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J. Biol. Chem., Vol. 277, Issue 41, 38373-38380, October 11, 2002

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G Protein-independent Activation of an Inward Na⁺ Current by Muscarinic Receptors in Mouse Pancreatic -Cells^*

Jean-François Rolland, Jean-Claude Henquin, and Patrick Gilon

From the Unité d'Endocrinologie et Métabolisme, University of Louvain, Faculty of Medicine, UCL 55.30, Avenue Hippocrate 55, B-1200 Brussels, Belgium

Received for publication, April 22, 2002, and in revised form, July 12, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Depolarization of pancreatic -cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped -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, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca²⁺ 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 inward rectifiers. 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 pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP--S) or guanosine-5'-O-(2-thiodiphosphate) (GDP--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 -cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic -cells triggers, by a G protein-independent mechanism, a selective Na⁺ current that explains the plasma membrane depolarization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 -cells. This stimulation involves two complementary pathways. Glucose generates a triggering signal, a rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_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 voltage-dependent Ca²⁺ channels, and Ca²⁺ influx (1-5). Glucose also produces amplifying signals that increase the efficacy of Ca²⁺ on exocytosis (6, 7).

Besides glucose, physiological agents such as hormones and neurotransmitters also modulate insulin secretion. A rich parasympathetic and sympathetic 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 parasympathetic 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 M₃ type, ACh triggers changes in phospholipid metabolism leading to formation of diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which mobilizes Ca²⁺ from intracellular Ca²⁺ stores. The resulting fall of the Ca²⁺ concentration in the endoplasmic reticulum activates a modest Ca²⁺ influx, through voltage-independent Ca²⁺ channels, which is commonly referred to as a capacitative Ca²⁺ entry. In addition, ACh depolarizes the plasma membrane of -cells. This depolarization is small and does not cause Ca²⁺ influx in unstimulated -cells. However, in the presence of stimulatory (depolarizing) concentrations of glucose, this additional depolarization by ACh enhances Ca²⁺ influx through voltage-dependent Ca²⁺ channels, leading to a sustained [Ca²⁺]_c elevation (10).

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 -cells by ACh is abrogated by omission of extracellular Na⁺ (12) and accompanied by increases in total Na⁺ content (13), ²²Na⁺ 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 -cells. Our study provides the first direct electrophysiological evidence for muscarinic, G protein-independent stimulation of an inward Na⁺ current in pancreatic -cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Cells-- The pancreas from NMRI mice killed by cervical dislocation was aseptically digested with collagenase in a bicarbonate-buffered solution containing 120 mmol/liter NaCl, 4.8 mmol/liter KCl, 2.5 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 24 mmol/liter NaHCO₃, 5 mmol/liter Hepes, 10 mmol/liter glucose, and 1 mmol/liter mg/ml bovine serum albumin (fraction V; Roche Molecular Biochemicals) and gassed with O₂/CO₂ (94:6%) to have a pH of 7.4. The islets were handpicked under a stereomicroscope. Single cells were obtained by incubating the islets for 5 min in a Ca²⁺-free medium containing 138 mmol/liter NaCl, 5.6 mmol/liter KCl, 1.2 mmol/liter MgCl₂, 5 mmol/liter Hepes, 3 mmol/liter glucose, and 1 mmol/liter 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 (Invitrogen) containing 10% heat-inactivated fetal calf serum and 10 mmol/liter glucose. All of the 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 -cell within an islet was continuously recorded at 37 °C with a high resistance (~200 M) intracellular microelectrode (16). -Cells were identified by the typical electrical activity that they display in the presence of 10 mmol/liter glucose.

Two criteria defined previously (17) were used to identify single -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, Lambrecht/Pfalz, Germany) and the software Pulsefit or an Axopatch 200 B patch-clamp amplifier (Axon Instruments, Foster City, CA) 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 M. 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. Voltage-clamp experiments were performed at room temperature (22-25 °C), whereas current-clamp experiments were carried out at 34-36 °C.

Solutions for Electrophysiological Recordings-- The standard extracellular solution used for membrane potential recordings with intracellular microelectrodes contained 122 mmol/liter NaCl, 4.7 mmol/liter KCl, 2.6 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 20 mmol/liter NaHCO₃, and 10 mmol/liter glucose. It was gassed with O₂/CO₂ (95%:5%) to maintain pH at 7.4.

