1

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

BY MUSCARINIC RECEPTORS IN MOUSE PANCREATIC ββββ-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;

[Ca2+]c, free cytosolic calcium concentration; DIDS, 4,4'-diisothiocyanostilbene-2,2'-

disulfonic acid; INa-ACh, Na+ current activated by ACh; IP3, inositol 1,4,5-trisphosphate;

[Na+]c, free cytosolic sodium concentration; PTX, pertussis toxin.

Address all correspondence toDr P. GilonUnité d’Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine,UCL 55.30, Av. Hippocrate 55,B-1200 Brussels, Belgium.Tel: + 32-2-764.94.33.Fax: + 32-2-764.55.32.E.mail: gilon@endo.ucl.ac.be

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 2, 2002 as Manuscript M203888200 by guest on M

arch 22, 2016http://w

ww

.jbc.org/D

ownloaded from

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 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 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.

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 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 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 M3 type, ACh

triggers changes in phospholipid metabolism leading to formation of diacylglycerol which

activates protein kinase C, and inositol 1,4,5-trisphosphate (IP3) 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 β-cells. This depolarization is small and does not cause Ca2+ influx in

unstimulated β-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).

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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), 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 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.

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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

CaCl2, 1.2 MgCl2, 24 NaHCO3, 5 Hepes, 10 glucose, 1 mg/ml bovine serum albumin (BSA;

fraction V; Roche Molecular Biochemicals, Mannheim, Germany), and gassed with O2/CO2

(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 Ca2+-free medium containing (in mmol/l)

138 NaCl, 5.6 KCl, 1.2 MgCl2, 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,

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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, INa-ACh was measured in cells kept hyperpolarized at -80 mV. ICa 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 (in mmol/l): 122 NaCl, 4.7 KCl, 2.6 CaCl2, 1.2 MgCl2, 20

NaHCO3, 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 O2/CO2 (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 K2SO4, 10 NaCl, 10 KCl, 3.7 MgCl2 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 INa-ACh, the pipette solution contained (in

mmol/l): 112 KCl, 5 KOH, 1 MgCl2, 3 MgATP, 0.1 Na2GTP and 10 HEPES (pH adjusted to

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

7

7.15 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 Na2GTP, 1 MgCl2 and 10 HEPES

(pH adjusted to 7.15 with 7 mmol/l HCl) (Int Sol F). For conventional whole-cell recordings

of ICa, the pipette solution contained (in mmol/l): 125 CsCl, 30 KOH, 1 MgCl2, 10 EGTA, 3

MgATP, 0.1 Na2GTP, 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 INa-ACh contained (in mmol/l): 120

NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, 24 NaHCO3, 5 HEPES (pH 7.4) and 10 glucose (Ext

Sol A). Ca2+-free solution was prepared by substituting MgCl2 for CaCl2 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+ 4.8 solution contained (in mmol/l): 135 N-methyl-D-glucamine (NMDG), 4.8 KCl,

2.5 CaCl2, 1.2 MgCl2, 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-D-

glucamine 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 CaCl2, 1.2 MgCl2,

5 HEPES (pH 7.4), 0.1 CdCl2, 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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

8

NMDG, 2.5 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4), 0.1 CdCl2, 0.25 tolbutamide (pH adjusted

to 7.4 with 96 mmol/l HCl) and 10 glucose (Ext Sol G). For recordings of ICa, the external

solution contained (in mmol/l): 125 NaCl, 4.8 KCl, 10 CaCl2, 1.2 MgCl2, 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 < 0.05.

RESULTS

Effects of ACh on the membrane potential of mouse pancreatic ββββ-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 ββββ-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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 (EC50) 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 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 ββββ-cells

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

10

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 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 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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 -

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 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 INa-ACh. At potentials less negative than the set equilibrium potential for Na+ (-30

and 0 mV when ENa was fixed at -60 mV; 0 mV when ENa 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 INa-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 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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

12

mV in Fig. 3C, 3F and 4 open squares, when ECl was at, -6, 0 and -14 mV, respectively).

