M4 Muscarinic Receptor Activation Modulates Calcium Channel Currents in Rat Intracardiac Neurons
J. Cuevas and
D. J. Adams
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101; and Department of Physiology and Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia
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ABSTRACT |
Cuevas, J. and Adams, D. J. M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 78: 1903-1912, 1997. Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-reponse relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 µM ACh was ~75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (
300 nM) and m4-toxin (
100 nM), but not by AF-DX 116 (300 nM) or m1-toxin (60 nM). The dose-response relationship obtained for antagonism of muscarine-induced inhibition of IBa by m4-toxin gave an IC50 of 11 nM. These results suggest that muscarinic agonist-induced inhibition of high-voltage-activated Ca2+ channels in rat intracardiac neurons is mediated by the M4 muscarinic receptor. M4 receptor activation shifted the voltage dependence and depressed maximal activation of Ca2+ channels but had no effect on the steady-state inactivation of Ca2+ channels. Peak Ca2+ channel tail current amplitude was reduced
30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinicreceptor-mediated inhibition. Both dihydropyridine- and
-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 µM guanosine 5
-O-(2-thiodiphosphate) trilithium salt (GDP-
-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.
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INTRODUCTION |
Four muscarinic receptor genes (m1-m4) have been detected in guinea pig and rat intrinsic cardiac neurons using in situ hybridization (Hassall et al. 1993
; Hoover et al. 1994
). These genes correspond to the pharmacologically defined M1-M4 muscarinic receptor subtypes (Hulme et al. 1990
). To date, three different muscarinic ACh receptor (mAChR)-induced voltage responses have been observed in guinea pig intracardiac neurons: two temporally distinct depolarizations and a slow hyperpolarization (Allen and Burnstock 1990
; Mihara et al. 1988
). The faster depolarization is believed to be associated with a decrease in K+ permeability, whereas the slower depolarization and the hyperpolarization with increases in a Cl
and a K+ conductance, respectively (Allen et al. 1994
). Acetylcholine (ACh) activation of muscarinic receptors also decreases the amplitude of the tetrodotoxin (TTX)-insensitive component of the action potential and Ca2+-dependent afterhyperpolarization in guinea pig cultured intracardiac neurons (Allen and Burnstock 1990
), consistent with a decrease in Ca2+ influx through voltage-dependent Ca2+ channels. Inhibition of depolarization-activated Ca2+ channel currents by muscarine has been observed in mammalian central and peripheral neurons, including sympathetic neurons of rat superior cervical ganglia (Mathie et al. 1992
; Wanke et al. 1987
) and rat hippocampal pyramidal neurons (Gähwiler and Brown 1987
). Muscarinic receptor-mediated Ca2+ channel inhibition has also been observed in amphibian parasympathetic neurons of the interatrial septum of the bullfrog heart (Tse et al. 1990
). However, muscarinic receptor modulation of voltage-dependent Ca2+ channels has not been investigated in parasympathetic neurons of mammalian intracardiac ganglia.
Both muscarinic and
-adrenergic receptor activation have been shown to suppress vagal-induced ACh release from rat cardiac parasympathetic nerve fibers (McDonough et al. 1986
; Manabe et al. 1991
; Wetzel et al. 1985
). The mechanism proposed to underlie
-adrenergic inhibition of ACh release is
-adrenoreceptor-induced attenuation of N-type Ca2+ channels, which has been described in isolated rat intracardiac neurons (Xu and Adams 1993
). However, the mechanism involved in muscarinic receptor-mediated inhibition of vagal-induced ACh release in rat parasympathetic cardiac neurons has not been investigated. Furthermore,in contrast to
-adrenoreceptor activation, which inhibits primarily N-type Ca2+ channels, muscarinic receptor activation has been shown to also modulate L-type Ca2+ channels in rat sympathetic neurons (Mathie et al. 1992
).
Both M1 and M4 muscarinic receptors appear to mediate ACh inhibition of Ca2+ channels in rat sympathetic neurons (Bernheim et al. 1992
). Precise identification of muscarinic receptor subtypes has been hampered by the lack of specific receptor antagonists. Recently, toxins highly selective for M1 and M4 muscarinic receptors have been isolated from the venom of the green mamba (Jolkkonen et al. 1994
;Max et al. 1993a
,b
), facilitating the separation of membrane responses evoked by activation of different muscarinicreceptor subtypes. The coupling of these receptors to Ca2+ channels has been shown to involve different signal transduction pathways in rat sympathetic neurons, which include both pertussis toxin-sensitive and -insensitive G-proteins (Beech et al. 1991
; Bernheim et al. 1991
, 1992
). In the present study, muscarinic ACh receptor modulation of depolarization-activated Ca2+ channels was investigated in isolated parasympathetic neurons from rat intracardiac ganglia. ACh reversibly inhibited Ca2+ channel currents via activation of an M4 muscarinic receptor, which is coupled to a pertussis toxin-sensitive G-protein and modulates multiple Ca2+ channel types in these neurons. A preliminary report of some of these results has been presented as an abstract (Cuevas and Adams 1995
).

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| FIG. 1.
