by incubation at 37 C in a humidified atmosphere of 95% O2-5% CO2 for 48–72 h before electrical recordings.

siRNAs against mouse HCN2 were purchased from Ambion (Austin, TX) (Table 4Go). For negative control, Ambion’s scrambled siRNA sequence (catalog no. 4624) or FAM-labeled scrambled sequence (catalog no. 4620) were employed. Genetic knockdown of HCN2 was achieved by transfection of siRNA directed against HCN2 mRNA using Oligofectamine (Invitrogen). Briefly, MIN6 cells were transfected with scrambled or anti-HCN2 siRNA at a final concentration of 30 nM in the presence of Optimem (Invitrogen). The Optimem was replaced with DMEM 4–6 h after transfection, and the cells were incubated for 48 h to allow for protein knockdown, at which time patch clamp measurements were performed to verify knockdown. Using FAM as a reporter, we observed a transfection efficiency of 75–85% at 48–72 h.


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  		Table 4. siRNA Primers for Mouse HCN2

 
Data Analysis
Results are represented as mean ± SEM. Data were analyzed with the Student’s t test or Wilcoxon matched-pairs test as appropriate. P < 0.05 was considered significant. Grubb’s Test was used to discard potential outliers. Capacitance data and siRNA knockdown of ß-Ih were analyzed by multiple and single measures comparison ANOVA, respectively, and Tukey’s posttest. For RT-PCR, negative controls used were RT-minus and H2O blanks.


   FOOTNOTES
 
Current address for R.A.L.: Laboratory for Stem Cell Engineering, Stem Cell Institute and the Department of Cell Biology and Human Anatomy, University of California, Davis, California.

This work was supported by grants to M.B.W (MOP 49521) and R.G.T. (MOP 77638) from the Canadian Institutes of Health Research. W.E.-K. was supported by a Novo-Nordisk Studentship from the Banting and Best Diabetes Centre and a Doctoral Research Award from the Canadian Diabetes Association (CDA). P.E.M is a CDA and Alberta Heritage Foundation for Medical Research Scholar and the Canada Research Chair in Islet Biology. R.A.L. received salary support from the Cardiac Arrhythmias Research & Education Foundation, Inc.

Disclosure Statement: The authors have nothing to declare.

First Published Online December 7, 2006

Abbreviations: db-cAMP, Dibutyryl-cAMP; FAM, fluorescein; fF, femtofarad; GSIS, glucose-stimulated insulin secretion; HCN, hyperpolarization-activated cyclic nucleotide-modulated; ß-Ih, slow-activating inward current; IBMX, 3-isobutyl-1-methylxanthine; KATP, ATP-sensitive potassium; KRBH, Krebs-Ringer bicarbonate-HEPES; Kv, voltage-dependent potassium; MIN, mouse insulinoma; pA, picoampere; pF, picofarad; qPCR, quantitative PCR; si, small interfering.

Received for publication June 22, 2006. Accepted for publication December 1, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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