Various solutions were used for patch-clamp recordings. In the perforated mode, the pipette solution contained 70 mmol/liter K₂SO₄, 10 mmol/liter NaCl, 10 mmol/liter KCl, 3.7 mmol/liter MgCl₂, and 5 mmol/liter 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 Me₂SO) was used at a final concentration of 300 µg/ml. Nystatin (stock solution of 10 mg/ml in Me₂SO) 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 112 mmol/liter KCl, 5 mmol/liter KOH, 1 mmol/liter MgCl₂, 3 mmol/liter MgATP, 0.1 mmol/liter Na₂GTP, and 10 mmol/liter Hepes (pH adjusted to 7.15 with 1 mmol/liter HCl) (Int Sol B). When needed, 10 mmol/liter 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/liter HCl) (Int Sol E). For experiments during which the equilibrium potential for Na⁺ was fixed at 60 or 20 mV, the pipette solution contained 107 mmol/liter NaCl, 10 mmol/liter NaOH, 3 mmol/liter MgATP, 0.1 mmol/liter Na₂GTP, 1 mmol/liter MgCl₂, and 10 mmol/liter Hepes (pH adjusted to 7.15 with 7 mmol/liter HCl) (Int Sol F). For conventional whole cell recordings of I_Ca, the pipette solution contained 125 mmol/liter CsCl, 30 mmol/liter KOH, 1 mmol/liter MgCl₂, 10 mmol/liter EGTA, 3 mmol/liter MgATP, 0.1 mmol/liter Na₂GTP, and 5 mmol/liter Hepes (pH 7.15) (Int Sol G). When specified, GTP--S or GDP--S was substituted for GTP in the pipette solution. When the conventional whole cell mode was used, ACh was applied 5 min after the rupture of the plasma membrane.

The standard extracellular solution used to monitor I_Na-ACh contained 120 mmol/liter NaCl, 4.8 mmol/liter KCl, 2.5 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 24 mmol/liter NaHCO₃, 5 mmol/liter Hepes (pH 7.4), and 10 mmol/liter glucose (Ext Sol A). Ca²⁺-free solution was prepared by substituting MgCl₂ for CaCl₂ of external solution A and was supplemented with 2 mmol/liter 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⁺ 4.8 solution contained 135 mmol/liter N-methyl-D-glucamine, 4.8 mmol/liter KCl, 2.5 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 5 mmol/liter Hepes (pH adjusted to 7.4 with 131 mmol/liter HCl), and 10 mmol/liter glucose (Ext Sol D). When needed, Na⁺-K⁺-free solution was prepared by substituting N-methyl-D-glucamine for KCl of external solution D (pH adjusted to 7.4 with 136 mmol/liter HCl) (Ext Sol E). For experiments during which the equilibrium potential for Na⁺ was fixed at 60 mV, the external solution contained 11 mmol/liter NaCl, 10 mmol/liter KCl, 180 mmol/liter N-methyl-D-glucamine, 2.5 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 5 mmol/liter Hepes (pH 7.4), 0.1 mmol/liter CdCl₂, 0.25 mmol/liter tolbutamide (pH adjusted to 7.4 with 172 mmol/liter HCl), and 10 mmol/liter glucose (Ext Sol F). For experiments during which the equilibrium potential for Na⁺ was fixed at 20 mV, the external solution contained 53 mmol/liter NaCl, 10 mmol/liter KCl, 100 mmol/liter N-methyl-D-glucamine, 2.5 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 5 mmol/liter Hepes (pH 7.4), 0.1 mmol/liter CdCl₂, 0.25 mmol/liter tolbutamide (pH adjusted to 7.4 with 96 mmol/liter HCl), and 10 mmol/liter glucose (Ext Sol G). For recordings of I_Ca, the external solution contained 125 mmol/liter NaCl, 4.8 mmol/liter KCl, 10 mmol/liter CaCl₂, 1.2 mmol/liter MgCl₂, 10 mmol/liter tetraethylammonium-Cl, 5 mmol/liter Hepes (pH 7.4), and 10 mmol/liter glucose (Ext Sol H).

Thapsigargin was obtained from Alomone Labs (Jerusalem, Israel). Unless otherwise stated, all of the other chemicals were from Sigma.

