G-PROTEIN-INDEPENDENT ACTIVATION OF AN INWARD Na + CURRENT

Transcription

1 JBC Papers in Press. Published on August 2, 2002 as Manuscript M G-PROTEIN-INDEPENDENT ACTIVATION OF AN INWARD Na + CURRENT BY MUSCARINIC RECEPTORS IN MOUSE PANCREATIC β-cells Jean-François Rolland, Jean-Claude Henquin and Patrick Gilon Short running title: Muscarinic activation of Na + current in β-cells. From the Unité d Endocrinologie et Métabolisme, University of Louvain, Faculty of Medicine, UCL 55.30, Av. Hippocrate 55, B-1200 Brussels, Belgium Abbreviations: ACh, acetylcholine; K + -ATP channels, ATP-sensitive potassium channels; [Ca 2+ ] c, free cytosolic calcium concentration; DIDS, 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid; I Na-ACh, Na + current activated by ACh; IP 3, inositol 1,4,5-trisphosphate; [Na + ] c, free cytosolic sodium concentration; PTX, pertussis toxin. Address all correspondence to Dr P. Gilon Unité d Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, UCL 55.30, Av. Hippocrate 55, B-1200 Brussels, Belgium. Tel: Fax: E.mail: gilon@endo.ucl.ac.be Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

2 2 ABSTRACT Depolarization of pancreatic β-cells is critical for stimulation of insulin secretion by acetylcholine, but remains unexplained. Using voltage-clamped β-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na + omission, but was not mimicked by nicotine and was insensitive to nicotinic antagonists, tetrodotoxin, DIDS, thapsigargin-pretreatment and external Ca 2+ and K + removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na + channels distinct from store-operated channels. No outward Na + current was produced by acetylcholine when the electrochemical Na + gradient was reversed, indicating that the channels are inwardrectifiers. No outward K + current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na + and K + was close to that expected for a Na + - selective membrane, suggesting that the channels opened by acetylcholine are specific for Na +. Overnight pretreatement with pertussis toxin, or addition of GTP-γ-S or GDP-β-S instead of GTP to the pipette solution did not alter this current, excluding involvement of G-proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in β-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic β-cells triggers, by a G-protein independent mechanism, a selective Na + current that explains the plasma membrane depolarization.

3 3 INTRODUCTION During the feeding periods, the increase in glycemia is limited in time and amplitude by the hypoglycemic action of insulin. Blood glucose itself is the main stimulator of insulin secretion by pancreatic β-cells. This stimulation involves two complementary pathways. Glucose generates a triggering signal, a rise in cytosolic Ca 2+ concentration ([Ca 2+ ] c ) 1, through the following sequence of events: the acceleration of cell metabolism increases the ATP/ADP ratio, which closes ATP-sensitive K + (K + -ATP) channels in the plasma membrane; the resulting decrease in K + conductance leads to membrane depolarization, opening of voltagedependent Ca 2+ channels and Ca 2+ influx (1-5). Glucose also produces amplifying signals that increase the efficacy of Ca 2+ on exocytosis (6, 7). Besides glucose, physiological agents such as hormones and neurotransmitters also modulate insulin secretion. A rich parasympathethic and sympathethic innervation enters the islets and ends close to the endocrine cells, allowing a fine neural tuning of the islet function (8, 9). Acetylcholine (ACh) is released by parasympathethic nerve endings, during the preabsorptive phase of feeding to enhance insulin secretion prior to the rise in plasma glucose, and during the absorptive phase (10). By binding to muscarinic receptors of the M 3 type, ACh triggers changes in phospholipid metabolism leading to formation of diacylglycerol which activates protein kinase C, and inositol 1,4,5-trisphosphate (IP 3 ) which mobilizes Ca 2+ from intracellular Ca 2+ stores. The resulting fall of the Ca 2+ concentration in the endoplasmic reticulum activates a modest Ca 2+ influx, through voltage-independent Ca 2+ channels, which is commonly referred to as a capacitative Ca 2+ entry. In addition, ACh depolarizes the plasma membrane of β-cells. This depolarization is small and does not cause Ca 2+ influx in unstimulated β-cells. However, in the presence of stimulatory (depolarizing) concentrations of glucose, this additional depolarization by ACh enhances Ca 2+ influx through voltagedependent Ca 2+ channels, leading to a sustained [Ca 2+ ] c elevation (10).

4 4 Although central for the increase in insulin secretion by ACh (10, 11), the depolarization has never been conclusively explained. Several observations suggest that a Na + current is involved. Thus, the depolarization of β-cells by ACh is abrogated by omission of extracellular Na + (12) and accompanied by increases in total Na + content (13), 22 Na + uptake (12, 14) and free cytosolic Na + concentration ([Na + ] c ) (15). These arguments, however, remain indirect and conflict with the general concept that nicotinic rather than muscarinic receptors mediate cholinergic effects on Na + conductance. In the present study, we used membrane potential recordings with microelectrode and both conventional and perforated whole-cell modes of the patch-clamp technique to identify and characterize the current by which ACh depolarizes the plasma membrane of mouse β-cells. Our study provides the first direct electrophysiological evidence for a muscarinic, G-protein-independent, stimulation of an inward Na + current in pancreatic β-cells.