Acetylcholine (ACh)-mediated inhibition of high-voltage-activated Ca2+ channel currents in rat parasympathetic neurons. A: family of Ba2+ currents evoked in the absence ( ) and presence ( ) of 100 µM ACh. Holding potential, 90 mV. B: whole cell current-voltage(I-V) relation of peak Ba2+ current amplitude obtained in the absence ( ) and presence ( ) of 100 µM ACh. Data points represent mean ± SE for 5 cells. C: ACh-mediated effects on the amplitude and time course of decay of Ca2+ channel currents. Superimposed traces of IBa elicited by depolarizing steps (3 s duration) to 0 mV from -90 mV in the absence (Control) and presence (+ACh) of 100 µM ACh. Solid lines represent best fit of the time course of current decay using either single (+ACh) or the sum of 2 (Control) exponential functions. Mecamylamine (3 µM) was coapplied with ACh to block nicotinic ACh receptor activation.
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METHODS |
Preparation
Isolated parasympathetic neurons, dissociated from neonatal rat intracardiac ganglia, were obtained and cultured as described previously (Fieber and Adams 1991
). Briefly, atria were dissected from neonatal rats (3-8 days old), killed by decapitation, and incubated in a Krebs solution containing 1 mg/ml collagenase (Type 2; Worthington Biomedical, Freehold, NJ) for 1 h at 37°C. Individual ganglia were dissected from the epicardial ganglion plexus, transferred to a culture dish containing a high glucose medium (Dulbecco's Modified Eagle Media, 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin), titurated with a fine bore pasteur pipette and plated onto 18-mm glass coverslips coated with laminin. The dissociated cells were incubated at 37°C in 95% O2-5% CO2 atmosphere and used for experiments within 24-72 h. At the time of experiments, the glass coverslip was transferred to a recording chamber (0.5 ml volume) and viewed at ×400 magnification using an inverted phase contrast microscope.
Current recording
Membrane currents were recorded from voltage-clamped neurons using the whole cell recording configuration of the patch-clamp technique (Hamill et al. 1981
). Patch pipettes (1-3 M
) were pulled from thin-walled borosilicate glass (GC150TF; Clark Electromedical Instruments, Reading, UK) and fire polished. Electrical access was achieved either by rupturing the membrane patch and dialyzing the cell, or through the perforated-patch method using amphotericin B (Rae et al. 1991
). The perforated-patch method was used to preserve the intracellular integrity of the neurons, thus maintaining functional muscarinic ACh responses lost after cell dialysis in these neurons. For perforated-patch experiments, a stock solution of amphotericin B (60 mg/ml) in dimethylsulfoxide (DMSO) was prepared and diluted in the pipette solution immediately before use yielding a final concentration of 360µg/ml amphotericin B in 0.6% DMSO. Antibiotic incorporation into the membrane patch was monitored by applying a
10-mV pulse at 1 Hz from a holding potential of
70 mV, and, in successful experiments, the appearance of a slow capacitive transient and a decrease in the series resistance (Rs) to <10 M
was observed. To minimize voltage error, Rs was typically compensated by 50% to
5 M
.
Depolarization-activated Ca2+ channel currents were elicited using voltage steps from
90 mV to more positive potentials applied every 10-20 s. Capacitive and leak currents were subtracted using the
P/4 pulse protocol, which assumes a linear relationship for these currents at voltages less than
90 mV (Xu and Adams 1992
). Membrane currents were amplified using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA), filtered at 10 kHz (
3 dB) with a 4-pole Bessel filter, and digitized at 50 kHz (Digidata 1200A, Axon Instruments). A PC Pentium/75 MHz computer running pClamp programs (Axon Instruments) was used to generate voltage pulses and to acquire and analyze data.
Experiments on dialyzed neurons were performed within 20 min of rupturing the membrane patch to minimize the effects of Ca2+ channel current "run-down," which has been reported using conventional whole cell recording configuration (Xu and Adams 1992
). During this time, Ca2+ channel current amplitude decreased by <10%. Calcium current run-down was not observed in neurons electrically accessed using the perforated-patch recording configuration.
The time course of inactivation of Ca2+ channel currents were fit using the pClamp program, Clampfit (Axon Instruments), and Boltzmann distributions for determining the voltage dependence of activation and inactivation were fit using SigmaPlot 3.0 (Jandel Scientific, San Rafael, CA). Both programs determined the best fit to the data using the minimum
2 method. Dose-response curves were obtained by measuring peak current amplitudes at each agonist/antagonist concentration, and the experimental data points were fit with the equation
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(1)
|
where I/Imax is the relative current, Io represents the nonreducible current, [A] is the agonist/antagonist concentration, IC50 is the half-maximal concentration, and k is the slope parameter. Data points represent mean ± SE. Statistical difference was determined using paired t-test for within group experiments, and unpaired t-test for between group experiments, and was considered significant if P < 0.05.