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 < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of ACh on the Membrane Potential of Mouse Pancreatic -Cells-- In the presence of 10 mmol/liter glucose, -cells within an islet display a rhythmic electrical activity characterized by alternating polarized silent phases and depolarized phases with bursts of action potentials (Fig. 1). The addition of 1 µmol/liter ACh induced a sustained and persistent depolarization with continuous spike activity in two of six islets. In the other islets, the initial period of sustained activity was followed by rapid oscillations of the membrane potential (Fig. 1). Similar effects of ACh on the -cell electrical activity have previously been observed in noncultured islets and are blocked by atropine (12, 18).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of ACh on the membrane potential of a mouse -cell. An isolated islet was perifused with a medium containing 10 mmol/liter glucose (G10) and stimulated with 1 µmol/liter ACh as indicated. This recording is representative of results obtained in six islets.

Muscarinic Receptor Activation Induces an Inward Current in -Cells-- The effect of ACh on the whole cell current was first studied in single -cells held hyperpolarized at 80 mV by the conventional whole cell configuration of the patch-clamp technique. Addition of ACh induced a sustained and reversible inward current, the amplitude of which increased with the concentration of the neurotransmitter (Fig. 2, A-D) to reach a maximum of 0.77 ± 0.15 pA/pF (n = 5) at 100 µmol/liter ACh. The half-maximal effective concentration (EC₅₀) estimated after fitting the data to a sigmoidal function was at 2.5 µmol/liter 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 developed rapidly, within ~1 s (Figs. 2B and 3G), in cells that were located very close to the inflow of solution and more slowly (Fig. 3, B and D) in cells that were located at some distance of this inflow.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   ACh induces an inward current by activating muscarinic receptors in mouse -cells. Single -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 under "Experimental Procedures." ACh and atropine were applied when indicated by the arrows. A-D, ACh induced a concentration-dependent inward current that was reversed or blocked by atropine. A-C are representative of three (A), three (B), and four (C) experiments. D shows the concentration-dependence of ACh-induced inward current. The values are the means ± S.E. of the amplitude of the current recorded in three to five cells for each ACh concentration. Fitting data points to a sigmoidal function yielded a half-maximal effective concentration (EC50) of 2.5 µmol/liter. E, pretreatment of intact -cells by thapsigargin (30 min, 1 µmol/liter) did not prevent ACh from activating an inward current. This trace is representative of five experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Characteristics of the inward current activated by ACh in mouse -cells. Single -cells were voltage-clamped at 80 mV using the conventional whole cell mode of the patch-clamp technique. The composition of the external (Ext Sol) and pipette solutions (Int Sol) used for these experiments is described under "Experimental Procedures." For the sake of clarity, their main characteristics are summarized on the left side of each panel. ACh (100 µmol/liter) was applied when indicated by the arrows. A and B, ACh induced an inward current in the absence of Ca2+ in the external and pipette solutions (Caout 0, Cain 0) (A) or when the Na+/K+ pump was blocked by removal of K+ from the external solution (Kout 0) (B). C and D, the inward current activated by ACh was abrogated by Na+ omission from the medium (Naout 0) (C) but unaffected by inhibition of voltage-dependent Na+ channels with 2 µmol/liter tetrodotoxin (D). E and F, ACh failed to induce an outward current when the driving force for Na+ was directed outwardly (Naout 0; Nain-rich) (E) or when the driving force for K+ was directed outwardly (Kout 0; Kin-rich) and, simultaneously, no Na+ current could occur (Naout 0; Nain 0) (F). G, 100 µmol/liter DIDS did not affect the inward current elicited by ACh. The traces are representative of at least five experiments.

The current elicited by ACh was completely suppressed or prevented by the muscarinic receptor antagonist, atropine (Fig. 2, A-C), but was not mimicked by 10 µmol/liter nicotine (n = 5, not shown), and was insensitive to nicotine or nicotinic antagonists. Thus, in the presence of 10 µmol/liter nicotine, 0.1 µmol/liter -bungarotoxin, or 100 µmol/liter hexamethonium, 100 µmol/liter ACh elicited a current that 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 -cells results from activation of muscarinic, but not nicotinic, receptors.