5 5 EXPERIMENTAL PROCEDURES Preparation of cells The pancreas from NMRI mice killed by cervical dislocation was aseptically digested with collagenase in a bicarbonate-buffered solution containing (in mmol/l) 120 NaCl, 4.8 KCl, 2.5 CaCl 2, 1.2 MgCl 2, 24 NaHCO 3, 5 Hepes, 10 glucose, 1 mg/ml bovine serum albumin (BSA; fraction V; Roche Molecular Biochemicals, Mannheim, Germany), and gassed with O 2 /CO 2 (94:6 %) to have a ph of 7.4. Islets were handpicked under a stereomicroscope. Single cells were obtained by incubating the islets for 5 min in a Ca 2+ -free medium containing (in mmol/l) 138 NaCl, 5.6 KCl, 1.2 MgCl 2, 5 Hepes, 3 glucose and 1 mmol/l EGTA (ph 7.4). After a brief centrifugation, this solution was replaced by culture medium, and the islets were disrupted by gentle pipetting through a siliconized glass pipette. The cells were plated on 22 mm-diameter glass coverslips. Intact islets and single islet cells were cultured for, respectively, 1 and 1-3 days in RPMI 1640 culture medium (GIBCO, Paisley, U.K.) containing 10 % heat-inactivated fetal calf serum and 10 mmol/l glucose. All solutions for tissue preparation and culture medium were supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. Electrophysiological recordings The membrane potential of a single β-cell within an islet was continuously recorded at 37 C with a high resistance (~200 MΩ) intracellular microelectrode (16). β-cells were identified by the typical electrical activity that they display in the presence of 10 mmol/l glucose. Two criteria defined previously (17) were used to identify single β-cells: a cell capacitance above 5 pf and the presence of a voltage-dependent Na + current that is inactivated at a holding potential of -70 mv but can be activated after a hyperpolarizing pulse to -140 mv. Patch-clamp measurements were carried out in both conventional and perforated whole-cell modes, using an EPC-9 patch-clamp amplifier (Heka Electronics,

6 6 Lambrecht/Pfalz, Germany) and the software Pulsefit, or an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA) and the software pclamp 8. Patch pipettes were pulled from borosilicate glass capillaries (World Precision Instruments, Inc., Hertfordshire, UK) to give a resistance of 4-5 MΩ. Except for the experiments performed to obtain the I-V curve, I Na-ACh was measured in cells kept hyperpolarized at -80 mv. I Ca was measured by applying 25 ms-depolarizations from -80 mv to +10 mv every 5 s. Voltageclamp experiments were performed at room temperature (22-25 C), whereas current-clamp experiments were carried out at C. Solutions for electrophysiological recordings The standard extracellular solution used for membrane potential recordings with intracellular microelectrodes contained (in mmol/l): 122 NaCl, 4.7 KCl, 2.6 CaCl 2, 1.2 MgCl 2, 20 NaHCO 3, and glucose as indicated in the legend. When necessary, a K + -free solution was prepared by substituting NaCl for KCl. These solutions were gassed with O 2 /CO 2 (95:5 %) to maintain ph at 7.4. Various solutions were used for patch-clamp recordings. In perforated mode, the pipette solution contained (in mmol/l): 70 K 2 SO 4, 10 NaCl, 10 KCl, 3.7 MgCl 2 and 5 Hepes (ph 7.1) (Int Sol A). The electrical contact was established by adding a pore-forming antibiotic, amphotericin B or nystatin, to the pipette solution. Amphotericin (stock solution of 60 mg/ml in DMSO) was used at a final concentration of 300 µg/ml. Nystatin (stock solution of 10 mg/ml in DMSO) was used at a final concentration of 200 µg/ml. The tip of the pipette was back-filled with antibiotic-free solution and the pipette was then filled with the amphotericin or nystatin-containing solution. The voltage clamp was considered satisfactory when the series conductance was >35-40 ns. In conventional whole-cell recordings of I Na-ACh, the pipette solution contained (in mmol/l): 112 KCl, 5 KOH, 1 MgCl 2, 3 MgATP, 0.1 Na 2 GTP and 10 HEPES (ph adjusted to

7 with 1 mmol/l HCl) (Int Sol B). When needed, 10 mmol/l EGTA was added to internal solution B (Int Sol C). Na + -rich/k + -free solution was prepared by substituting NaCl for KCl, and NaOH for KOH of internal solution B (Int Sol D). Na + -free/k + -rich solution was prepared by increasing KCl and KOH concentrations of internal solution B to 125 and 30, respectively (ph adjusted to 7.15 with 18 mmol/l HCl) (Int Sol E). For experiments during which the equilibrium potential for Na + was fixed at -60 mv or 20 mv, the pipette solution contained (in mmol/l): 107 NaCl, 10 NaOH, 3 MgATP, 0.1 Na 2 GTP, 1 MgCl 2 and 10 HEPES (ph adjusted to 7.15 with 7 mmol/l HCl) (Int Sol F). For conventional whole-cell recordings of I Ca, the pipette solution contained (in mmol/l): 125 CsCl, 30 KOH, 1 MgCl 2, 10 EGTA, 3 MgATP, 0.1 Na 2 GTP, and 5 HEPES (ph 7.15) (Int Sol G). When specified, GTP-γ-S or GDP-β-S was substituted for GTP in the pipette solution. When the conventional whole-cell mode was used, ACh was applied 5 minutes after rupture of the plasma membrane. The standard extracellular solution used to monitor I Na-ACh contained (in mmol/l): 120 NaCl, 4.8 KCl, 2.5 CaCl 2, 1.2 MgCl 2, 24 NaHCO 3, 5 HEPES (ph 7.4) and 10 glucose (Ext Sol A). Ca 2+ -free solution was prepared by substituting MgCl 2 for CaCl 2 of external solution A, and was supplemented with 2 mmol/l EGTA (Ext Sol B). When needed, Na + 145/K + -free solution was prepared by substituting NaCl for KCl of external solution A (Ext Sol C). Na + - free/k solution contained (in mmol/l): 135 N-methyl-D-glucamine (NMDG), 4.8 KCl, 2.5 CaCl 2, 1.2 MgCl 2, 5 HEPES (ph adjusted to 7.4 with 131 mmol/l HCl) and 10 glucose (Ext Sol D). When needed, Na + -K + -free solution was prepared by substituting N-methyl-Dglucamine for KCl of external solution D (ph adjusted to 7.4 with 136 mmol/l HCl) (Ext Sol E). For experiments during which the equilibrium potential for Na + was fixed at -60 mv, the external solution contained (in mmol/l): 11 NaCl, 10 KCl, 180 NMDG, 2.5 CaCl 2, 1.2 MgCl 2, 5 HEPES (ph 7.4), 0.1 CdCl 2, 0.25 tolbutamide (ph adjusted to 7.4 with 172 mmol/l HCl) and 10 glucose (Ext Sol F). For experiments during which the equilibrium potential for Na + was fixed at -20 mv, the external solution contained (in mmol/l): 53 NaCl, 10 KCl, 100