Solutions and materials
The bath solution (physiological salt solution, PSS) used in these experiments contained (in mM) 140 NaCl, 2.5 CaCl2, 1.2 MgCl2, 7.7 D-glucose, and 10 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid (HEPES), adjusted to pH 7.4 with NaOH. Ca2+ channel currents were obtained in the presence of 300 nM TTX, and Ba2+ (5 mM) was used as the charge carrier to maximize Ca2+ channel current amplitude, and to minimize any Ca2+-dependent current rundown (Xu and Adams 1992
). The pipette solution used for perforated-patch experiments contained (in mM) 75 Cs2SO4, 55 CsCl, 5 MgSO4, and 10 HEPES, pH adjusted to 7.2 with N-methyl-D-glucamine; whereas that used for conventional (dialyzed) whole cell recordings contained (in mM) 140 CsCl, 2 MgCl2, 2 1,2-bis(2-aminophenoxy)ethane-N,N,N
,N
-tetraacetic acid (BAPTA) cesium salt, 2 Mg2 ATP, 0.1 guanosine 5
-triphosphate (GTP) sodium salt, and 10 HEPES, pH adjusted to 7.2 with Cs-OH. The osmolality of the solutions (280-290 mmol/kg) was monitored with a vapor pressure osmometer (Wescor 5500, Logan, UT). Muscarinic ACh-evoked responses were elicited by pressure ejection of either ACh, muscarine, or oxotremorine-M at the concentrations indicated from an extracellular pipette position ~50 µm from the cell soma. Mecamylamine (3 µM) was coapplied with ACh to inhibit nicotinic ACh receptor activation (Fieber and Adams 1991
). The recording chamber was continually perfused (~2 ml/min) with the indicated bath solutions at 22-23°C. In a series of experiments, external Na+ (70 mM) was replaced isosmotically by tetraethylammonium ions (TEA) to block outward K+ channel currents and optimize the recording of Ca2+ channel tail currents. External TEA, however, shifts the ACh dose-response curve to the right by ~36-fold, without reducing the maximum response attainable (Caulfield 1991
). Therefore a maximally effective concentration of 100 µM ACh was used in these whole cell experiments. Pharmacological characterization of the muscarinic receptor subtype was carried out using specific muscarinic receptor antagonists: pirenzepine, AF-DX 116, and the green mamba (Dendroaspis angusticepcep) toxins, m1-toxin and m4-toxin. Muscarinic receptor antagonists were bath applied and/or pressure ejected together with the agonists.
In one series of experiments, 100 µM guanosine 5
-O-(2-thiodiphosphate) trilithium salt (GDP-
-S) was substituted for GTP in the pipette solution, and in another series, neurons were preincubated in 200 ng/ml of pertussis toxin (Bordetella pertussis; PTX) for 24 h immediately before the experiments.

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| FIG. 2.
Dose dependence of ACh-mediated inhibition of high-voltage-activated Ca2+ channel currents. A: Ba2+ currents evoked in response to +100-mV depolarization from a holding potential of 90 mV in the absence (Control) and presence ACh and ACh + tetraethylammonium (TEA). Currents were evoked by voltage steps from 90 to +10 mV. Mecamylamine (3 µM) was coapplied with ACh. B: relative peak whole cell IBa amplitude obtained at +10 mV in the absence ( ) and presence ( ) of 70 mM TEA, plotted as a function of ACh concentration. Current amplitude was measured isochronally at the time of the peak of control IBa (10 ms after the onset of the voltage step). Data points represent the mean ± SE for 6 cells. The curve represents a best fit of the data obtained in the absence of TEA by Eq. 1 with an IC50 = 5.6 nM ACh and slope, k = 0.6.
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All chemical reagents used were of analytic grade. The following drugs were used: GDP-
-S and GTP obtained from Boehringer Mannheim (Indianapolis, IN); AChCl, amphotericin B, (±)muscarine chloride, and mecamylamine hydrochloride from Sigma Chemical (St Louis, MO); nimodipine, oxotremorine methiodide (oxotremorine-M), and pirenzepine dihydrochloride from Research Biochemicals International (Natick, MA);
-conotoxin GVIA (
-CgTX, Conus geographus), TTX, and PTX from Calbiochem (San Diego, CA); and MT-3 (Dendroaspis angusticeps) from Alomone Labs. (Jerusalem, Israel). AF-DX 116, m1-toxin, and m-4 toxin were a generous gift from Dr. L. T. Potter (University of Miami School of Medicine, Miami, FL). m4-toxin is identical to the recently described muscarinic toxin 3 (MT-3) in amino acid sequence and selectivity for muscarinic m4 receptors (Jolkkonen et al. 1994
).
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RESULTS |
Muscarinic ACh receptor activation modified the active membrane electrical properties of all rat intracardiac neurons studied (n > 100). Muscarinic receptor activation depolarizes neurons and increases the number of action potentials elicited by depolarizing current pulses without significantly altering action-potential duration (Cuevas et al. 1997
). The actions of ACh on resting membrane potential and discharge characteristics have been shown to be mediated by M1 muscarinic receptor activation. In the present study, we examine the effects of ACh on high-voltage-activated Ca2+ channels.

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| FIG. 3.
ACh-mediated inhibition of Ca2+ channel currents is antagonized by M4, but not by M1 or M2 muscarinic receptor antagonists. A: whole cell I-V relations of peak Ba2+ currents obtained in the absence ( ) of ACh, the presence of 100 µM ACh alone ( ), and in the presence of 100 µM ACh + 300 nM AF-DX 116 ( ). B: I-V relations obtained from the same neuron before ( ) and after 10 min incubation in 1 U/ml of m1-toxin ( ). C: superimposed whole cell IBa elicited by depolarization to +10 mV from 70 mV in the absence (Control) or presence of 100 µM ACh, and in the presence of 100 µM ACh together with 100 nM pirenzepine. D: whole cell I-V relations of peak IBa obtained in the absence of ACh ( ), presence of 100 µM ACh alone ( ), and in the presence of 100 µM ACh + 300 nM pirenzepine ( ).