In another series of experiments, -cells were voltage-clamped in the perforated whole cell mode and treated with 1 µmol/liter thapsigargin, which completely emptied the endoplasmic reticulum in Ca²⁺, as indicated by the suppression of Ca²⁺ 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 -Cells-- The ionic specificity of the current was first evaluated in the standard whole cell mode by removing Ca²⁺, Na⁺, or K⁺ from the bath or pipette solutions. Addition of ACh to a Ca²⁺-free medium elicited an inward current, indicating that the latter was not carried by Ca²⁺ (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 nonspecific 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 Cd²⁺ to block voltage-dependent Ca²⁺ channels (and avoid [Ca²⁺]_c overload or activation of Ca²⁺-dependent currents) and tolbutamide to block K⁺-ATP channels (and avoid a large outward current through these channels). However, even under these conditions, the 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 -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 100 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 whether 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.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   The current activated by ACh in mouse -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 -cells were voltage-clamped in conventional whole cell mode at various potentials (130, 100, 60, 30, and 0 mV when ENa+ was set at 60 mV and 100, 60, 20, and 0 mV when ENa+ was set at 20 mV) around the reversal potential for Na+. Each point shows the mean ± S.E. of the current amplitude elicited by 100 µmol/liter of ACh at each potential in three to five cells.

An increase in Cl permeability is expected to depolarize the plasma membrane because the equilibrium potential for Cl in -cells has been estimated to be above the threshold for activation of voltage-dependent Ca²⁺ 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 mV in Figs. 3 (C and F) 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 -cells (22). Thus, in the presence of 100 µmol/liter DIDS, 100 µmol/liter 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 -cells are known or assumed to be transduced by a G protein, and it has been reported that in neurons and cardiomyocytes, several muscarinic receptor subtypes modulate ionic channels, such as Ca²⁺ and K⁺ channels, through pertussis toxin-sensitive 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 -cells with pertussis toxin (250 ng/ml), the amplitude of I_Na-ACh was 109 ± 7% (n = 6) of that observed in untreated 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--S or GDP--S, which are, respectively, nonhydrolyzable activator and inhibitor of G proteins. Fig. 5 shows the maximum inward current (I_Na-ACh) elicited by a 1-min application of 100 µmol/liter ACh to -cells voltage-clamped at 80 mV and dialyzed for 5 min with a solution containing 100 µmol/liter GTP, 10 µmol/liter GTP--S, or 4 mmol/liter GDP--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.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Activation of INa-ACh in mouse -cells does not involve G proteins. Single mouse -cells were voltage-clamped at 80 mV and dialyzed with a pipette solution (Int Sol B; see "Experimental Procedures" for compositions) containing 100 µmol/liter GTP (Control), 10 µmol/liter GTP--S, or 4 mmol/liter GDP--S. Each column represents the mean ± S.E. of the current amplitude elicited by 100 µmol/liter ACh in nine (Control), five (GTP--S), and four (GDP--S) cells.

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 Ca²⁺ current in pancreatic -cells (23). Fig. 6A shows representative whole cell Ca²⁺ current traces recorded with a pipette solution containing 100 µmol/liter GTP. The current (I_Ca) was evoked by 25-ms depolarizing pulses from 80 to 10 mV before (control), during (ACh), and after (wash) application of 100 µmol/liter ACh to the bath. Fig. 6B summarizes the changes of the normalized peak Ca²⁺ current produced by ACh in the presence of different guanine nucleotides in the pipette solution. With 100 µmol/liter GTP in the pipette solution (control), ACh reversibly inhibited the current. This inhibitory effect was abolished when the pipette solution contained 4 mmol/liter GDP--S and became irreversible when 10 µmol/liter GTP--S was included in the pipette. The spontaneous decrease in the current amplitude recorded with GDP--S reflects rundown (23). These control experiments thus show that the guanine nucleotides were effective in our recording conditions.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Inhibition of ICa by ACh in mouse -cells involves G proteins. Single -cells were dialyzed with a pipette solution (Int Sol G; see "Experimental Procedures" for compositions) containing 100 µmol/liter GTP (Control), 10 µmol/liter GTP--S, or 4 mmol/liter GDP--S, and submitted to a 25-ms depolarization to +10 mV from a holding potential of 80 mV. A, representative voltage-dependent Ca2+ currents recorded with a pipette solution containing 100 µmol/liter 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 ICa recorded with different guanine nucleotides in the pipette solution. Open squares, GTP; closed triangles, GTP--S; closed circles, GDP--S. To facilitate comparisons, ICa was normalized in each individual experiment by dividing the peak current at each time by the maximum peak current at time 0. The traces are the means ± S.E. of results obtained in six cells for each experimental condition.