8 8 NMDG, 2.5 CaCl 2, 1.2 MgCl 2, 5 HEPES (ph 7.4), 0.1 CdCl 2, 0.25 tolbutamide (ph adjusted to 7.4 with 96 mmol/l HCl) and 10 glucose (Ext Sol G). For recordings of I Ca, the external solution contained (in mmol/l): 125 NaCl, 4.8 KCl, 10 CaCl 2, 1.2 MgCl 2, 10 tetraethylammonium-cl, 5 HEPES (ph 7.4) and 10 glucose (Ext Sol H). Thapsigargin was obtained from Alomone Labs (Jerusalem, Israel). Unless otherwise stated, all other chemicals were from Sigma (St. Louis, MO). Presentation of results The experiments are illustrated by means or representative traces of results obtained with the indicated number of cells from at least three different cultures. The statistical significance of differences between means was assessed by unpaired Student's t test. Differences were considered significant at P < RESULTS Effects of ACh on the membrane potential of mouse pancreatic β-cells In the presence of 10 mmol/l glucose, β-cells within an islet display a rhythmic electrical activity characterized by the alternance of polarized silent phases and depolarized phases with bursts of action potentials (Fig. 1A). Addition of 1 µmol/l ACh induced a sustained and persistent depolarization with continuous spike activity in 2/6 islets. In the other islets, the initial period of sustained activity was followed by rapid oscillations of the membrane potential (Fig. 1A). Similar effects of ACh on the β-cell electrical activity have previously been observed in non-cultured islets, and are blocked by atropine (12, 18). Muscarinic receptor activation induces an inward current in β-cells The effect of ACh on the whole-cell current was first studied in single β-cells held hyperpolarized at -80 mv by the conventional whole-cell configuration of the patch-clamp

9 9 technique. Addition of ACh induced a sustained and reversible inward current the amplitude of which increased with the concentration of the neurotransmitter (Fig. 2A-D) to reach a maximum of 0.77 ± 0.15 pa/pf (n = 5) at 100 µmol/l ACh. The half-maximal effective concentration (EC 50 ) estimated after fitting the data to a sigmoidal function was at 2.5 µmol/l ACh (Fig. 2D). The kinetics of activation of the current by ACh could not be established reliably because the characteristics of our perifusion system (chamber volume of ~0.8 ml and flow rate of ~0.5 ml/min) preclude fast solution exchange. However, it was repeatedly noted that the current activated by ACh developped rapidly, within ~1 sec (Figs. 2B, 3G), in cells that were located very close to the inflow of solution, and more slowly (Fig. 3B, D) in cells that were located at some distance of this inflow. The current elicited by ACh was completely suppressed or prevented by the muscarinic receptor antagonist, atropine (Fig. 2A-C), but was not mimicked by 10 µmol/l nicotine (n = 5, not shown), and was insensitive to nicotine or nicotinic antagonists. Thus, in the presence of 10 µmol/l nicotine, 0.1 µmol/l α-bungarotoxine or 100 µmol/l hexamethonium, 100 µmol/l ACh elicited a current which was, respectively, 90 ± 8 % (n = 11), 111 ± 11 % (n = 6) or 107 ± 6 % (n = 7) of the current activated by ACh in the absence of nicotinic agents. These experiments show that the ACh-induced inward current in β-cells results from activation of muscarinic, but not nicotinic, receptors. In another series of experiments, β-cells were voltage-clamped in the perforated whole-cell mode and treated with 1 µmol/l thapsigargin, which completely emptied the endoplasmic reticulum in Ca 2+, as indicated by the suppression of Ca 2+ mobilization by ACh (n = 5; not shown). Subsequent application of ACh elicited a current of the same amplitude (0.77 ± 0.09 pa/pf, n = 11) as that measured in the whole-cell configuration (Fig. 2E). The ACh-induced inward current, therefore, is not a store-operated current. Characteristics of the current activated by ACh in β-cells

10 10 The ionic specificity of the current was first evaluated in the standard whole-cell mode by removing Ca 2+, Na + or K + from the bath or pipette solutions. Addition of ACh to a Ca 2+ -free medium elicited an inward current indicating that the latter was not carried by Ca 2+ (Fig. 3A). Omission of K + from the perifusion medium did not prevent the current elicited by ACh indicating that the current does not involve changes in Na + pump activity (Fig. 3B). By contrast, omission of extracellular Na + abrogated the current, which suggests that it is carried by Na + (Fig. 3C). The current was nevertheless insensitive to tetrodotoxin indicating that voltage-gated Na + channels are not involved (Fig. 3D). When the electrochemical gradient for Na + was reversed (Na + -rich pipette solution and Na + -free bath medium) to permit an outward current, ACh was ineffective (Fig. 3E) suggesting that Na + flows only in the inward direction. A non-specific cationic current carrying both Na + and K + could also depolarize the plasma membrane because it usually has a reversal potential close to 0 mv. The experiments performed above did not allow us to exclude the possibility that ACh activates such a current because either the membrane was clamped close to the equilibrium potential of K + (Fig. 3C) or no K + was present in the pipette and bath solutions (Fig. 3E). To address that question, two series of experiments were thus performed. In the first series, Na + was omitted from both pipette and bath solutions, whereas K + was present at a high concentration in the pipette solution only (Fig. 3F). Under these conditions where the equilibrium potential for K + was infinitely negative and no Na + current could occur, ACh did not activate any outward current. In the second series of experiments, the Na + versus K + specificity of the current activated by ACh was evaluated by measuring its reversal potential with pipette and external solutions selected to have very different equilibrium potentials for Na + and K +, i.e. -60 or 20 mv for Na +, and infinitely positive potentials for K +. These experiments were performed in the presence of Cd 2+ to block voltage-dependent Ca 2+ channels (and avoid [Ca 2+ ] c overload or activation of Ca 2+ -dependent currents), and tolbutamide to block K + -ATP channels (and avoid a large outward current through these channels). However, even under these conditions, the