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Muscarinic ACh-mediated attenuation of Ca2+ channel currents
Ca2+ channel currents were isolated by inhibiting depolarization-activated Na+ currents with extracellular TTX (300 nM), and K+ channels with intracellular Cs+ and extracellular TEA and Ba2+ (5 mM). Ba2+ was used as the charge carrier through open Ca2+ channels in most experiments for reasons discussed in METHODS. The effect of ACh on the Ba2+ current-voltage (I-V) relationship was examined using brief (100 ms) step depolarizations of 10-mV increments (
50 to +90 mV) from a holding potential of
90 mV. Figure 1A shows a family of high-voltage-activated Ba2+ currents (IBa) obtained in the absence (Control) and presence of 100 µM ACh. Focal application of ACh reversibly depressed the peak amplitude and slowed the activation of IBa at all test potentials. Figure 1B shows the average I-V relationship obtained for five neurons. Under control conditions, IBa was activated at approximately
30 mV and I-V relation was maximal at 0 mV, reversing at approximately +40 mV. In the presence of ACh, the I-V relationship exhibited a similar voltage dependence, but the peak IBa amplitude was reduced at all voltages. At 0 mV, IBa was decreased by 75%, from
963.3 ± 99.5 pA to
236.5 ± 39.6 pA (n = 5), in the absence and presence of ACh, respectively. ACh inhibition of Ca2+ channel current occurred to a similar degree when Ca2+ was the charge carrier (data not shown).
Figure 1C shows representative Ca2+ channel currents evoked by a 3 s step depolarization to 0 mV from a holding potential of
90 mV in the absence and presence of 100 µM ACh + 3 µM mecamylamine. Under control conditions, the time-dependent inactivation of IBa was biphasic, and best fit by the sum of two exponential functions with time constants of 90 ms (
f) and 1.40 s (
s). In the presence of ACh, the inward current decay was best fit by a single exponential function with a time constant of 1.90 s (
s). Bath application of 100 µM Cd2+ completely blocked the depolarization-activated Ba2+ current both in the absence and the presence of ACh (data not shown). In four similar experiments, the time course of IBa decay was best fit by the sum of two exponential functions with mean time constants of 99 ± 9 ms (
f) and 1.35 ± 0.09 s (
s). In all neurons studied, the decay of IBa obtained in the presence of 100 µM ACh was best fit by a single exponential function with
s = 1.60 ± 0.21 s (n = 4). The effect of ACh on the time course of decay of IBa was reversible on wash out (data not shown).
ACh concentration-dependent inhibition of IBa
Focal application of the muscarinic receptor agonists, ACh, muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly inhibited whole cell IBa amplitude. The dose-response relationship for ACh-induced inhibition of Ca2+ channel currents was examined both in the absence and presence of external TEA. Figure 2A shows superimposed Ba2+ currents evoked by depolarizing pulses to +10 mV from
90 mV obtained in the absence (control) and presence of different ACh concentrations (0.1 nM to 1 µM) and 100 µM ACh plus 70 mM TEA. Relative peak IBa amplitude plotted as a function of ACh concentration is shown in Fig. 2B. The dose-reponse relationship obtained for inhibition of IBa by ACh was best fit by Eq. 1 with a half-maximal inhibitory ACh concentration of 6 nM. Maximal inhibition of IBa amplitude was obtained with 100 µM ACh in the absence and presence of 70 mM TEA. Muscarine (5 µM) and oxotremorine-M (5 µM) reversibly depressed peak IBa amplitude at 0 mV by 55 ± 4% and 60 ± 3% (n = 3), respectively, compared with control in the presence of external TEA (data not shown).
Inhibition of ACh modulation of IBa by muscarinic receptor antagonists
Figure 3A shows peak I-V relationships for whole cell IBa obtained in the absence and presence of focally applied 100 µM ACh + 3 µM mecamylamine. The ACh-induced decrease in IBa was not affected by bath application of 100 nM AF-DX 116, a selective M2 muscarinic receptor antagonist (Hulme et al. 1990
). AF-DX 116 (300 nM) alone had no effect on either IBa amplitude or time course (data not shown). Figure 3B shows the I-V relationships obtained in the absence (Control) and presence of bath-applied m1-toxin (60 nM), an irreversible antagonist of the M1 muscarinic receptor (Max et al. 1993a
). Focally applied ACh reduced peak IBa amplitude at all voltages tested in the presence of m1-toxin. ACh-mediated inhibition of peak IBa was, however, partially antagonized by bath application of pirenzepine. Figure 3C shows superimposed inward Ba2+ currents evoked in response to step depolarizations from
90 to 0 mV in the absence and presence of 100 µM ACh and ACh plus 100 nM pirenzepine. In three similar experiments, ACh-induced inhibition of IBa was reduced by 46% in the presence of 100 nM pirenzepine, decreasing from 65 ± 7% (1 µM ACh) to 35 ± 3% (1 µM ACh + 100 nM pirenzepine). In the presence of 300 nM pirenzepine, ACh-induced inhibition of IBa was depressed by 81% (n = 3), and this antagonism was observed at all voltages tested (Fig. 3D).
The antagonism of muscarine-induced inhibition of IBa was examined using m4-toxin, a specific competitive antagonist of the M4 muscarinic ACh receptor (Jolkkonen et al. 1994
; Max et al. 1993b
). Figure 4A shows superimposed Ba2+ currents evoked in response to a step depolarization to +10 mV from
90 mV obtained in the absence (control) and presence of muscarine and m4-toxin. IBa amplitude was reduced by ~50% in the presence of 5 µM muscarine, and this inhibition was anatgonized by m4-toxin. The doseresponse relationship for m4-toxin antagonism of muscarine-induced inhibition of IBa is shown in Fig. 4B. Antagonism of muscarine-induced IBa inhibition by m4-toxin was half-maximal at 11 nM and maximally effective with 1 µM m4-toxin. In four similar experiments, oxotremorine-M inhibition of IBa was completely and reversibly abolished by bath application of 60 nM m4-toxin.