Impact of a Depolarizing Current Equivalent to I_Na-ACh on the -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 -cells perifused at 34-36 °C with 6 mmol/liter glucose, a concentration that is subthreshold in islets (24). A depolarizing current corresponding to the average I_Na-ACh induced by 100 µmol/liter ACh (0.77 pA/pF) and adjusted to cell size (0.77 pA × 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/liter ACh was first tested and compared with that of depolarizing currents corresponding to I_Na-ACh elicited by 1 (0.23 pA/pF), 10 (0.61 pA/pF), and 100 µmol/liter ACh (0.77 pA/pF). The addition of 1 µmol/liter ACh triggered 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/liter ACh (a in Fig. 7B) elicited electrical activity similar to that produced by 1 µmol/liter ACh itself. Injection of larger currents corresponding to I_Na-ACh induced by 10 and 100 µmol/liter ACh (b and c, respectively, in Fig. 7B) augmented the frequency of the electrical activity. These results indicate that the small I_Na-ACh is sufficient to depolarize the membrane potential beyond the threshold for the activation of voltage-dependent Ca²⁺ channels.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Injection of a depolarizing current mimics the ACh effects on the -cell membrane potential. The membrane potential of a single mouse -cell was monitored in current-clamp mode of the patch-clamp technique. The concentration of glucose of the medium was 6 mmol/liter 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/liter ACh (0.77 pA × 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/liter ACh. A current was then injected at increasing amplitudes corresponding to those of the inward currents elicited by 1 (0.23 pA × 8.7 pF = 2 pA in this cell; period a), 10 (0.61 × 8.7 pF = 5 pA; period b), and 100 µmol/liter ACh (0.77 × 8.7 pF = 7 pA; period c). Injection of the smallest current mimicked the effect of 1 µmol/liter ACh. These traces are representative of four (A) and five (B) experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

By activating muscarinic receptors, ACh induces a number of effects in pancreatic -cells that culminate in an increase in insulin secretion (10). Among these effects, a glucose-dependent 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 -cells. It further shows that the activation of the current is not mediated by G proteins.

Acetylcholine Activates an Inward Na⁺ Current in -Cells-- Our data demonstrate that ACh activates an inward current that is attributed to Na⁺ influx because of its 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 Ca²⁺, K⁺, or Cl to the current could be obtained. Thus, the ACh-induced current was not affected by removal of extracellular Ca²⁺. 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 -cells (22).

Activation of this Na⁺ current by ACh may explain the increase in total Na⁺ content (13), ²²Na⁺ uptake (12, 14), and [Na⁺]_c (15) that the neurotransmitter induces in islet cells and the abrogation of all of these effects in a Na⁺-free medium. The amplitude of the Na⁺ current elicited by ACh is small but is sufficient to account for the 15-mmol/liter increase in [Na⁺]_c occurring in -cells after 15 min of stimulation with 100 µmol/liter ACh (15, 25). Indeed, 100 µmol/liter ACh activated a mean inward current of 0.77 pA/pF, which corresponds to 6.15 pA for an average -cell capacitance of 7.9 ± 0.08 pF (estimated from 644 -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 fmol of Na⁺. For an intracellular space of 620 fl/-cell (26), this would result in a [Na⁺]_c increase by 92 mmol/liter, which is well above the 15 mmol/liter 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 -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 nonselective cationic channels (28, 29), classically mediate cholinergic effects on Na⁺ currents. Such channels are clearly not responsible for the ACh-induced inward current in -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 cells expressing M₃ receptors (35).