11 11 reversal potential could not be reliably estimated by voltage ramp protocols because of the smallness of the current. Therefore, the effect of ACh was tested in separate β-cells held at selected fixed potentials between -130 and 0 mv (Fig. 4). Experiments at potentials more negative than -130 mv or more positive than 0 mv could not be performed because of instability of the seal. Pipette and external solutions were first selected to have an equilibrium potential for Na + at 60 mv. At potentials more negative than -60 mv, the amplitude of the inward current induced by ACh increased with the driving force for Na + (larger at -130 than mv) and displayed a slope conductance of 6.6 ps/pf (Fig. 4: open squares). At the set equilibrium potential for Na + (-60 mv), ACh did not induce any current. To test if the reversal potential of the current activated by ACh strictly followed the equilibrium potential of Na +, the equilibrium potential of Na + was then fixed at -20 mv. At this potential, ACh failed to activate any current. However, at potentials more negative than -20 mv, ACh induced an inward current with an amplitude proportional to the driving force for Na + and with a slope conductance of 5.3 ps/pf (Fig. 4: filled circles). All these results clearly indicate selectivity for Na + without contribution of K +. Because of this ionic characteristic, the current will be referred to as I Na-ACh. At potentials less negative than the set equilibrium potential for Na + (-30 and 0 mv when E Na was fixed at -60 mv; 0 mv when E Na was fixed at -20 mv), ACh did not activate any outward current (Fig. 4), which, together with the results of Fig. 3E, suggests that the channels responsible for I Na-ACh are inward rectifiers. An increase in Cl - permeability is expected to depolarize the plasma membrane because the equilibrium potential for Cl - in β-cells has been estimated to be above the threshold for activation of voltage-dependent Ca 2+ channels (19, 20). Activation of a Cl - current by ACh has not been directly tested by omitting Cl - from the medium. However, this possibility can also be discarded for two reasons. First, ACh did not elicit any current when the membrane was clamped at a potential different from the equilibrium potential of Cl - and under conditions where no Na + current occurred (e.g. at holding potentials of -80, -80 and -60

12 12 mv in Fig. 3C, 3F and 4 open squares, when E Cl was at, -6, 0 and -14 mv, respectively). Second, I Na-ACh was unaffected by DIDS, a blocker of the volume-activated current (21) that carries Cl - and possibly other ions in β-cells (22). Thus, in the presence of 100 µmol/l DIDS, 100 µmol/l ACh elicited a current that was 112 ± 10 % (n = 14) of the current activated by ACh in the absence of the blocker (Fig. 3G). Activation of I Na-ACh does not involve G-proteins Muscarinic effects of ACh in β-cells are known or assumed to be transduced by a G-protein, and it has been reported that, in neurones and cardiomyocytes, several muscarinic receptor subtypes modulate ionic channels, such as Ca 2+ and K + channels, through pertussis toxinsensitive G-proteins of the G i or G o class (10). However, after permanent inactivation of G i - or G o -proteins by overnight pretreatment of β-cells with pertussis toxin (250 ng/ml), the amplitude of I Na-ACh was 109 ± 7 % (n = 6) of that observed in non-treated cells. To test the possible involvement of all kinds of G-proteins in the activation of I Na-ACh, GTP in the pipette solution (control conditions) was replaced by GTP-γ-S or GDP-β-S that are, respectively, non-hydrolysable activator and inhibitor of G-proteins. Fig. 5 shows the maximum inward current (I Na-ACh ) elicited by 1 min application of 100 µmol/l ACh to β-cells voltage-clamped at -80 mv and dialyzed for 5 min with a solution containing 100 µmol/l GTP, 10 µmol/l GTP- γ-s or 4 mmol/l GDP-β-S. In all conditions, I Na-ACh was reversible upon washout of the neurotransmitter, and its amplitude was similar with the three nucleotides suggesting that activation of I Na-ACh does not involve G-proteins. To ascertain that our experimental conditions were adequate to identify the involvement of a G-protein, we tested the GTP analogues on the ACh-induced inhibition of voltage-dependent Ca 2+ current in pancreatic β-cells (23). Fig. 6A shows representative whole-cell Ca 2+ current traces recorded with a pipette solution containing 100 µmol/l GTP. The current (I Ca ) was evoked by 25 ms depolarizing pulses from -80 to 10 mv before

13 13 (control), during (ACh) and after (wash) application of 100 µmol/l ACh to the bath. Fig. 6B summarizes the changes of the normalized peak Ca 2+ current produced by ACh in the presence of different guanine nucleotides in the pipette solution. With 100 µmol/l GTP in the pipette solution (control), ACh reversibly inhibited the current. This inhibitory effect was abolished when the pipette solution contained 4 mmol/l GDP-β-S, and became irreversible when 10 µmol/l GTP-γ-S was included in the pipette. The spontaneous decrease in current amplitude recorded with GDP-β-S reflects rundown (23). These control experiments thus show that the guanine nucleotides were effective in our recording conditions. Impact of a depolarizing current equivalent to I Na-ACh on the β-cell membrane potential Because I Na-ACh is small, it was important to verify that a current of a similar amplitude was sufficient to elicit electrical activity. These experiments were performed in β-cells perifused at C with 6 mmol/l glucose, a concentration that is subthreshold in islets (24). A depolarizing current corresponding to the average I Na-ACh induced by 100 µmol/l ACh (0.77 pa/pf) and adjusted to cell size (0.77 pa x capacitance of the tested cell) was injected in the current-clamp mode. Such a current elicited an electrical activity in all tested cells, and its removal was accompanied by an immediate repolarization (Fig. 7A). In other experiments shown in Fig. 7B, the depolarizing effect of 1 µmol/l ACh was first tested and compared to that of depolarizing currents corresponding to I Na-ACh elicited by 1 (0.23 pa/pf), 10 (0.61 pa/pf) and 100 µmol/l ACh (0.77 pa/pf). Addition of 1 µmol/l ACh triggerred electrical activity with action potentials that ceased upon washout of the neurotransmitter. Subsequent injection of a current with an amplitude similar to that of I Na-ACh induced by 1 µmol/l ACh (a in Fig. 7B) elicited electrical activity similar to that produced by 1 µmol/l ACh itself. Injection of larger currents corresponding to I Na-ACh induced by 10 and 100 µmol/l ACh (b and c, respectively, in Fig. 7B) augmented the frequency of the electrical activity. These results