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| FIG. 4.
Dose-dependent antagonism of muscarine-induced inhibition of depolarization-activated Ca2+ channel currents by m4-toxin. A: superimposed Ba2+ currents obtained in the absence (Control) and presence 5 µM muscarine and muscarine coapplied with m4-toxin. Currents were evoked by step depolarization to +10 mV from a holding potential of 90 mV. B: dose-response relationship for the displacement of muscarine-induced inhibition by varying concentrations of m4-toxin. Relative peak whole cell IBa amplitude plotted as a function of m4-toxin concentration. Data points represent the mean ± SE for 6 cells. The curve represents a best fit of the data by Eq. 1 with half-maximal concentration (IC50) = 11 nM m4-toxin and k = 0.8.
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Block of ACh-mediated inhibition of Iba by GDP-
-S and PTX
ACh-mediated inhibition of IBa observed in neurons dialyzed with an intracellular pipette solution containing both 2 mM BAPTA and 0.1 mM GTP was similar to that observed in neurons electrically accessed using the perforated-patch method. Figure 5A shows representative currents in response to a step depolarization from
70 to 0 mV recorded from a dialyzed neuron in the absence and presence of 100 µM ACh. In dialyzed whole cell recordings, maximal inhibition of IBa by ACh occurred at +10 mV. IBa amplitude decreased by ~72% in the presence of ACh, from
840 ± 32 pA (control) to
237 ± 13 pA (ACh; n = 4). Figure 5B shows depolarization-activated IBa recorded from a neuron dialyzed with a pipette solution containing 0.1 mM GDP-
-S. Peak IBa amplitude was similar to that recorded from neurons dialyzed with the control pipette solution; however, bath or focal application of ACh had no effect on peak IBa amplitude or activation kinetics. Mean IBa amplitudes, obtained in the absence and presence of 100 µM ACh, were
980 ± 52 and
879 ± 32 pA (n = 3), respectively. In the presence of GDP-
-S, the rate of decay of IBa was decreased in some neurons (Fig. 5B), but ACh failed to inhibit IBa in the presence of GDP-
-S in all neurons examined.

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| FIG. 5.
ACh-mediated inhibition of Ca2+ channels is abolished by pretreatment with pertussis toxin (PTX) or intracellular guanosine 5 -O-(2-thiodiphosphate) trilithium salt (GDP- -S). Superimposed Ba2+ currents elicited by step depolarizations from 90 to 0 mV from a neuron dialyzed with either normal pipette solution (A), or a pipette solution containing 100 µM GDP- -S (B), in the absence (Control) and presence of 100 µM ACh (+ACh). The rate of decay of IBa was decreased in the presence of GDP- -S, but this was not consistently observed in all neurons. C: superimposed IBa evoked by step depolarizations ( 70 to 0 mV), in the absence (Control) and presence of ACh, from a neuron incubated for 24 h in 200 ng/ml PTX. D: bar graph of the mean peak IBa amplitude obtained on depolarization from 90 to 0 mV in the presence of 100 µM ACh, for neurons dialyzed with a normal pipette solution (n = 4), with a pipette solution containing 100 µM GDP- -S (n = 3), or preincubated in PTX(n = 3). The peak IBa amplitude in each condition has been normalized to control (absence of ACh). Asterisk denotes significant difference (P < 0.01).
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Ba2+ currents elicited by voltage steps from
70 to +10 mV in a neuron preincubated in PTX (200 ng/ml, 24 h) are shown in Fig. 5C. ACh failed to inhibit IBa in PTX-treated neurons, whereby IBa amplitude was
1.12 ± 0.11 nA and
1.04 ± 0.09 nA (n = 3), in the absence and presence of ACh, respectively. A summary of the peak IBa amplitudes elicited on depolarization to +10 mV, normalized to their respective control values, under the different experimental conditions is presented in Fig. 5D. ACh decreased IBa by72 ± 1% (n = 4) in neurons dialyzed with control pipette solution, which was statistically significant (P < 0.03). The peak IBa amplitude obtained in the presence of ACh in dialyzed neurons was not statistically different to IBa amplitude obtained using the perforated-patch whole cell recording configuration. In neurons dialyzed with GDP-
-S, IBa was decreased by 10 ± 2% (n = 4), whereas in neurons preincubated in PTX, IBa was reduced by only 7 ± 5% (n = 3). Neither of these ACh-evoked decreases in IBa were statistically significant.
Effects of ACh on the voltage dependence of steady-state inactivation and activation
The effect of ACh on steady-state inactivation of Ca2+ channels in rat intracardiac neurons was studied using a double pulse protocol. Neurons were initially held at
90 mV, and 4-s prepulses from
120 to +10 mV were applied in 10-mV increments before a voltage step to +20 mV to activate (open) the available Ca2+ channels. The steady-state inactivation of IBa exhibited a sigmoidal dependence on voltage and was best fit with a single Boltzmann function according to the equation
A fit of the mean relative current [IBa/IBa(max)]-voltage relationship for three neurons exhibited half-maximal steady-state inactivation (Vh) at
57 mV under control conditions and
54 mV in the presence of ACh (n = 3). The slope parameter (k) was
13 mV both in the absence and presence of ACh (data not shown).