The channels activated by ACh in -cells have not been identified, but several of their properties could be established. Voltage-dependent Na⁺ channels are present in mouse -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 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 nonspecific cationic channel allowing flow of both Na⁺ and K⁺ or Ca²⁺. 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 Ca²⁺ content of the endoplasmic reticulum in -cells activates conductances for Ca²⁺ and perhaps other ionic species including Na⁺ (25, 39). Even if such a mechanism slightly contributes to the current, it is not responsible for I_Na-ACh, because ACh still activated the inward current after emptying of the endoplasmic reticulum Ca²⁺ stores by thapsigargin. This is consistent with our previous report that thapsigargin and cyclopiazonic acid, which empty the endoplasmic reticulum in Ca²⁺ 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 [Ca²⁺]_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 -cells with GTP--S or GDP--S, two guanine nucleotide analogues that, respectively, activate and inhibit G proteins. However, both analogues were effective in our experimental conditions as shown by their modulation of ACh-induced inhibition of the voltage-dependent Ca²⁺ current (23). These observations unexpectedly indicate that activation of I_Na-ACh does not involve G proteins in -cells. There is growing evidence that various seven-transmembrane metabotropic receptors can also activate transduction systems without the 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 inositol 1,4,5-trisphosphate receptors by the protein Homer (42) are two examples of G protein-independent events linked to activation of metabotropic glutamate receptor.

Role of I_Na-ACh in the Control of -Cell Membrane Potential by ACh-- Apart from the increase in Na⁺ conductance, all of the plausible mechanisms by which ACh might depolarize the -cell membrane can be excluded. First, ACh does not decrease the -cell membrane K⁺ conductance. Unlike glucose and sulfonylureas, ACh does not inhibit the efflux of ⁸⁶Rb⁺ (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 -cell membrane because of the high equilibrium potential for Cl (19, 20), is not involved. Thus, ACh has no effect on ⁸⁶Cl 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 Ca²⁺ channels. On the contrary, we confirm here that high concentrations of the neurotransmitter rather inhibit Ca²⁺ currents through voltage-dependent Ca²⁺ channels (23). It is possible, however, that the small capacitative Ca²⁺ entry induced by ACh, via a lowering of intracellular Ca²⁺ stores, slightly contributes to the depolarization. Fourth, blockade of the Na⁺/K⁺ pump is known to depolarize -cells (24). However, several arguments suggest that ACh does not inhibit the Na⁺/K⁺ pump. A depolarizing effect of ACh persisted after inhibition 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.² Moreover, ACh slightly increased initial ⁸⁶Rb⁺ 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 -cells. Our proposal is in complete agreement with the observation that this depolarization is abrogated by extracellular Na⁺ omission but insensitive to tetrodotoxin (12). 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/liter of ACh (i.e. 0.23 ± 0.02 pA/pF) elicited electrical activity in cells perifused with a subthreshold glucose concentration. Because the effect of a given current augments with the resistance of the membrane and because 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 Ca²⁺ channels eventually leads to a sustained increase in [Ca²⁺]_c that largely contributes to the insulin-releasing action of ACh (10).

	FOOTNOTES

^* This work was supported by Grant 3.4552.98 from the Fonds de la Recherche Scientifique Médicale (Brussels), Grant 1.5.121.00 from the Fonds National de la Recherche Scientifique (Brussels), Grant 2.4599.01 from the Fonds de la Recherche Fondamentale Collective (Brussels), Grant ARC 00/05-260 from the General Direction of Scientific Research of the French Community of Belgium, and the Interuniversity Poles of Attraction Program (P5/3-20), Federal Office for Scientific, Technical and Cultural Affairs of Belgium.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Senior Research associate of the Fonds National de la Recherche Scientifique (Brussels). To whom correspondence should be addressed: Unité d'Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, UCL 55.30, Av. Hippocrate 55, B-1200 Brussels, Belgium. Tel.: 32-2-764-95-79; Fax: 32-2-764-55-32; E-mail: gilon@endo.ucl.ac.be.

Published, JBC Papers in Press, August 2, 2002, DOI 10.1074/jbc.M203888200

² J.-F. Rolland, J.-C. Henquin, and P. Gilon, unpublished data.

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_c, free cytosolic calcium concentration; ACh, acetylcholine; K⁺-ATP channels, ATP-sensitive potassium channels; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; I_Na-ACh, Na⁺ current activated by ACh; [Na⁺]_c, free cytosolic sodium concentration; F, farad; GTP--S, guanosine-5'-O-(3-thiotriphosphate); GDP--S, guanosine-5'-O-(2-thiodiphosphate).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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