14 14 indicate that the small I Na-ACh is sufficient to depolarize the membrane potential beyond the threshold for the activation of voltage-dependent Ca 2+ channels.

15 15 DISCUSSION By activating muscarinic receptors, ACh induces a number of effects in pancreatic β-cells, which culminate in an increase in insulin secretion (10). Among these effects, a glucosedependent depolarization of the plasma membrane plays a central role. Experiments using intracellular microelectrodes and tracer fluxes have led to the suggestion that a Na + current underlies the ACh-mediated depolarization (12). This proposal has, however, remained incompletely convincing because Na + currents are classically activated by nicotinic rather than muscarinic receptors, and because the predicted ionic mechanism has not received direct electrophysiological support. The present study eventually succeeded in identifying an inward current that is specifically carried by Na + and is responsible for the muscarinic depolarization of pancreatic β-cells. It further shows that the activation of the current is not mediated by G- proteins. Acetylcholine activates an inward Na + current in β-cells Our data demonstrate that ACh activates an inward current that is attributed to Na + influx because of it suppression either when Na + was removed from the extracellular medium or when the membrane potential of the cell was close to the equilibrium potential of Na +. On the other hand, no contribution of Ca 2+, K + or Cl - to the current could be obtained. Thus, the AChinduced current was not affected by removal of extracellular Ca 2+. When no Na + current could occur, no inward or outward current was evoked by ACh even when the equilibrium potential of K + was infinitely negative or positive, or when the membrane potential was clamped away from the equilibrium potential of Cl -. The current elicited by ACh was also insensitive to DIDS, a blocker of the Cl - -mediated, volume-activated current in β-cells (22). Activation of this Na + current by ACh may explain the increase in total Na + content (13), 22 Na + uptake (12, 14) and [Na + ] c (15) that the neurotransmitter induces in islet cells, and the abrogation of all these effects in a Na + -free medium. The amplitude of the Na + current

16 16 elicited by ACh is small, but sufficient to account for the 15 mmol/l increase in [Na + ] c occurring in β-cells after 15 min of stimulation with 100 µmol/l ACh (15, 25). Indeed, 100 µmol/l ACh activated a mean inward current of 0.77 pa/pf, which corresponds to 6.15 pa for an average β-cell capacitance of 7.9 ± 0.08 pf (estimated from 644 β-cells tested in the present study). Assuming that the current remains stable over 15 min, the current charge amounts to 5537 pcb, which corresponds to ~ 57 fmoles of Na +. For an intracellular space of 620 fl per β-cell (26), this would result in a [Na + ] c increase by 92 mmol/l, which is well above the 15 mmol/l measured. Activation of the Na + pump obviously tends to correct the [Na + ] c rise. The reverse mechanism, an inhibition of the Na + pump, has been proposed to explain the increase in [Na + ] c produced by ACh in sheep Purkinje fibers (27). The explanation does not hold for β-cells in which ACh still increases [Na + ] c after blockade of the pump by ouabain (15). This is fully compatible with the persistence of the ACh-induced inward current in a K + - free medium, another situation where the Na + pump is blocked. Nicotinic receptors, which are non-selective cationic channels (28, 29), classically mediate cholinergic effects on Na + currents. Such channels are clearly not responsible for the ACh-induced inward current in β-cells because the muscarinic antagonist, atropine, completely prevented the current and the rise in [Na + ] c (15), whereas the ACh-activated current was not mimicked by nicotine, and was insensitive to nicotinic antagonists. Activation of a Na + conductance by muscarinic receptors is unusual but has occasionally been reported in cardiac myocytes (30, 31), smooth muscle cells of the gastro-intestinal tract (32, 33), chromaffin cells (34), and Chinese hamster ovary (CHO) cells expressing M 3 receptors (35). The channels activated by ACh in β-cells have not been identified but several of their properties could be established. Voltage-dependent Na + channels are present in mouse β-cells but they are inactivated at the holding potential of -80 mv that we used (36). We can discard the possibility that such channels mediate the effect of ACh because the Na + current evoked by the neurotransmitter did not require any voltage change and was insensitive to

