G protein-independent activation of an inward Na(+) current by muscarinic receptors in mouse pancreatic beta-cells.

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-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 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 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-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-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 beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.

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 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 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.
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 sig-nal, a rise in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] c ), 1 through the following sequence of events: the acceleration of cell metabolism increases the ATP/ADP ratio, which closes ATP-sensitive K ϩ (K ϩ -ATP) channels in the plasma membrane; the resulting decrease in K ϩ conductance leads to membrane depolarization, opening of voltage-dependent Ca 2ϩ channels, and Ca 2ϩ influx (1)(2)(3)(4)(5). Glucose also produces amplifying signals that increase the efficacy of Ca 2ϩ 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 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, which mobilizes Ca 2ϩ from intracellular Ca 2ϩ stores. The resulting fall of the Ca 2ϩ concentration in the endoplasmic reticulum activates a modest Ca 2ϩ influx, through voltage-independent Ca 2ϩ channels, which is commonly referred to as a capacitative Ca 2ϩ entry. In addition, ACh depolarizes the plasma membrane of ␤-cells. This depolarization is small and does not cause Ca 2ϩ influx in unstimulated ␤-cells. However, in the presence of stimulatory (depolarizing) concentrations of glucose, this additional depolarization by ACh enhances Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels, leading to a sustained [Ca 2ϩ ] 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 ϩ current is involved. Thus, the depolarization of ␤-cells by ACh is abrogated by omission of extracellular Na ϩ (12) and accompanied by increases in total Na ϩ content (13), 22 Na ϩ uptake (12,14), and free cytosolic Na ϩ concentration ([Na ϩ ] c ) (15). These arguments, however, remain indirect and conflict with the general concept that nicotinic rather than muscarinic receptors mediate cholinergic effects on Na ϩ 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 electrophysi- ological evidence for muscarinic, G protein-independent stimulation of an inward Na ϩ current in pancreatic ␤-cells.

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 120 mmol/liter NaCl, 4.8 mmol/ liter KCl, 2.5 mmol/liter CaCl 2 , 1.2 mmol/liter MgCl 2 , 24 mmol/liter NaHCO 3 , 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 O 2 /CO 2 (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 Ca 2ϩ -free medium containing 138 mmol/liter NaCl, 5.6 mmol/liter KCl, 1.2 mmol/liter MgCl 2 , 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 g/ml streptomycin.
Electrophysiological Recordings-The membrane potential of a single ␤-cell within an islet was continuously recorded at 37°C with a high resistance (ϳ200 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 ϩ current that is inactivated at a holding potential of Ϫ70 mV but can be activated after a hyperpolarizing pulse to Ϫ140 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 Ϫ80 mV. I Ca was measured by applying 25-ms depolarizations from Ϫ80 mV to ϩ10 mV every 5 s. Voltageclamp experiments were performed at room temperature (22-25°C), whereas current-clamp experiments were carried out at 34 -36°C.
Various solutions were used for patch-clamp recordings. In the perforated mode, the pipette solution contained 70 mmol/liter K 2 SO 4 , 10 mmol/liter NaCl, 10 mmol/liter KCl, 3.7 mmol/liter MgCl 2 , 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 Me 2 SO) was used at a final concentration of 300 g/ml. Nystatin (stock solution of 10 mg/ml in Me 2 SO) was used at a final concentration of 200 g/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 Ͼ35-40 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 MgCl 2 , 3 mmol/liter MgATP, 0.1 mmol/liter Na 2 GTP, 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 ϩ -rich/K ϩ -free solution was prepared by substituting NaCl for KCl and NaOH for KOH of internal solution B (Int Sol D). Na ϩ -free/K ϩ -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 ϩ was fixed at Ϫ60 or Ϫ20 mV, the pipette solution contained 107 mmol/liter NaCl, 10 mmol/liter NaOH, 3 mmol/ liter MgATP, 0.1 mmol/liter Na 2 GTP, 1 mmol/liter MgCl 2 , 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 MgCl 2 , 10 mmol/ liter EGTA, 3 mmol/liter MgATP, 0.1 mmol/liter Na 2 GTP, 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.