Second, INa-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 INa-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 Ca2+ and K+ channels, through pertussis toxin-

sensitive G-proteins of the Gi or Go class (10). However, after permanent inactivation of Gi-

or Go-proteins by overnight pretreatment of β-cells with pertussis toxin (250 ng/ml), the

amplitude of INa-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 INa-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 (INa-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, INa-ACh was reversible upon washout of the

neurotransmitter, and its amplitude was similar with the three nucleotides suggesting that

activation of INa-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 Ca2+ current in pancreatic β-cells (23). Fig. 6A shows representative

whole-cell Ca2+ current traces recorded with a pipette solution containing 100 µmol/l GTP.

The current (ICa) was evoked by 25 ms depolarizing pulses from -80 to 10 mV before

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 Ca2+ 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 INa-ACh on the ββββ-cell membrane potential

Because INa-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

34-36°C with 6 mmol/l glucose, a concentration that is subthreshold in islets (24). A

depolarizing current corresponding to the average INa-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 INa-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 INa-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 INa-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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

14

indicate that the small INa-ACh is sufficient to depolarize the membrane potential beyond the

threshold for the activation of voltage-dependent Ca2+ channels.

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 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 β-cells. It further shows that the activation of the current is not mediated by G-

proteins.

Acetylcholine activates an inward Na+ current in ββββ-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 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 β-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 these effects in a Na+-free medium. The amplitude of the Na+ current

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 M3 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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 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

INa-ACh.

Mechanisms of activation of INa-ACh

As in other cells (37, 38), lowering the Ca2+ content of the endoplasmic reticulum in β-cells

activates conductances for Ca2+ and perhaps other ionic species including Na+ (25, 39). Even

if such a mechanism slightly contributes to, it is not responsible for INa-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 INa-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 INa-ACh was unaffected by inactivation of Gi/Go-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 Ca2+ current (23). These observations unexpectedly

indicate that activation of INa-ACh does not involve G-proteins in β-cells. There is growing

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 IP3 receptors by the

protein Homer (42) are two examples of G-protein-independent event linked to activation of

metabotropic glutamate receptor.

Role of INa-ACh in the control of ββββ-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 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 β-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 β-cells (24). However, several arguments suggest that

ACh does not inhibit the Na+/K+ pump. A depolarizing effect of ACh persisted after inhibition

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

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 86Rb+ uptake

(14), which is opposite to the effect observed after blockade of the pump with ouabain (48,

49).

Activation of INa-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 INa-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. 0.23 ± 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 Ca2+ channels eventually leads to a sustained

increase in [Ca2+]c that largely contributes to the insulin-releasing action of ACh (10).

Acknowledgments: 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 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.