The voltage dependence of activation was examined in dialyzed neurons by measuring tail current amplitude, using a double pulse protocol. Neurons were held at
90 mV, and brief steps to various test potentials were applied before a hyperpolarizing voltage step to
100 mV. Figure 6A shows Ba2+ currents obtained in the absence and presence of ACh in response to voltage steps to the indicated potentials and the ensuing tail currents elicited on repolarization to
100 mV. The corresponding I-V relationship obtained for the tail currents of the neuron shown in Fig. 6A are shown in Fig. 6B. Ca2+ channels exhibit sigmoidal activation at potentials positive to
40 mV in both the absence and presence of ACh. Data points were best fit using a two-component Boltzmann distribution
where i1 and i2 represent the fraction contributed by each component to the final function. For control, i1 and i2 were 0.45 and 0.55, respectively, whereas in the presence of ACh, i1 and i2 were 0.19 and 0.82, respectively. Half-maximal activation of the first component (Vh1) shifted from
7 mV in the absence (Control) to
2 mV in the presence of ACh, whereas the second component (Vh2) remained relatively constant, +26 mV (Control) and +27 mV (+ACh). The asymptotic maximums predicted from these fits were
2.8 nA for control and
1.9 nA in the presence of ACh, which corresponds to an ~30% reduction of IBa at +90 mV. The effects of ACh on the voltage dependence of Ca2+ channel activation were completely reversible on wash out.

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| FIG. 6.
Effects of muscarinic receptor activation on the voltage dependence of Ca2+ channel activation. A: whole cell Ba2+ currents evoked by step depolarizations (40 ms duration) to +10 (i), +50 (ii), or +90 mV (iii) from a holding potential of 90 mV in the absence (C) and presence of 100 µM ACh (+ACh). Tail currents recorded after repolarization to 100 mV are displayed on an expanded time scale. B: I-V relation of the Ba2+ tail currents obtained in the absence (control, ; recovery, ) and presence ( ) of 100 µM ACh. C: relative Ba2+ tail current amplitude at 100 mV, normalized to maximum Ba2+ current amplitudes in the absence (control, ) and presence ( ) of 100 µM ACh at the various test potentials. Data points are plotted as a function of the voltage of the step depolarization and represent mean ± SE for 4 experiments. Curves represent best fit of the data using double Boltzmann distributions with i1, i2, Vh1, Vh2, k1, and k2 equal to 0.70, 0.30, 9.7 mV, +35.9 mV, 7.7, and 17.6 for control and 0.71, 0.29, +1.5 mV, +53.2 mV, 13.5, and 14.0 in the presence of ACh, respectively.
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Ca2+ channel activation curves obtained for four similar experiments are shown in Fig. 6C. Data points represent mean IBa normalized to maximum IBa both in the absence (Control) and presence of ACh (+ACh) and were best fit by a two-component Boltzmann distribution. The relative contributions of the two components of IBa were not markedly changed by ACh; however, in the presence of ACh, Vh1 was shifted by +11.2 mV and Vh2 by +17.3 mV in the normalized Ca2+ channel activation curves.
Muscarinic ACh modulation of different Ca2+ channel subtypes
The Ca2+ channel subtype(s) modulated by ACh was examined by determining the amount of ACh-induced inhibition of IBa in the presence of specific Ca2+ channel antagonists. Rat parasympathetic cardiac neurons have been shown to contain at least three distinct types of Ca2+ channels, which may be classified pharmacologically: 1) a dihydropyridine-sensitive Ca2+ channel, 2) an
-CgTX GVIA-sensitive Ca2+ channel, and 3) a dihydropyridine- and
-CgTX-insensitive Ca2+ channel (Xu and Adams 1992
). Figure 7A shows inward Ba2+ currents obtained in the absence and presence of ACh, during bath application of PSS, and PSS containing either the dihydropyridine antagonist, nimodipine (10 µM),
-CgTX-GVIA (300 nM), or Cd2+ (100 µM). A plot of IBa amplitude, measured 10 ms after onset of step depolarization to 0 mV, during sequential exposure to different Ca2+ channel antagonist is shown in Fig. 7B. In the presence of either nimodipine,
-CgTX, or nimodipine +
-CgTX exposure, ACh was able to further inhibit IBa. ACh inhibited ~65% of the inward current remaining during bath application of 10 µM nimodipine, and ~15% of the current that remained in the presence of either
-CgTX or nimodipine +
-CgTX (n = 3). Bath application of 100 µM Cd2+ completely inhibited IBa, and the subsequent addition of ACh had no further effect.

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| FIG. 7.
Differential modulation of Ca2+ channel subtypes by ACh. A: whole cell Ba2+ currents evoked by step depolarizations to +5 mV from a holding potential of 70 mV in the absence and presence of the indicated Ca2+ channel antagonists. Nimodipine (10 µM; i), -conotoxin-GVIA ( -CgTX; 300 nM; ii), -CgTX + nimodipine, and Cd2+ (100 µM) (iii) were bath applied, and ACh (100 µM) was pressure ejected from an extracellular pipette. B: Ba2+ current amplitude plotted as a function of time during sequential bath perfusion with normal physiological salt solution (PSS) or PSS containing the indicated Ca2+ channel antagonists in the absence ( ) and presence ( ) of 100 µM ACh. IBa current amplitude was measured 10 ms from the onset of the step depolarization.