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 Ͻ 0.05.

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 mol/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).
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 Ϫ80 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 Ϯ 0.15 pA/pF (n ϭ 5) at 100 mol/liter ACh. The half-maximal effective concentration (EC 50 ) estimated after fitting the data to a sigmoidal function was at 2.5 mol/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 ϳ0.8 ml and flow rate of ϳ0.5 ml/min) preclude fast solution exchange. However, it was repeatedly noted that the current activated by ACh developed rapidly, within ϳ1 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.
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 mol/liter nicotine (n ϭ 5, not shown), and was insensitive to nicotine or nicotinic antagonists. Thus, in the presence of 10 mol/liter nicotine, 0.1 mol/liter ␣-bungarotoxin, or 100 mol/liter hexamethonium, 100 mol/liter ACh elicited a current that was, respectively, 90 Ϯ 8% (n ϭ 11), 111 Ϯ 11% (n ϭ 6), or 107 Ϯ 6% (n ϭ 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 voltageclamped in the perforated whole cell mode and treated with 1 mol/liter thapsigargin, which completely emptied the endoplasmic reticulum in Ca 2ϩ , as indicated by the suppression of Ca 2ϩ mobilization by ACh (n ϭ 5; not shown). Subsequent application of ACh elicited a current of the same amplitude (0.77 Ϯ 0.09 pA/pF, n ϭ 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 Ca 2ϩ , Na ϩ , or K ϩ from the bath or pipette solutions. Addition of ACh to a Ca 2ϩ -free medium elicited an inward current, indicating that the latter was not carried by Ca 2ϩ (Fig. 3A). Omission of K ϩ from the perifusion medium did not prevent the current elicited by ACh, indicating that the current does not involve changes in Na ϩ pump activity (Fig. 3B). By contrast, omission of extracellular Na ϩ abrogated the current, which suggests that it is carried by Na ϩ (Fig. 3C). The current was nevertheless insensitive to tetrodotoxin, indicating that voltage-gated Na ϩ channels are not involved (Fig. 3D). When the electrochemical gradient for Na ϩ was reversed (Na ϩ -rich pipette solution and Na ϩ -free bath medium) to permit an outward current, ACh was ineffective (Fig. 3E), suggesting that Na ϩ flows only in the inward direction.
A nonspecific cationic current carrying both Na ϩ and K ϩ 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 ϩ (Fig. 3C) or no K ϩ 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 ϩ was omitted from both pipette and bath solutions, whereas K ϩ was present at a high concentration in the pipette solution only (Fig. 3F). Under these conditions where the equilibrium potential for K ϩ was infinitely negative and no Na ϩ current could occur, ACh did not activate any outward current. In the second series of experiments, the Na ϩ versus K ϩ 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 ϩ and K ϩ , i.e. Ϫ60 or Ϫ20 mV for Na ϩ , and infinitely positive potentials for K ϩ . These experi- ments were performed in the presence of Cd 2ϩ to block voltagedependent Ca 2ϩ channels (and avoid [Ca 2ϩ ] c overload or activation of Ca 2ϩ -dependent currents) and tolbutamide to block K ϩ -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 Ϫ130 and 0 mV (Fig. 4). Experiments at potentials more negative than Ϫ130 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 ϩ at Ϫ60 mV. At potentials more negative than Ϫ60 mV, the amplitude of the inward current induced by ACh increased with the driving force for Na ϩ (larger at Ϫ130 than Ϫ100 mV) and displayed a slope conductance of 6.6 pS/pF (Fig. 4, open squares). At the set equilibrium potential for Na ϩ (Ϫ60 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 ϩ , the equilibrium potential of Na ϩ was then fixed at Ϫ20 mV. At this potential, ACh failed to activate any current. However, at potentials more negative than Ϫ20 mV, ACh induced an inward current with an amplitude proportional to the driving force for Na ϩ and with a slope conductance of 5.3 pS/pF (Fig. 4, filled circles). All these results clearly indicate selectivity for Na ϩ without contribution of K ϩ . 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 ϩ (Ϫ30 and 0 mV when E Na was fixed at Ϫ60 mV; 0 mV when E Na was fixed at Ϫ20 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 Ϫ permeability is expected to depolarize the plasma membrane because the equilibrium potential for Cl Ϫ in ␤-cells has been estimated to be above the threshold for activation of voltage-dependent Ca 2ϩ channels (19,20). Activation of a Cl Ϫ current by ACh has not been directly tested by omitting Cl Ϫ 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 Ϫ and under conditions where no Na ϩ current occurred (e.g. at holding potentials of Ϫ80, Ϫ80, and Ϫ60 mV in Figs. 3 (C and F) and 4 (open squares), when E Cl was at, Ϫ6, 0, and Ϫ14 mV, respectively). Second, I Na-ACh was unaffected by DIDS, a blocker of the volume-activated current (21) that carries Cl Ϫ and possibly other ions in ␤-cells (22). Thus, in the presence of 100 mol/liter DIDS, 100 mol/liter ACh elicited a current that was 112 Ϯ 10% (n ϭ 14) of 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 mol/liter) was applied when indicated by the arrows. A and B, ACh induced an inward current in the absence of Ca 2ϩ in the external and pipette solutions (Ca out 0, Ca in 0) (A) or when the Na ϩ /K ϩ pump was blocked by removal of K ϩ from the external solution (K out 0) (B). C and D, the inward current activated by ACh was abrogated by Na ϩ omission from the medium (Na out 0) (C) but unaffected by inhibition of voltage-dependent Na ϩ channels with 2 mol/liter tetrodotoxin (D). E and F, ACh failed to induce an outward current when the driving force for Na ϩ was directed outwardly (Na out 0; Na in -rich) (E) or when the driving force for K ϩ was directed outwardly (K out 0; K in -rich) and, simultaneously, no Na ϩ current could occur (Na out 0; Na in 0) (F). G, 100 mol/liter DIDS did not affect the inward current elicited by ACh. The traces are representative of at least five experiments.

FIG. 4. The current activated by ACh in mouse ␤-cells is rectifying inwardly.
The reversal potential for Na ϩ was fixed at Ϫ60 mV (open squares) or Ϫ20 mV (closed circles) by using external (Ext Sol) and pipette solutions (Int Sol) with appropriate concentrations of Na ϩ (see "Experimental Procedures" for compositions). Single ␤-cells were voltage-clamped in conventional whole cell mode at various potentials (Ϫ130, Ϫ100, Ϫ60, Ϫ30, and 0 mV when E Na ϩ was set at Ϫ60 mV and Ϫ100, Ϫ60, Ϫ20, and 0 mV when E Na ϩ was set at Ϫ20 mV) around the reversal potential for Na ϩ . Each point shows the mean Ϯ S.E. of the current amplitude elicited by 100 mol/liter of ACh at each potential in three to five cells. 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 Ca 2ϩ and K ϩ 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 Ϯ 7% (n ϭ 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 mol/liter ACh to ␤-cells voltage-clamped at Ϫ80 mV and dialyzed for 5 min with a solution containing 100 mol/liter GTP, 10 mol/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.
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 2ϩ current in pancreatic ␤-cells (23). Fig. 6A shows representative whole cell Ca 2ϩ current traces recorded with a pipette solution containing 100 mol/liter GTP. The current (I Ca ) was evoked by 25-ms depolarizing pulses from Ϫ80 to 10 mV before (control), during (ACh), and after (wash) application of 100 mol/liter ACh to the bath. Fig. 6B summarizes the changes of the normalized peak Ca 2ϩ current produced by ACh in the presence of different guanine nucleotides in the pipette solution. With 100 mol/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 mol/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. 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 mol/liter ACh (0.77 pA/pF) and adjusted to cell size (0.77 pA ϫ 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 mol/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 mol/liter ACh (0.77 pA/pF). The addition of 1 mol/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 mol/liter ACh (a in Fig. 7B) elicited electrical activity similar to that produced by 1 mol/ liter ACh itself. Injection of larger currents corresponding to I Na-ACh induced by 10 and 100 mol/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 Ca 2ϩ channels. DISCUSSION 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 ϩ current underlies the ACh-mediated depolarization (12). This proposal has, however, remained incompletely convincing because Na ϩ 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 ϩ 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 ϩ Current in ␤-Cells-Our data demonstrate that ACh activates an inward current that is attributed to Na ϩ influx because of its suppression either when Na ϩ was removed from the extracellular medium or when the membrane potential of the cell was close to the equilibrium potential of Na ϩ . On the other hand, no contribution of Ca 2ϩ , K ϩ , or Cl Ϫ to the current could be obtained. Thus, the ACh-induced current was not affected by removal of extracellular Ca 2ϩ . When no Na ϩ current could occur, no inward or outward current was evoked by ACh, even when the equilibrium potential of K ϩ was infinitely negative or positive or when the membrane potential was clamped away from the equilibrium potential of Cl Ϫ . The current elicited by ACh was also insensitive to DIDS, a blocker of the Cl Ϫ -mediated, volumeactivated current in ␤-cells (22).
Activation of this Na ϩ current by ACh may explain the increase in total Na ϩ content (13), 22 Na ϩ uptake (12,14), and [Na ϩ ] c (15) that the neurotransmitter induces in islet cells and the abrogation of all of these effects in a Na ϩ -free medium. The amplitude of the Na ϩ current elicited by ACh is small but is sufficient to account for the 15-mmol/liter increase in [Na ϩ ] c occurring in ␤-cells after 15 min of stimulation with 100 mol/ liter ACh (15,25). Indeed, 100 mol/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 Ϯ 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 ϳ57 fmol of Na ϩ . For an intracellular space of 620 fl/␤-cell (26), this would result in a [Na ϩ ] c increase by 92 mmol/liter, which is well above the 15 mmol/liter measured. Activation of the Na ϩ pump obviously tends to correct the [Na ϩ ] c rise. The reverse mechanism, an inhibition of the Na ϩ pump, has been proposed to explain the increase in [Na ϩ ] c produced by ACh in sheep Purkinje fibers (27). The explanation does not hold for ␤-cells in which ACh still increases [Na ϩ ] c after blockade of the pump by ouabain (15). This is fully compatible with the persistence of the AChinduced inward current in a K ϩ -free medium, another situation where the Na ϩ pump is blocked.
Nicotinic receptors, which are nonselective cationic channels (28,29), classically mediate cholinergic effects on Na ϩ currents. Such channels are clearly not responsible for the AChinduced inward current in ␤-cells because the muscarinic antagonist atropine completely prevented the current and the rise in [Na ϩ ] c (15), whereas the ACh-activated current was not mimicked by nicotine and was insensitive to nicotinic antagonists. Activation of a Na ϩ 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 M 3 receptors (35).
The channels activated by ACh in ␤-cells have not been identified, but several of their properties could be established. Voltage-dependent Na ϩ channels are present in mouse ␤-cells, but they are inactivated at the holding potential of Ϫ80 mV that we used (36). We can discard the possibility that such channels mediate the effect of ACh because the Na ϩ current evoked by the neurotransmitter did not require any voltage change and was insensitive to tetrodotoxin, as are the AChinduced increases in Na ϩ uptake (12) and [Na ϩ ] c (15). Our results also indicate that the current is not carried by a nonspecific cationic channel allowing flow of both Na ϩ and K ϩ or Ca 2ϩ . The Na ϩ channels activated by ACh display inward rectifying properties as shown by their inability to carry an 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 mol/liter ACh (0.77 pA ϫ 10.4 pF ϭ 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 mol/liter ACh. A current was then injected at increasing amplitudes corresponding to those of the inward currents elicited by 1 (0.23 pA ϫ 8.7 pF ϭ 2 pA in this cell; period a), 10 (0.61 ϫ 8.7 pF ϭ 5 pA; period b), and 100 mol/liter ACh (0.77 ϫ 8.7 pF ϭ 7 pA; period c). Injection of the smallest current mimicked the effect of 1 mol/liter ACh. These traces are representative of four (A) and five (B) experiments.