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

20

1. Ashcroft, F. M., and Rorsman, P. (1989) Prog. Biophys. Mol. Biol. 54, 87-143

2. Gilon, P., Ravier, M. A., Jonas, J. C., and Henquin, J. C. (2002) Diabetes 51 (Suppl 1),

S144-S151

3. Maechler, P., and Wollheim, C. B. (2001) Nature 414, 807-812

4. Matschinsky, F. M. (1996) Diabetes 45, 223-241

5. Seino, S., Iwanaga, T., Nagashima, K., and Miki, T. (2000) Diabetes 49, 311-318

6. Aizawa, T., Komatsu, M., Asanuma, N., Sato, Y., and Sharp, G. W. (1998) Trends

Pharmacol. Sci. 19, 496-499

7. Henquin, J. C. (2000) Diabetes 49, 1751-1760

8. Ahren, B. (2000) Diabetologia 43, 393-410

9. Brunicardi, F. C., Sun, Y. S., Druck, P., Goulet, R. J., Elahi, D., and Andersen, D. K.

(1987) Am. J. Surg. 153, 34-40

10. Gilon, P., and Henquin, J. C. (2001) Endocr. Rev. 22, 565-604

11. Satin, L. S., and Kinard, T. A. (1998) Endocrine 8, 213-223

12. Henquin, J. C., Garcia, M. C., Bozem, M., Hermans, M. P., and Nenquin, M. (1988)

Endocrinology 122, 2134-2142

13. Saha, S., and Hellman, B. (1991) Eur. J. Pharmacol. 204, 211-215

14. Gagerman, E., Sehlin, J., and Taljedal, I. B. (1980) J. Physiol. 300, 505-513

15. Gilon, P., and Henquin, J. C. (1993) FEBS Lett. 315, 353-356

16. Meissner, H. P. (1990) Methods Enzymol. 192, 235-246

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

21

17. Rolland, J. F., Henquin, J. C., and Gilon, P. (2002) Diabetes 51, 376-384

18. Gilon, P., Nenquin, M., and Henquin, J. C. (1995) Biochem. J. 311, 259-267

19. Eberhardson, M., Patterson, S., and Grapengiesser, E. (2000) Cell Signal. 12, 781-786

20. Sehlin, J. (1978) Am. J. Physiol. 235, E501-E508

21. Best, L., Brown, P. D., Sheader, E. A., and Yates, A. P. (2000) J. Membr. Biol. 177,

169-175

22. Kinard, T. A., and Satin, L. S. (1995) Diabetes 44, 1461-1466

23. Gilon, P., Yakel, J., Gromada, J., Zhu, Y., Henquin, J. C., and Rorsman, P. (1997) J.

Physiol. 499, 65-76

24. Henquin, J. C., and Meissner, H. P. (1982) J. Physiol. 332, 529-552

25. Miura, Y., Gilon, P., and Henquin, J. C. (1996) Biochem. Biophys. Res. Commun. 224,

67-73

26. Heimberg, H., De Vos, A., Pipeleers, D., Thorens, B., and Schuit, F. (1995) J. Biol.

Chem. 270, 8971-8975

27. Iacono, G., and Vassalle, M. (1989) Am. J. Physiol. 256, H1407-H1416

28. Hosey, M. M. (1992) FASEB J. 6, 845-852

29. Hucho, F. (1986) Eur. J. Biochem. 158, 211-226

30. Matsumoto, K., and Pappano, A. J. (1989) J. Physiol. 415, 487-502

31. Shirayama, T., Matsumoto, K., and Pappano, A. J. (1993) J. Pharmacol. Exp. Ther. 265,

641-648

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

22

32. Inoue, R., and Isenberg, G. (1990) Am. J. Physiol. 258, C1173-C1178

33. Benham, C. D., Bolton, T. B., and Lang, R. J. (1985) Nature 316, 345-347

34. Inoue, M., Sakamoto, Y., and Imanaga, I. (1995) Eur. J. Pharmacol. 276, 123-129

35. Carroll, R. C., and Peralta, E. G. (1998) EMBO J. 17, 3036-3044

36. Plant, T. D. (1988) Pflugers Arch. 411, 429-435

37. Putney, J. W., Jr. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14669-14671

38. Tepel, M., Wischniowski, H., and Zidek, W. (1994) Biochim. Biophys. Acta 1220, 248-

252

39. Worley, J. F., III, McIntyre, M. S., Spencer, B., and Dukes, I. D. (1994) J. Biol. Chem.

269, 32055-32058

40. Wess, J., Blin, N., Mutschler, E., and Bluml, K. (1995) Life Sci. 56, 915-922

41. Heuss, C., and Gerber, U. (2000) Trends Neurosci. 23, 469-475

42. Brzostowski, J. A., and Kimmel, A. R. (2001) Trends Biochem. Sci. 26, 291-297

43. Guerineau, N. C., Bossu, J. L., Gahwiler, B. H., and Gerber, U. (1995) J. Neurosci. 15,

4395-4407

44. Olesen, S. P., Davies, P. F., and Clapham, D. E. (1988) Circ. Res. 62, 1059-1064

45. Brown, L. D., Kim, K. M., Nakajima, Y., and Nakajima, S. (1993) Brain Res. 612, 200-

209

46. Nenquin, M., Awouters, P., Mathot, F., and Henquin, J. C. (1984) FEBS Lett. 176, 457-

461

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

23

47. Hermans, M. P., Schmeer, W., Gerard, M., and Henquin, J. C. (1991) Biochim. Biophys.