17 17 tetrodotoxin, as are the ACh-induced increases in Na + uptake (12) and [Na + ] c (15). Our results also indicate that the current is not carried by a non-specific cationic channel allowing flow of both Na + and K + or Ca 2+. The Na + channels activated by ACh display inward rectifying properties as shown by their inability to carry an outward current when the electrochemical gradient for Na + was reversed. Because the inward current activated by ACh is a specific Na + current, we termed it I Na-ACh. Mechanisms of activation of I Na-ACh As in other cells (37, 38), lowering the Ca 2+ content of the endoplasmic reticulum in β-cells activates conductances for Ca 2+ and perhaps other ionic species including Na + (25, 39). Even if such a mechanism slightly contributes to, it is not responsible for I Na-ACh, because ACh still activated the inward current after emptying of the endoplasmic reticulum Ca 2+ stores by thapsigargin. This is consistent with our previous report that thapsigargin and cyclopiazonic acid, which empty the endoplasmic reticulum in Ca 2+ more efficiently than does ACh, did not mimic or prevent the [Na + ] c rise elicited by ACh (25). The fact, that neither pretreatment with thapsigargin nor inclusion of a high concentration of EGTA in the pipette solution prevented ACh from inducing an inward current, also excludes the possibility that activation of I Na-ACh is secondary to a rise in [Ca 2+ ] c. Although it is classically admitted that muscarinic receptors are coupled to G-proteins (40), activation of I Na-ACh was unaffected by inactivation of G i /G o -proteins by pertussis toxin pretreatment, or infusing β-cells with GTP-γ-S or GDP-β-S, two guanine nucleotide analogues that, respectively, activate and inhibit G-proteins. Both analogues were however effective in our experimental conditions as shown by their modulation of ACh-induced inhibition of the voltage-dependent Ca 2+ current (23). These observations unexpectedly indicate that activation of I Na-ACh does not involve G-proteins in β-cells. There is growing

18 18 evidence that various seven transmembrane metabotropic receptors can also activate transduction systems without involvement of G-proteins (41, 42). In particular, muscarinic agonists have been found to activate, in a G-protein-independent way, a Na + current in ventricular myocytes (31), a cationic current in CA3 pyramidal cells (43), and a K + current in aortic endothelial cells (44, 45). The transduction mechanisms have not been identified but direct or indirect (via adaptor proteins) interactions between the receptor and effector proteins have tentatively been proposed. Activation of cationic conductance by a SRC-tyrosine kinase in CA3 pyramidal neurons (41) and facilitation of the stimulation of IP 3 receptors by the protein Homer (42) are two examples of G-protein-independent event linked to activation of metabotropic glutamate receptor. Role of I Na-ACh in the control of β-cell membrane potential by ACh Apart from the increase in Na + conductance, all plausible mechanisms by which ACh might depolarize the β-cell membrane can be excluded. First, ACh does not decrease the β-cell membrane K + conductance. Unlike glucose and sulfonylureas, ACh does not inhibit the efflux of 86 Rb + (a tracer of K + ) (12, 46) and does not reduce K + -ATP (17) or other K + currents (this study). Second, an increase in Cl - permeability, which would depolarize the β-cell membrane because of the high equilibrium potential for Cl - (19, 20), is not involved. Thus, ACh has no effect on 86 Cl - efflux from mouse islets (14, 47) and its depolarizing effect is not influenced by omission of extracellular Cl - (47). Third, there is no evidence that ACh directly activates Ca 2+ channels. On the contrary, we confirm here that high concentrations of the neurotransmitter rather inhibit Ca 2+ currents through voltage-dependent Ca 2+ channels (23). It is possible, however, that the small capacitative Ca 2+ entry induced by ACh, via a lowering of intracellular Ca 2+ stores, slightly contributes to the depolarization. Fourth, blockade of the Na + /K + pump is known to depolarize β-cells (24). However, several arguments suggest that ACh does not inhibit the Na + /K + pump. A depolarizing effect of ACh persisted after inhibition

19 19 of the Na + /K + ATPase by omission of extracellular K +, and reactivation of the Na + /K + pump after its blockade (by K + removal or ouabain) induced a transient repolarization that was not suppressed by ACh (unpublished data). Moreover, ACh slightly increased initial 86 Rb + uptake (14), which is opposite to the effect observed after blockade of the pump with ouabain (48, 49). Activation of I Na-ACh is therefore the most plausible mechanism of ACh-induced depolarization of β-cells. Our proposal is in complete agreement with the observation that this depolarization is abrogated by extracellular Na + omission but insensitive to tetrodotoxin (8). We further show here that the amplitude of I Na-ACh is sufficient to explain the effects of ACh on the membrane potential. Thus, injection of a current with an amplitude similar to that activated by 1 µmol/l of ACh (i.e ± 0.02 pa/pf) elicited electrical activity in cells perifused with a subthreshold glucose concentration. Since the effect of a given current augments with the resistance of the membrane and since the latter increases with the glucose concentration (closure of K + -ATP channels), it can be anticipated that even smaller currents could depolarize the plasma membrane in the presence of stimulating glucose concentrations. The subsequent activation of voltage-dependent Ca 2+ channels eventually leads to a sustained increase in [Ca 2+ ] c that largely contributes to the insulin-releasing action of ACh (10). Acknowledgments: This work was supported by grant from the Fonds de la Recherche Scientifique Médicale (Brussels), grant from the Fonds National de la Recherche Scientifique (Brussels), grant from the Fonds de la Recherche Fondamentale Collective (Brussels), grant ARC 00/ from the General Direction of Scientific Research of the French Community of Belgium, and by the Interuniversity Poles of Attraction Programme (P5/3-20) - Federal Office for Scientific, Technical and Cultural Affairs of Belgium. P.G. is Senior Research associate of the Fonds National de la Recherche Scientifique, Brussels.

20 20 1. Ashcroft, F. M., and Rorsman, P. (1989) Prog. Biophys. Mol. Biol. 54, Gilon, P., Ravier, M. A., Jonas, J. C., and Henquin, J. C. (2002) Diabetes 51 (Suppl 1), S144-S Maechler, P., and Wollheim, C. B. (2001) Nature 414, Matschinsky, F. M. (1996) Diabetes 45, Seino, S., Iwanaga, T., Nagashima, K., and Miki, T. (2000) Diabetes 49, Aizawa, T., Komatsu, M., Asanuma, N., Sato, Y., and Sharp, G. W. (1998) Trends Pharmacol. Sci. 19, Henquin, J. C. (2000) Diabetes 49, Ahren, B. (2000) Diabetologia 43, Brunicardi, F. C., Sun, Y. S., Druck, P., Goulet, R. J., Elahi, D., and Andersen, D. K. (1987) Am. J. Surg. 153, Gilon, P., and Henquin, J. C. (2001) Endocr. Rev. 22, Satin, L. S., and Kinard, T. A. (1998) Endocrine 8, Henquin, J. C., Garcia, M. C., Bozem, M., Hermans, M. P., and Nenquin, M. (1988) Endocrinology 122, Saha, S., and Hellman, B. (1991) Eur. J. Pharmacol. 204, Gagerman, E., Sehlin, J., and Taljedal, I. B. (1980) J. Physiol. 300, Gilon, P., and Henquin, J. C. (1993) FEBS Lett. 315, Meissner, H. P. (1990) Methods Enzymol. 192,