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DISCUSSION |
Muscarinic agonists reversibly inhibited high-voltage-activated Ca2+ channel currents in parasympathetic neurons of neonatal rat intracardiac ganglia. ACh inhibited peak IBa amplitude in a dose-dependent manner with a half-maximal inhibitory concentration of 6 nM and maximal inhibition obtained with 100 µM ACh, in the absence and presence of 70 mM TEA. ACh (100 µM) reduced peak IBa amplitude by ~75% and altered Ca2+ channel activation, whereas steady-state inactivation was unaffected. Ba2+ currents recorded in the absence and presence of muscarinic agonists were completely abolished by external Cd2+ (100 µM), indicating that muscarinic agonists are modulating Ca2+ channel currents. Muscarinic-mediated inhibition of Ca2+ channel currents has been reported in autonomic neurons, including rat sympathetic (Bernheim et al. 1992
; Wanke et al. 1987
) and bullfrog parasympathetic (Tse et al. 1990
) neurons. In sympathetic neurons from rat superior cervical ganglia (SCG), muscarinic inhibition of ICa was shown to be sensitive to pirenzepine (<120 nM), suggesting that the IBa inhibition was mediated by activation of the M1 muscarinic receptor subtype (Wanke et al. 1987
). However, a recent study in rat SCG sympathetic neurons showed that the activation of both M1 and M4 receptor subtypes mediate the inhibition of Ca2+ channel currents (Bernheim et al. 1992
). In NG108-15 neuroblastoma × glioma hybrid cells transfected with cDNA encoding for m1-m4 muscarinic AChR subtypes, M2 and M4 receptor activation inhibited Ba2+ currents, whereas M1 and M3 receptor activation had no effect(Higashida et al. 1990
). The discrepancy regarding thereceptor subtype involved may have arisen, in part, from the similar pharmacological profiles of the muscarinic receptors, particularly M1 and M4. For example, the pirenzepine concentrations used by Wanke et al. (1987)
to inhibit M1 receptors may also antagonize M4 receptors in rat sympathetic neurons, particularly because similar pirenzepine concentrations block M4 receptor-mediated depression of ICa in transfected NG108-15 cells (Caulfield and Brown 1991
).
In the present study, the use of selective muscarinic receptor antagonists suggest that M4 receptor activation inhibits Ca2+ channel currents in rat intracardiac neurons. The M2 muscarinic receptor antagonist, AF-DX 116 (300 nM), failed to inhibit ACh-induced attenuation of Ca2+ channel currents. Given that the Kd for inhibition of M2 muscarinic receptors by AD-FX 116 was shown to be 200 nM in Chinese Hamster Ovary (CHO) cells transfected with the m2 muscarinic receptor gene (Hulme et al. 1990
), it is unlikely that the attenuation of Ca2+ channel currents in rat intracardiac neurons is mediated by M2 muscarinic receptors. ACh-evoked inhibition of IBa was, however, antagonized by pirenzepine (300 nM), in contrast to observations in bullfrog intracardiac neurons where pirenzepine
100 µM had no effect (Tse et al. 1990
). Pirenzepine at a concentration of 300 nM has been shown to inhibit the activation of both M1 (Kd = 16 nM) and M4 (Kd = 80 nM) muscarinic receptor subtypes (Caulfield and Brown 1991
; Hulme et al. 1990
). However, the selective M1 muscarinic receptor antagonist, m1-toxin, failed to inhibit ACh-mediated attenuation of IBa at a concentration sufficient to antagonize M1 muscarinic receptor-mediated inhibition of IM in these neurons (Cuevas et al. 1997
). Further evidence suggesting that ACh-induced inhibition of IBa is mediated by M4 receptor activation is the observation that m4-toxin, specific for the m4/M4 muscarinic receptor subtype (Jolkkonen et al. 1994
; Max et al. 1993b
), antagonizes muscarine-induced depression of Ca2+ channel currents in a dose-dependent manner with a half-maximally effective concentration of 11 nM. Together these data suggest that ACh-induced attenuation of Ca2+ channel currents in cardiac neurons is mediated exclusively by M4 muscarinic receptors.
Muscarinic agonist attenuation of Ca2+ channel currents via the M4 receptor activation is mediated by a PTX-sensitive G-protein. ACh-induced depression of IBa was abolished by either intracellular dialysis with GDP-
-S or preincubation of the neurons with PTX. In contrast, ACh modulation of IM and discharge activity that is mediated by M1 muscarinic receptor activation in rat intracardiac neurons was not inhibited by PTX (J. Cuevas and D. J. Adams, unpublished observations). A PTX-sensitive effector coupling of muscarinic receptor activation to the inhibition of Ca2+ channel currents has been previously described in rat sympathetic neurons (Bernheim et al. 1992
; Wanke et al. 1987
) and bullfrog parasympathetic neurons (Tse et al. 1990
). Furthermore, only the M4 receptor subtype has been shown to inhibit ICa via a PTX-sensitive pathway (Bernheim et al. 1992
; Higashida et al. 1990
). In rat intracardiac neurons, PTX-sensitive G-proteins have been shown to mediate norepinephrine inhibition of Ca2+ channel currents (Xu and Adams 1993
). The attenuation of IBa by norepinephrine and ACh was not additive (data not shown), suggesting a convergence in the signal transduction pathways, similar to that reported in NG 108-15 cells transfected with the M4 receptor, where ACh and norepinephrine have mutually occlusive interactions (Higashida et al. 1990
).