Acta 1092, 205-210

48. Sehlin, J., and Taljedal, I. B. (1974) J. Physiol. 242, 505-515

49. Henquin, J. C. (1980) Biochem. J. 186, 541-550

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

24

FIGURE LEGENDS

Fig.1. Effects of ACh on the membrane potential of mouse ββββ-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 ββββ-

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 concentration-

dependent 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 concentration-

dependence 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 (EC50) 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 ββββ-cells. Single β-

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 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

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-D: The inward current

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

25

activated by ACh was abrogated by Na+ omission fom the medium (Naout 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 (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). 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 ββββ-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 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.M of the current amplitude

elicited by 100 µmol/l of ACh at each potential in 3 to 5 cells.

Fig.5: Activation of INa-ACh in mouse ββββ-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 ICa by ACh in mouse ββββ-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

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

26

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/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 ICa 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, ICa 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 ββββ-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.

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Fig.1

-20

-40

-60

A

Vm (m

V)

G10ACh 1 µmol/l

2 min

by guest on March 22, 2016 http://www.jbc.org/ Downloaded from

0

0.5

[ACh] (mol/l)E

D

C

20 s

1pA/

pF1p

A/pF

1pA/

pF

20 s

1pA/

pF

B

A

Atrop 10 µmol/lACh 100 µmol/l

ACh 100 µmol/lAtrop 10 µmol/l

ACh 0.1 µmol/l

20 s

20 s

ACh 100 µmol/l

EC50 : 2.5 µmol/lI Na-

ACh (

pA/p

F)

10-8 10-7 10-6 10-5 10-4 10-3

Fig.2

Thapsigargin-Pretreated cell

Ext Sol AInt Sol A

Ext Sol AInt Sol B

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

C

F

E

B

A

D

ACh 100 µmol/l

1 pA

/pF

20 s

Caout 0Cain 0

Kout 0

Naout 0

Tetrodotoxin

Naout 0Nain-rich

Kout 0, Naout 0Kin -rich, Nain 0

DIDSG

Ext Sol CInt Sol B

Ext Sol EInt Sol E

Ext Sol DInt Sol D

Ext Sol DInt Sol B

Ext Sol AInt Sol B

Ext Sol BInt Sol C

Ext Sol AInt Sol B

Fig.3

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

-150 -100 -50 0

-0.50

-0.25

I Na-

ACh (

pA/p

F)

Vm (mV)

Ext Sol FInt Sol F Ext Sol G

Int Sol F

Fig.4

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

0

0.3

0.6

Fig.5

Control GTP-γ-S GDP-β-S

I Na-

ACh (

pA/p

F)Ext Sol AInt Sol B

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

-1.0

-0.8

-0.6

Fig.61 min

Control

ACh 100µM

Wash

B

A

-80

+10

Control

100p

A

ACh 100 µmol/l

Vm (m

V)I C

a

GTP-γ-S

GDP-β-SI Ca (

norm

aliz

ed)

Ext Sol HInt Sol G

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

0

-40

-80

1 min

0

-25

-50

-75

1 min

Vm (m

V)Vm

(mV)

I (pA/pF)

0.77

0

ACh 1 µmol/l ab

cI (pA/pF)

0

0.77

Fig.7

A

B

Ext Sol AInt Sol A

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Jean-François Rolland, Jean-Claude Henquin and Patrick Gilonreceptors in mouse pancreatic beta-cells

G-protein-independent activation of an inward Na+ current by muscaring

published online August 2, 2002J. Biol. Chem.

10.1074/jbc.M203888200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/early/2002/08/02/jbc.M203888200.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on March 22, 2016

http://ww

w.jbc.org/

Dow

nloaded from