21 Rolland, J. F., Henquin, J. C., and Gilon, P. (2002) Diabetes 51, Gilon, P., Nenquin, M., and Henquin, J. C. (1995) Biochem. J. 311, Eberhardson, M., Patterson, S., and Grapengiesser, E. (2000) Cell Signal. 12, Sehlin, J. (1978) Am. J. Physiol. 235, E501-E Best, L., Brown, P. D., Sheader, E. A., and Yates, A. P. (2000) J. Membr. Biol. 177, Kinard, T. A., and Satin, L. S. (1995) Diabetes 44, Gilon, P., Yakel, J., Gromada, J., Zhu, Y., Henquin, J. C., and Rorsman, P. (1997) J. Physiol. 499, Henquin, J. C., and Meissner, H. P. (1982) J. Physiol. 332, Miura, Y., Gilon, P., and Henquin, J. C. (1996) Biochem. Biophys. Res. Commun. 224, Heimberg, H., De Vos, A., Pipeleers, D., Thorens, B., and Schuit, F. (1995) J. Biol. Chem. 270, Iacono, G., and Vassalle, M. (1989) Am. J. Physiol. 256, H1407-H Hosey, M. M. (1992) FASEB J. 6, Hucho, F. (1986) Eur. J. Biochem. 158, Matsumoto, K., and Pappano, A. J. (1989) J. Physiol. 415, Shirayama, T., Matsumoto, K., and Pappano, A. J. (1993) J. Pharmacol. Exp. Ther. 265,

22 Inoue, R., and Isenberg, G. (1990) Am. J. Physiol. 258, C1173-C Benham, C. D., Bolton, T. B., and Lang, R. J. (1985) Nature 316, Inoue, M., Sakamoto, Y., and Imanaga, I. (1995) Eur. J. Pharmacol. 276, Carroll, R. C., and Peralta, E. G. (1998) EMBO J. 17, Plant, T. D. (1988) Pflugers Arch. 411, Putney, J. W., Jr. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, Tepel, M., Wischniowski, H., and Zidek, W. (1994) Biochim. Biophys. Acta 1220, Worley, J. F., III, McIntyre, M. S., Spencer, B., and Dukes, I. D. (1994) J. Biol. Chem. 269, Wess, J., Blin, N., Mutschler, E., and Bluml, K. (1995) Life Sci. 56, Heuss, C., and Gerber, U. (2000) Trends Neurosci. 23, Brzostowski, J. A., and Kimmel, A. R. (2001) Trends Biochem. Sci. 26, Guerineau, N. C., Bossu, J. L., Gahwiler, B. H., and Gerber, U. (1995) J. Neurosci. 15, Olesen, S. P., Davies, P. F., and Clapham, D. E. (1988) Circ. Res. 62, Brown, L. D., Kim, K. M., Nakajima, Y., and Nakajima, S. (1993) Brain Res. 612, Nenquin, M., Awouters, P., Mathot, F., and Henquin, J. C. (1984) FEBS Lett. 176,

23 Hermans, M. P., Schmeer, W., Gerard, M., and Henquin, J. C. (1991) Biochim. Biophys. Acta 1092, Sehlin, J., and Taljedal, I. B. (1974) J. Physiol. 242, Henquin, J. C. (1980) Biochem. J. 186,

24 24 FIGURE LEGENDS Fig.1. Effects of ACh on the membrane potential of mouse β-cells. Isolated islets were perifused with a medium containing 10 mmol/l glucose (G) and stimulated with 1 µmol/l ACh as indicated. This recording is representative of results obtained in 6 islets. Fig.2: ACh induces an inward current by activating muscarinic receptors in mouse β- cells. Single β-cells were voltage-clamped at 80 mv using the conventional (A-D) or the perforated (E) whole-cell mode of the patch-clamp technique. The composition of the external (Ext Sol) and pipette solutions (Int Sol) used is described in experimental procedures. ACh and atropine were applied when indicated by the arrows. A-D: ACh induced a concentrationdependent inward current that was reversed or blocked by atropine. Traces A-C are representative of 3 (A), 3 (B) and 4 (C) experiments. Panel D shows the concentrationdependence of ACh-induced inward current. Values are means +/- S.E.M of the amplitude of the current recorded in 3 to 5 cells for each ACh concentration. Fitting data points to a sigmoidal function yielded a half-maximal effective concentration (EC 50 ) of 2.5 µmol/l. E: pretreatment of intact β-cells by thapsigargin (30 min, 1 µmol/l) did not prevent ACh from activating an inward current. This trace is representative of 5 experiments. Fig.3: Characteristics of the inward current activated by ACh in mouse β-cells. Single β- cells were voltage-clamped at -80 mv using the conventional whole-cell mode of the patchclamp technique. The composition of the external (Ext Sol) and pipette solutions (Int Sol) used for these experiments is described in experimental procedures. For the sake of clarity, their main characteristics are summarized on the left of each panel. ACh (100 µmol/l) was applied when indicated by the arrows. A-B: ACh induced an inward current in the absence of Ca 2+ in the external and pipette solutions (Ca out 0, Ca in 0) (A), or when the Na + /K + pump was blocked by removal of K + from the external solution (K out 0) (B). C-D: The inward current