Intracellular dialysis of the voltage-clamped neurons with a pipette solution containing the Ca2+ chelator BAPTA, failed to inhibit muscarinic agonist-induced inhibition of IBa, suggesting that cytosolic Ca2+ is not involved in the signal transduction pathway coupling muscarinic receptors to suppression of Ca2+ channels. In contrast, a BAPTA-sensitive pathway has been implicated in the slow muscarinic inhibition of Ca2+ channels observed in rat sympathetic neurons (Bernheim et al. 1992
). However, the inhibition mediated by this pathway has been attributed to M1 and not M4 muscarinic receptor activation and is PTX insensitive (Bernheim et al. 1992
). Previous studies have demonstrated that neither diacylglycerol, protein kinase C, nor adenylate cyclase activation have any significant effects on Ca2+ channel currents in rat intracardiac neurons (Xu and Adams 1993
). Activation of a PTX-sensitive G-protein(s) has been proposed to inhibit ICa via direct interaction with Ca2+ channels (see Hille 1994
). This putative mechanism had been proposed for the muscarinic receptor inhibition of Ca2+ channel currents in bullfrog intracardiac neurons (Tse et al. 1990
) and is consistent with the results of the present study.
Under control conditions, Ca2+ channel currents reached their peak within ~10 ms and exhibited a biphasic time course of decay (see Xu and Adams 1992
). In the presence of ACh, the activation of Ca2+ channel currents was slowed, and the rapidly inactivating component of IBa decay was abolished. The decay of IBa was best fit by a single exponential function (
s) similar to that described by norepinephrine modulation of Ca2+ currents in rat intracardiac neurons (Xu and Adams 1993
). Steady-state inactivation of voltagedependent Ca2+ channels in rat intracardiac neurons was unaffected by ACh. The voltage dependence of Ca2+ channel activation was shifted to more positive potentials by ACh. In the absence and presence of ACh, the voltage dependence of activation was best fit by the sum of two Boltzmann distributions, and ACh shifted the half-maximal potential for the components by +11.2 and +17.3 mV, respectively. In bullfrog intracardiac neurons, the voltage dependence of Ca2+ channels was similarly shifted to more positive potentials in the presence of ACh (Tse et al. 1990
). However, a shift of Ca2+ channel gating to more positive potentials, which require stronger depolarizations to open, is not sufficient to account for the ACh-induced inhibition of IBaobserved in rat intracardiac neurons (see Fig. 6) (Bean 1989
). Peak Ca2+ channel tail current amplitude obtained in the presence of ACh was reduced by
30%, relative to control at +90 mV, indicating a voltage-independent component of the attenuation of Ca2+ channel activation. Similarly, ACh has been shown to inhibit Ca2+ channels through both voltage-dependent and -independent mechanisms in rat sympathetic neurons (Mathie et al. 1992
).
Application of ACh inhibited primarily the
-CgTX GVIA-sensitive Ca2+ channels that are responsible for the rapidly inactivating component of IBa.
-CgTX GVIA-sensitive (N-type) Ca2+ channels constitute ~75% of the total Ca2+ channel current (Xu and Adams 1992
), but ACh also depressed dihydropyridine-sensitive (L-type) and
-CgTX GVIA- and dihydropyridine-insensitive Ca2+ channel currents. Norepinephrine also inhibited N-type Ca2+ channels in rat intracardiac neurons via a PTX-sensitive G-protein mechanism (Xu and Adams 1993
). In rat sympathetic neurons, however, the M4 receptor-mediated, PTX-sensitive pathway inhibits N-type Ca2+ channels exclusively (Bernheim et al. 1992
; Mathie et al. 1992
; Wanke et al. 1987
).
Functional significance
Cholinergic (vagal) innervation of mammalian intracardiac ganglia primarily involves axosomatic synapses(Ellison and Hibbs 1976
), and therefore the investigation of ACh modulation of voltage-dependent Ca2+ channels in the cell soma is relevant to autonomic transmission. ACh-mediated inhibition of Ca2+ channel currents may regulate transmitter release from both intrinsic and extrinsic presynaptic nerve terminals within intracardiac ganglia and to cardiac muscle. M4 muscarinic receptor inhibition of a heterogeneous population of Ca2+ channels may be physiologically significant, given that neurally evoked transmitter release in rat parasympathetic ganglia is not inhibited by
-CgTX GVIA but is suppressed by Cd2+ block of Ca2+ channels (Seabrook and Adams 1989
). The stimulation of muscarinic receptors on sympathetic nerve terminals in the heart reduce the release of norepinephrine (Foldes et al. 1989
; Löffelholz and Muscholl 1969
; Manabe et al. 1991
; Vizi et al. 1989
), whereas atropine enhances vagal-stimulated ACh release in the intact mammalian myocardium (Löffelholz and Muscholl 1969
; Wetzel et al. 1985
). In sympathetic neurons,
-adrenergic inhibition of ICa may underlie depression of norepinephrine release and provide negative feedback regulation (Lipscombe et al. 1989
). Given that acetylcholine is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this muscarinic ACh-mediated Ca2+ channel inhibitory mechanism suggests that it may also provide negative feedback regulation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Lincoln Potter for purifying and supplying the m1- and m4-toxins.
This research was supported by National Heart, Lung, and Blood Institute Grant HL-35422 and National Health and Medical Research Council of Australia Grant 961138.
 |
FOOTNOTES |
Present address of J. Cuevas: Dept. of Biology, University of California, San Diego, La Jolla, CA 92093.
Address for reprint requests: D. J. Adams, Dept. of Physiology and Pharmacology, University of Queensland, Brisbane, QLD 4072, Australia.
E-mail: dadams{at}plpk.uq.edu.au
Received 29 January 1997; accepted in final form 20 June 1997.
 |
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