25 25 activated by ACh was abrogated by Na + omission fom the medium (Na out 0) (C), but unaffected by inhibition of voltage-dependent Na + channels with 2 µmol/l tetrodotoxin (D). E-F: ACh failed to induce an outward current when the driving force for Na + was directed outwardly (Na out 0; Na in -rich) (E), or when the driving force for K + was directed outwardly (K out 0; K in -rich) and, simultaneously, no Na + current could occur (Na out 0; Na in 0) (F). F: 100 µmol/l DIDS did not affect the inward current elicited by ACh. The traces are representative of at least 5 experiments. Fig.4: The current activated by ACh in mouse β-cells is rectifying inwardly. The reversal potential for Na + was fixed at 60 mv (open squares) or 20 mv (closed circles) by using external (Ext Sol) and pipette solutions (Int Sol) with appropriate concentrations of Na + (see experimental procedures for compositions). Single β-cells were voltage-clamped in conventional whole-cell mode at various potentials (-150, -100, -60, -30 and 0 mv when E Na+ was set at 60 mv, and 100, -60, -20 and 0 mv when E Na+ was set at 20 mv) around the reversal potential for Na +. Each point shows the mean +/- S.E.M of the current amplitude elicited by 100 µmol/l of ACh at each potential in 3 to 5 cells. Fig.5: Activation of I Na-ACh in mouse β-cells does not involve G-proteins. Single mouse β- cells were voltage-clamped at -80 mv and dialysed with a pipette solution (Int Sol B; see experimental procedures for compositions) containing 100 µmol/l GTP (control), 10 µmol/l GTP-γ-S or 4 mmol/l GDP-β-S. Each column represents the mean +/- S.E.M of the current amplitude elicited by 100 µmol/l ACh in 9 (Control), 5 (GTP-γ-S) and 4 (GDP-β-S) cells. Fig.6: Inhibition of I Ca by ACh in mouse β-cells involves G-proteins. Single β-cells were dialysed with a pipette solution (Int Sol G; see experimental procedures for compositions) containing 100 µmol/l GTP (control), 10 µmol/l GTP-γ-S or 4 mmol/l GDP-β-S, and

26 26 submitted to a 25 ms-depolarization to +10 mv from a holding potential of -80 mv. A: Representative voltage-dependent Ca 2+ currents recorded with a pipette solution containing 100 µmol/l GTP before (Control), during (ACh 100 µmol/l) and after (Wash) addition of ACh to the perifusion medium. B: Time-course of the effect of ACh on the peak I Ca recorded with different guanine nucleotides (GTP, open squares; GTP-γ-S, closed triangles; GDP-β-S, closed circles) in the pipette solution. To facilitate comparisons, I Ca was normalized in each individual experiment by dividing the peak current at each time by the maximum peak current at time zero. Traces are means ± SE of results obtained in 6 cells for each experimental condition. Fig.7: Injection of a depolarizing current mimics the ACh effects on the β-cell membrane potential. The membrane potential of a single mouse β-cell was monitored in current-clamp mode of the patch-clamp technique. The concentration of glucose of the medium was 6 mmol/l throughout. No current (0) was injected into the cell except for the periods indicated by upward deflections of the traces above each panel. In A, injection of a current with an amplitude corresponding to that elicited by 100 µmol/l ACh (0.77 pa x 10.4 pf = 8 pa in this cell) elicited an electrical activity characterized by action potentials on top of a plateau phase. In B, the cell was first stimulated with 1 µmol/l ACh. A current was then injected, at increasing amplitudes corresponding to those of the inward currents elicited by 1 (0.23 pa x 8.7 pf = 2 pa in this cell; period a), 10 (0.61 x 8.7 pf = 5 pa; period b) and 100 µmol/l ACh (0.77 x 8.7 pf = 7 pa; period c). Injection of the smallest current mimicked the effect of 1 µmol/l ACh. These traces are representative of 4 (A) and 5 (B) experiments.

27 A G10 ACh 1 µmol/l Fig.1 2 min Vm (mv)

28 A ACh 0.1 µmol/l Ext Sol A Int Sol B B C D I Na-ACh (pa/pf) E 1pA/pF 1pA/pF 1pA/pF 1pA/pF s 20 s 20 s 20 s ACh 100 µmol/l Atrop 10 µmol/l [ACh] (mol/l) ACh 100 µmol/l Atrop 10 µmol/l ACh 100 µmol/l EC 50 : 2.5 µmol/l Thapsigargin- Pretreated cell Ext Sol A Int Sol A Fig.2

29 A Ca out 0 Ca in 0 ACh 100 µmol/l Ext Sol B Int Sol C B K out 0 Ext Sol C Int Sol B C D E Na out 0 Tetrodotoxin Na out 0 Na in -rich Ext Sol D Int Sol B Ext Sol A Int Sol B Ext Sol D Int Sol D F K out 0, Na out 0 K in -rich, Na in 0 Ext Sol E Int Sol E G DIDS Ext Sol A Int Sol B 1 pa/pf 20 s Fig.3

30 Vm (mv) Ext Sol F Int Sol F Ext Sol G Int Sol F I Na-ACh (pa/pf) Fig.4

31 Ext Sol A Int Sol B 0.6 I Na-ACh (pa/pf) Control GTP-γ-S GDP-β-S Fig.5

32 A Vm (mv) Ext Sol H Int Sol G I Ca 100pA B -0.6 Control ACh 100 µmol/l ACh 100µM Wash GTP-γ-S I Ca (normalized) -0.8 Control GDP-β-S min Fig.6

33 A Ext Sol A Int Sol A I (pa/pf) Vm (mv) -40 B min ACh 1 µmol/l a b c I (pa/pf) Vm (mv) min Fig.7

34 G-protein-independent activation of an inward Na+ current by muscaring receptors in mouse pancreatic beta-cells Jean-François Rolland, Jean-Claude Henquin and Patrick Gilon J. Biol. Chem. published online August 2, 2002 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts