215

Journal of Physiology (1991), 434, pp. 215-237 With 9 figures Printed in Great Britain

ACETYLCHOLINE-EVOKED CURRENTS IN CULTURED NEURONES DISSOCIATED FROM RAT PARASYMPATHETIC CARDIAC GANGLIA

BY LYNNE A. FIEBER* AND DAVID J. ADAMS From the Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami,. FL 33101, USA

(Received 26 April 1990) SUMMARY

1. The properties of acetylcholine (ACh)-activated ion channels of parasympathetic neurones from neonatal rat cardiac ganglia grown in tissue culture were examined using patch clamp recording techniques. Membrane currents evoked by ACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, and unaffected by atropine, suggesting that the ACh-induced currents are mediated by nicotinic receptor activation. 2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currents exhibited strong inward rectification and a reversal (zero current) potential of -3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changing the main permeant cation or by removal of divalent cations from the intracellular or extracellular solutions. Unitary ACh-activated currents exhibited a linear I-V relationship with slope conductances of 32 pS in cell-attached membrane patches and 38 pS in excised membrane patches with symmetrical CsCl solutions. 3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependent manner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hexamethonium (IC50 1 UtM), as well as by the neuromuscular relaxant, d-tubocurarine (Kd = 3 /M). Inhibition of ACh-evoked currents by hexamethonium could not be described by a simple blocking model for drug-receptor interaction. 4. The amplitude of the ionic current through the open channel was dependent on the extracellular Na+ concentration. The direction of the shift in reversal potential upon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptor channel is cation selective and the magnitude suggests a high cation to anion permeability ratio. The cation permeability (Px/PNa) followed the ionic selectivity sequence Cs+(1 06) > Na+(1P0) > Ca2+(093). Anion substitution experiments showed a relative anion permeability, PCi/pNa 1< 005. 5. The nicotinic ACh-activated channels described mediate the responses of postganglionic parasympathetic neurones of the mammalian heart to vagal stimulation. * Present address: Department of Cell Biology and Physiology, Washington University, 660 S. Euclid Avenue, St Louis, MO 63108, USA.

MS 8455

216

L. A. FIEBER AND D. J. ADAMS INTRODUCTION

The mammalian cardiac ganglion, located in the atrial subepicardium, mediates the vagal innervation of the heart and is proposed to play a role in the control of the heart beat (Moravec & Moravec, 1987; Gagliardi, Randall, Bieger, Wurster, Hopkins & Armour, 1988). To date detailed electrophysiological studies of postsynaptic responses in parasympathetic neurones of the heart have been confined to the amphibian preparations of the frog and mudpuppy (Dennis, Harris & Kuffler, 1971; Roper, 1976; Hartzell, Kuffler, Stickgold & Yoshikami, 1977; Conner & Parsons, 1983). The characterization of the postsynaptic response of rat intracardiac neurones to the neurotransmitter, acetylcholine (ACh), released upon vagal stimulation is important for understanding how postsynaptic neuronal responses modulate cholinergic innervation of the heart. Although many structural similarities exist between neuronal and muscle nicotinic receptors (Lindstrom, Schoepfer & Whiting, 1987; Steinbach & Ifune, 1989), there is considerable pharmacological evidence of functional differences between the two types. Whereas the nicotinic receptor in skeletal muscle consists of five subunits (2a, /l, &, y or e), four ACh-binding subunits (a2, a3l a4, a5) and three non-ACh-binding subunits (/82, 83, /4) of the neuronal nicotinic receptor have been identified. It appears that a functional neuronal receptor channel consists of at least one ACh-binding and one non-ACh-binding subunit (Ballivet, Nef, Couturier, Rungger, Bader, Bertrand & Cooper, 1988; Papke, Boulter, Patrick & Heinemann, 1989). Electrophysiological studies of membrane currents evoked in response to exogenous ACh in mammalian cultured central and peripheral neurones provide evidence of numerous types of neuronal nicotinic receptor channels (O'Lague, Potter & Furshpan, 1978; Ogden, Gray, Colquhoun & Rang, 1984; Aracava, Deshpande, Swanson, Rapoport, Wonnacott, Lunt & Albuquerque, 1987; Lipton, Aizenman & Loring, 1987; Mathie, Cull-Candy & Colquhoun, 1987). Although the pharmacological actions of ganglionic blocking agents on neurally evoked postsynaptic currents in rat parasympathetic submandibular ganglia (Ascher, Large & Rang, 1979; Rang, 1982) and amphibian parasympathetic cardiac ganglia (Hartzell et al. 1977; Lipscombe & Rang, 1988) have been described, neuronal nicotinic receptor function has not been investigated extensively in isolated neurones. A foundation for quantitative comparison of the functional properties of neuronal and muscle nicotinic receptor channels is lacking. In this paper we investigate the pharmacology and ionic permeability of the neuronal nicotinic receptor channel in cultured neurones dissociated from rat parasympathetic cardiac ganglia to permit comparison with neuronal nicotinic receptor channels in amphibian and rat autonomic ganglia. The findings presented provide a basis for future studies of the molecular structure of the receptor channel. Preliminary reports of some of these results have appeared (Adams, Fieber & Konishi, 1987; Fieber & Adams, 1988). METHODS

Preparation and 8olutions Neonatal rats were killed by cervical dislocation (decapitation) prior to removal of atria. A culture of neurones was made from dissociated cells of the neonatal rat cardiac ganglion plexus by dissecting out individual ganglia following enzyme treatment. Dissected atria containing ganglia

NEURONAL NICOTINVIC RECEPTOR CHANNEL

217

were incubated in collagenase (1 mg/ml, Worthington-Biomedical) for 1 h at 37 °C, transferred to a sterile culture dish containing culture medium (Dulbecco's Modified Eagle Medium with 10 mmglucose, 10 % (v/v) fetal calf serum, 100 U/ml penicillin and 0 1 mg/ml streptomycin), triturated with a fine-bore Pasteur pipette, then plated onto 18 mm glass cover-slips coated with laminin. The dissociated cells were incubated at 37 °C in a 95 % air, 5 % CO2 atmosphere. Electrophysiological recordings were made from neurones maintained in tissue culture for 48-72 h. At the time of experiments, the glass cover-slip was transferred to a low-volume (0 5 ml) recording chamber and viewed at 400 x magnification using an inverted, phase contrast microscope (Nikon Diaphot). Experiments were conducted at room temperature (22-23 °C). The extracellular solution (physiological salt solution, PSS) consisted of (mM): 140 NaCl, 3 KCl, 2-5 CaCl2, 1-2 MgCl2, 7-7 glucose, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)-NaOH, pH 7-2. The relative cation permeability of the ACh-activated channel was investigated by replacement of NaCl with an osmotically equivalent amount of the following test compounds: N-methylglucamine chloride, CsCl, CaCl2 and mannitol. Mannitol was chosen over salts of large positively charged ions such as, N-methylglucamine, as a Na+ substitute because the amplitude of the ACh-evoked current in the presence of N-methylglucamine was less than predicted for the extracellular Na' concentration ([Na+]O) (see Adams, Nonner, Dwyer & Hille, 1981; Sanchez, Dani, Siemen & Hille, 1986). To investigate anion selectivity, reversal potentials were measured in external solutions containing Cl-, gluconate or S04 2- as the major anion. Reversal potential measurements were corrected for differences in junction potential between the bath solution and the indifferent electrode (0-15 M-KCl-agar bridge) but not for differences in Na+ activity coefficients. Liquid junction potential measurements to the indifferent electrode were made with respect to a reference electrode (saturated KCl, reverse sleeve junction; Corning X-EL 47619). The osmotic activity of the solutions was monitored with a vapour pressure osmometer (Wescor 5500). The intracellular pipette solution contained (mM): 140 CsCl, 2 MgATP, 10 HEPESCsOH, pH 7-2 (adjusted after addition of ATP), and either 2 CS4BAPTA or 1 CS2EGTA. In a series of experiments the free internal Mg2+ concentration ([Mg2J]i) was reduced to negligible levels by replacing MgATP with Na2ATP in the intracellular pipette solution containing EGTA. Agonistinduced responses were investigated using a pressure application device (Picospritzer II, General Valve Corp., NY, USA) by focal application of ACh to the neurone from an extracellular pipette (> 20 MQ resistance) containing 100 ,tM-AChCl in the appropriate extracellular solution. Agonist was applied during continuous bath perfusion and exchange of the external solution in the recording chamber was complete within 10 s. The pressure ejection pipette was positioned approximately 30 ,tm from the soma mem')rane to evoke maximal responses to agonist under control conditions (100 ms, 10 lbf in-2 = 44-5 N). To minimize receptor desensitization, a delay of > 30 s between agonist applications was maintained. This protocol, however, does not eliminate fast nicotinic receptor desensitization (T = 50-100 ms) occurring during application of 100 /LM-ACh (Dilger & Brett, 1990). The receptor antagonists atropine, K-bungarotoxin, hexamethonium, mecamylamine and d-tubocurarine were bath applied at the concentrations stated. The antagonist concentration at the cell surface during pressure application of agonist alone will be equal to or lower than that in the bulk solution. The apparent dose-response curves thus may be shifted towards higher antagonist concentrations. All chemical reagents were of analytical grade. Acetylcholine chloride, atropine sulphate, ethyleneglycol-bis-(/-aminoethylether) NN,N',N'-tetraacetic acid (EGTA), hexamethonium bromide, laminin, mecamylamine hydrochloride, (-)nicotine di-(+)tartrate and d-tubocurarine chloride were obtained from Sigma Chemical Co. (St Louis, MO, USA). 1,2-Bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA, tetracaesium salt) was obtained from Molecular Probes, Inc. (Eugene, OR, USA). K-Bungarotoxin, a fraction of the venom a-bungarotoxin (a-BTX) proposed to bind to a site unique to the neuronal nicotinic receptor (see Loring & Zigmond, 1988), was obtained from Calbiochem (San Diego, CA, USA).

Electrical recordings Agonist-induced responses of cultured intracardiac neurones were studied under current and voltage clamp modes using the whole-cell recording configuration of the patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Only isolated cardiac neurones that generated an action potential in response to the injection of depolarizing current pulses were studied. Membrane current and voltage were monitored using a List patch clamp amplifier (L/M EPC-7), filtered at 5 kHz with a low-pass Bessel filter (4-pole, Ithaco 4302) and recorded on videotape using

218

L. A. FIEBER ANVD D. J. AD)AMS

an analog-to-digital recorder adaptor (PCM- 1; Medical Systems, NY, UTSA). The membrane current and voltage were monitored continuously on a digital oscilloscope (Nicolet 3091). The cell capacitance (Cm) was determined for each cell from the compensation of the capacity transient in response to a 10 mV voltage step. No compensation of the series resistance (RJ) was made. However, given that R0 was usually < 6 MQ and the maximum amplitude of whole-cell currents evoked by agonist was - t nA, then the voltage error due to R. would be < 6 mV. WVhole-cell an(l single-channel currents and voltage were displayed using a chart recorder (Gould 2200S; DC125 Hz bandwidth) for analysis. For single-channel recording, patch pipettes (thin-walled borosilicate glass, Clark Electromedical, UK) of 1-2 MQ resistance were made and coated with Sylgard (Dow Corning Corp., Midland, MI, USA) to within lOm of the tip to reduce extraneous electrical noise due to 100 capacitative coupling. Single-channel currents were recorded from cell-attached and excised (inside-out) membrane patches with a pipette solution containing 140 mM-CsCl, 10 mM-HEPESCsOH, pH 7-2. To obtain an excised membrane patch, a cell-attached patch was pulled slowly away from the soma membrane, carried briefly through the solution-air interface and the bathing solution exchanged for one corresponding to the pipette solution. Experiments were performed in pairs, the first of each pair in the absence of agonist and the second with the same patch pipette solution, but containing 10 /tM-ACh. It is possible that single-channel currents obtained from the latter patches were subject to desensitization. When single-channel activity was observed in a cellattached membrane patch, the patch was evaluated based on the dependence of single-channel events on the presence of ACh in the patch pipette. A small conductance channel (< 7 pS) was observed in approximately one-third of membrane patches in the absence of ACh in symmetrical CsCl solutions, but was not studied. Single-channel currents were analysed to obtain histograms of current amplitude by either direct measurement using the digital storage oscilloscope and/or by sampling videotape records at 20 kHz (Tecmar Labmaster DMA interface) for analysis on a PC 80286 computer using the pCLAMP programs (Axon Instruments Inc., CA, USA). Analysis of receptor-antagonist interactions For experiments investigating actions of antagonists of the neuronal nicotinic receptor, it was assumed that the receptor-channel interaction obeyed the law of mass action. It was also assumed that the agonist (or blocker)-receptor interaction equilibrates rapidly and that the observed physiological response resulting from agonist-receptor binding is proportional to receptor occupancy: k+1

A(agonist) + R(receptor channel) = AR k_I

where AR is proportional to the response, i.e., the observed ionic current. At equilibrium, the forward and reverse rates are equal such that k+l[A][R] = k_JAR]. If p is the fraction of receptors occupied to give AR, then R represents the complement (1 -p), and solving for p gives 1 p= KJ[A] where Ka= k+1 Kd (1 +Ka[A]) k 1 Kd I

Since p is proportional to the observed response,

control

( t1oA]

on

d

where C is a constant. In the case of competitive antagonists, [A] becomes [B], the concentration of blocker, and the equation is I

1

|

C

d1B x

+K-l[B 'control = This equation allows calculation of the half-inhibitory concentration of drug, IC50, or for this purpose referred to as the dissociation constant Kd, by plotting '/Icontrol versus log[B] in the (

.NEURONAL NICOTINIC RECEPTOR CHANNEL

219

presence of a fixed concentration of agonist, and the Hill coefficient (n,,) from the slope of the curve (see Limbird, 1986). In fitting this equation for responses modulated by antagonists, the Kd, nH and end-point of the curve were variables, and only the origin of the curve (absence of antagonist; 100%) and the requirement for a single affinity site were constrained. I)etermination of relative cation permeabilitie8 Relative permeability estimates for cations were calculated using the Goldman-Hodgkin-Katz (GHK) equation (see Hille, 1975). The form of the equation used to determine the predicted shifts in reversal potential (AErev) expected for Na+ substitution experiments was RT /PNarNa+Jo\ AErev = F lnt p i

where RT/F is 25-3 mV at 22 °C, P.a/PCs is the permeability ratio for Na+, and [Cs'] and [Na'] are the ion concentrations of the internal and external solutions, respectively. To determine the relative permeability to Ca2", a GHK voltage equation was derived to include Ca2' and activity coefficients (see Meves & Vogel, 1973; Lewis, 1979). Activity coefficients of the salts were obtained from Robinson & Stokes (1959) and Butler (1968). Under conditions where [Ca2+]i = 0, [Na+]i = 0, [Cs]. = 0, the net currents contributed by the individual ions are: ic

= Ca

iNa

and where

=

4Pa ]2)= 4Pa Ca V([ Ca V(( I- exp (2 V) (1- exp(V')r) (I1+exp (V);] Na

Vle;p()

ics = PC. V

[1 I-epex(V)(V)

V =F RT

When the membrane potential equals the reversal potential (Em = Erev): = ° 0i

=

iCa + iNa + iCs,

xi = 0 = (1

+

ep

V)a ) + PNa[Na+]o-PCj,[Cs+]i exp (V),

4PCa[Ca2+]+ (PNa[Na+]o-PCs[Cs+]i exp (V)) (1 + exp (V)) = 0. Let X = exp (V), then 4Pca[Ca

Solving for

+ PNa[Na ]O+ PNa[NaJ]OX

P5[Cs] ]iX

PCs[1]X= 0.

PCa Na

PCa

PNa

(PC/ PNa) [CsJ]X(1 +X)[Naa ] (1 + X)

4[Ca2 ]0 ={(PCa/PNa) rCs ]1x- rNa ]0} (1 +X) 4[Ca +]0

In isotonic Ca2+ external solution, i.e.

[Na]o

=

0

PCa (PCs/PNa){ICS ]iX(l +X)} 4[Ca ] PNa _N2

220

L. A. FIEBER AiND D. J. ADAMS RESULTS

Rat parasympathetic cardiac neurones grown in tissue culture range in diameter from 20 to 35 ,um and axonal processes originating from the cell soma indicate that neurones are usually uni- or bipolar. Although most neurones had a single nucleus some binucleate neurones were observed similar to those observed in cultures of intracardiac neurones from guinea-pig heart (Hassall & Burnstock, 1986). Nonneuronal cells were most likely a combination of glia, fibroblasts and Schwann cells (Kobayashi, Hassall & Burnstock, 1986), and constituted as much as 60% of the total number of cultured cells. Cultured neurones exhibited no spontaneous action potentials and had resting membrane potentials in the range of -50 to -65 mV, and a mean input resistance of 720+ 35 MQ (S.E.M., n - 21).

Acetylcholine-evoked currents in intracardiac neurones Acetylcholine applied from a pressure ejection pipette evoked either a single action potential or a train of action potentials in the neurone when recorded in current clamp mode (Fig. 1A), whereas pressure ejection of PSS (without ACh) produced no response. Under voltage clamp conditions, ACh evoked a transient inward current in cultured cardiac neurones, with the amplitude dependent on the membrane potential (Fig. 1 B). The amplitude of the ACh-induced current at a holding potential of -70 mV was approximately -1 nA, and normalized to the cell capacitance (22 + 5 pF), the current density was approximately 45 pA/pF. The membrane current response followed a 100 ms pulse of ACh from the pressure ejection pipette with an average latency of 10 ms and reached its peak amplitude before the cessation of the pulse. The half-time of decay of the ACh-induced current was 10+0 13 s (S.E.M., n = 20 cells) at -70 mV and was independent of membrane p6tential over the range -150 to + 50 mV. Desensitization of ACh-evoked currents was apparent from the marked reduction of current amplitude observed in response to repeated agonist applications < 20 s apart. A current-voltage (I-V) relationship for the ACh-induced response obtained in PSS is shown in Fig. 1 C. The reversal (zero current) potential under these conditions averaged -3+0-1 mV (S.E.M., n = 25). ACh-evoked currents obtained at negative holding potentials were larger than at the corresponding positive potentials. Inward rectification occurred with either Cs' or K+ as the major intracellular cation and in the absence of external Ca2+. In six cells dialysed with an intracellular pipette solution containing no added Mg2+ (and 10 mM-EGTA), inward rectification of the I-V relationship also was observed (Fig. 1D).

Voltage dependence of ACh-induced single-channel currents To investigate the mechanism(s) underlying the inward rectification of the macroscopic I-V curve for ACh-evoked currents, the I-V relationship for single AChinduced channels was determined in cell-attached and excised membrane patches. Typical records of ACh-induced single-channel currents from cell-attached and inside-out membrane patches are shown in Fig. 2A and B respectively. The corresponding single-channel I-V relationships are shown in Fig. 2 C and D. Unitary

NE URONAL NVICOTINIC RECEPTOR CHANNEL

221

I-V relationships determined from cell-attached patches exhibited slight inward rectification, whereas the I-V relationship derived from unitary currents obtained in excised patches was linear over a 200 mV range. The mean single-channel conductance in cell-attached membrane patches was 32 pS, and 38 pS in excised B

A

ACh

V +30 mV +10

0 -10

AI

-30

-J 20 mV 1 s

j 100 pA 1 s C

-120 -90 -60 -30

pA/pF

r 10

D

pA/pF

rF 1o 50

mV

-10

-20 -30 -40 -50 -60

*-70

Fig. 1. Excitatory response of rat cultured cardiac neurones to exogenous ACh. A, voltage record of action potentials evoked in response to a 20 ms pulse of ACh (arrow-head) applied from an extracellular pipette. The resting membrane potential was -54 mV. Temperature 22 'C. B, whole-cell currents evoked by ACh in PSS at the membrane potentials indicated. Arrow-head indicates a 100 ms pulse of ACh (< 100 imM). C, current-voltage relationship for peak current amplitude evoked by ACh in PSS. Each data point represents mean current density (pA/pF) + S.E.M. from eight cells. D, current-voltage relationship for peak current amplitude evoked by ACh in PSS using a Mg2+-free intracellular pipette solution. Each data point represents mean current density (pA/pF)+s.E.M. from five cells.

L. A. FIEBER AND D. J. ADAMS

222 A

Cell attached

+100 mV

-c

+60

.

-c

-c

-60

@ jl3 ~~1 pA 200 ms B

Inside-out

+60 mV 9

-c

0 -20

-c f

-60

-c -c

I 2 pA 100 ms

Fig. 2. For legend see facing page.

patches with symmetrical CsCl solutions. In symmetrical BaCl2 solutions, the AChinduced single-channel I-V relationship also was linear and unitary currents exhibited two distinct conductance levels of 20 and 66 pS (n = 3 patches; L. A. Fieber & D. J. Adams, unpublished observations). Since the conductance of the single ACh-gated channel in excised patches appears voltage independent, the number of channels open (NPo, where N is the number of functional channels and PO is the open probability) at the peak of the ACh-evoked current was calculated to be > 103 per neurone. A semilogarithmic plot of NPo versus membrane potential indicates that an e-fold decrease in NPo occurs with approximately 100 mV depolarization.

NEURONAL N\ICOTINIC RECEPTOR CHANNEL C

pA 2

D

223

pA 3 2

-100

-50

50 , X 1

100 ,mV

-100

-50

50

., ,.. -1

100

'mV ~~~~~~~~~~~~~~~-1 -2

-2 -3 -3

-4

Fig. 2. ACh-activated single-channel currents obtained in cell-attached and excised membrane patches from rat cardiac neurones. A, ACh-induced single-channel currents obtained from a cell-attached patch at the potentials indicated applied across the membrane patch. The resting membrane potential was -40 mV. The closed state of the channel is indicated (c). Temperature 23 'C. B, ACh-induced single-channel currents obtained in symmetrical CsCl solution from an inside-out membrane patch at the membrane potentials indicated. C, single-channel current amplitudes obtained from a cell-attached patch plotted as a function of the membrane potential. Each data point represents the average from > sixty events+ S.E.M. These data suggest rectification at positive membrane potentials and a slope conductance of 32 pS was obtained for the linear portion of the I-V curve. D, single-channel current amplitudes from excised patches plotted as a function of the membrane potential. Each data point represents the average current arnplitude from ) twenty events from each of three different patches + S.E.M. The I-V relationship was fitted by linear regression (P < 001) with a slope conductance of 38 pS.

Acetylcholine activates neuronal nicotinic receptors The effect of bath-applied K-bungarotoxin (K-BTX), which has been shown to inhibit ACh-evoked currents in neurones of rat sympathetic ganglia (Sah, Loring & Zigmond, 1987) and retinal ganglion cells (Lipton et al. 1987) is shown in Fig. 3A. K-BTX (0 2 ,UM) attenuated the amplitude of ACh-evoked currents by > 95 %, and this inhibition was maintained during the 20 min wash-out that followed exposure to K-BTX. Cultured neurones in the same dish, examined 30-60 min later, responded to ACh by producing currents of normal magnitude, suggesting that inhibition of AChevoked currents by K-BTX is slowly reversible. Pre-treatment of cultured neurones with K-BTX prior to agonist application inhibited the response to ACh, suggesting that block of ACh-evoked currents by K-BTX did not require the binding of ACh and opening of ACh receptor channels. The effects of the muscarinic receptor antagonist, atropine, and the nicotinic receptor agonist, nicotine, were investigated to determine the receptor specificity of the ACh-induced response in rat cardiac neurones. The time course and peak amplitude of ACh-evoked currents were unchanged during bath application of PSS containing 1 ,aM-atropine (Fig. 3B). Furthermore, the I-V relationship for whole-cell currents evoked by ACh was similar in the absence and presence of atropine (Fig.

L. A. FIEBER AND D. J. ADAMS

224

3C). The ACh-evoked inward current also could be mimicked by a brief pulse of nicotine (100 1am) applied to the soma (not shown). These results suggest that nicotinic receptors alone mediate the excitatory response to ACh. Antagonists of the nicotinic receptor channel The pharmacological properties of the nicotinic receptor channel were investigated to characterize the neuronal nicotinic receptor and to permit a comparison with A

ACh

V Control

nA - 0.5

C 0.2

-120

,uM-K-BTX--

-80

40

-40

200 pA 1 s

B

*-0.5

Atropine

Control

+10 mV

--1.0

--1.5

0 -10

- -2.0

_

-30

it, J 200 pA 1 s

Fig. 3. Effects of K-bungarotoxin (K-BTX) and atropine on ACh-evoked whole-cell currents. A, inhibition of ACh-evoked current amplitude by 0-2 ,UM-K-BTX, bath applied. Holding potential was - 120 mV. Arrow-head indicates the application of a brief pulse (100 ms) of 100 /LM-ACh. B, whole-cell currents evoked by ACh in the absence (control) and presence of bath-applied atropine (1 /tM). (7 I-V relationship for ACh-induced currents shown in B obtained in the absence (@) and presence (A) of 1 /IM-atropine.

nicotinic receptor channels of peripheral autonomic neurones and the vertebrate motor endplate. The actions of the ganglionic blocking agents mecamylamine and hexamethonium (C6). and the neuromuscular relaxant, d-tubocurarine (d-TC), on ACh-evoked currents in parasympathetic neurones were examined. Whole-cell ACh-evoked currents obtained in the absence and in the presence of 0 1 and I 0 /tM-mecamylamine are shown in Fig. 4A. Mecamylamine (0 1yaM) reduced the amplitude but did not alter the time course of the inward currents evoked by ACh. Inhibition by higher mecamylamine concentrations (> 1 ,tM) was voltage dependent, whereby a greater proportional block was observed at hyperpolarized potentials than

NEURONAL NICOTINIC RECEPTOR CHANNEL

225

at depolarized potentials (not shown). The dose-response relationship obtained for inhibition of ACh-evoked currents by mecamylamine is shown in Fig. 4B. Very low concentrations of mecamylamine (1 nm) occasionally potentiated the peak current amplitude relative to the control level. Since potentiation was not consistently A

Control

0.1 jiM-mecamylamine

Control

1 .0 gM-mecamylamine

J 500 pA 1s

B

n 120E 100,

80-

'

60-

o 4020-

m 0 a,

-9

-8

-7

-6

-5

[Mecamylamine] (log M) Fig. 4. Dose-response relationship for inhibition of ACh-evoked currents by mecamylamine. A, representative records of ACh-evoked currents obtained at - 120 mV at the concentrations of mecamylamine indicated. Arrow-head indicates a 100 ms pulse of ACh (< 100 UM). B, dose-response relationship for inhibition of ACh-evoked inward currents by mecamylamine. Each data point represents the mean current amplitude + S.E.M. measured at - 120 mV in three different neurones. The curve of best fit to the data had a Kd = 37 nm and nH = -0-7 (see Methods).

observed, neurones in which it occurred are not included in the average data for 1 nmmecamylamine in the dose-response curve. A Kd of 37 nm and Hill coefficient of -0 7 were calculated from the fit of the dose-response relationship (continuous line, Fig. 4B). Complete block of the ACh-evoked current was not achieved, even at concentrations far exceeding the Kd for mecamylamine inhibition. Whole-cell currents evoked by ACh under control conditions and following bath application of either 3- or 100 jtM-d-TC are shown in Fig. 5A. Low concentrations of d-TC (< 3 aUm) reversibly depressed the peak current amplitude without affecting the time course of ACh-evoked responses, and the current was inhibited to the same PHY 434

L. A. FIEBER AND D. J. ADAMS

226

degree at each membrane potential. Voltage-dependent block was observed, however, in the presence of higher concentrations of d-TC (> 10 ,UM) and the rate of decay of ACh-induced currents was enhanced. For example, the half-time of decay of the AChevoked current was reduced by an average of 30 % (n = 3 cells) in the presence of A

Control

3 pM-d-TC

Control

100 IM-d-TC

J 200 pA 1 s

B -o

100-

,-.

E 80-

tCD 600

° 40C)

0 0

a) 20-

CU) C

a) C. a) 0L

0

- -7

-6

-5

-4

-3

[d-TC] (log M) Fig. 5. Dose-response relationship for inhibition of ACh-evoked currents by dtubocurarine (d-TC). A, representative records of ACh-evoked currents obtained at -120 mV in the presence of 3- and 100 /mM-dTC. B, dose-response relationship for d-TC inhibition of ACh-evoked currents. Each point represents the mean relative current amplitude + S.E.M. determined in at least five neurones at 120 mV. The fitted curve had a Kd = 3 /M and nH = -1. -

100 JtM-d-TC (see Fig. 5A). The dose-response relationship was best fitted assuming uniform affinity site for binding of antagonist with a Kd of 3 /tM and Hill slope of -1 (Fig. 5B). Inhibition of ACh-evoked currents by 300 ,tM-d-TC was incomplete, similar to that observed in 30 /tM-mecamylamine. Bath application of the nicotinic ganglionic blocker, C6, reversibly inhibited inward currents evoked by ACh (Fig. 6A). The rate of decay of ACh-induced currents was increased by approximately 55 % in the presence of 0l tM-C6. The blockade of ACh-evoked currents by C6 exhibited a marked voltage dependence whereby the a

NEURONAL NICOTINIC RECEPTOR CHANNEL

227

degree of inhibition increased with membrane hyperpolarization (Fig. 6B). The dose-response curve for block of ACh-induced currents by C6 was not fitted by a model that assumes a simple interaction between receptor and antagonist molecule (see Fig. 6C). Fifty per cent block (IC50) of the current amplitude by C6 occurred in the micromolar range. B

A

pA r 100

10 ,UM-C6

Control

-30 mV -100

-70

--

-120

10 PM-C6

-200

-300

7

-400 Control

j 200 pA

-500

1 s

C 100 -

0

V

80 -

a.)

C

60 -

-0 0

0)

CD

40 -

T

20 -

a)

0C

C

n_-

-8

-7

-6 [C61 (log M)

-5

-4

Fig. 6. Inhibition of ACh-evoked currents by hexamethonium (C6). A, whole-cell AChevoked currents obtained in the absence and presence of external 1O0UM-C6. B, I-V relationship for inhibition of ACh-evoked currents in the absence (@) and presence of either 10/tM-C6 (V) or 30 /tM-C6 (V). Recovery upon wash-out of hexamethonium is indicated by 0. C, dose-response relationship for inhibition of ACh-evoked currents by hexamethonium. Each data point represents the mean relative current amplitude+ S.E.M. determined at -120 mV in at least five neurones. The data for C6 inhibition of AChevoked currents were not well described by a simple model for receptor-antagonist interaction (see Methods).

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L. A. FIEBER AND D. J. ADAMS

228

Ionic selectivity of the nicotinic receptor channel The neuronal nicotinic receptor channel has been assumed to be a non-selective cation channel analogous to the nicotinic receptor channel in skeletal muscle. The reversal potential (-3 mV) of the ACh-activated current obtained in PSS (see Fig. A

pA r 100

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4

2 Na+

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2 Na+i

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.

0-

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0

100

200

300

[Na+l (mM) on whole-cell of substitution ACh-induced currents in rat cardiac Effects [Na']0 Fig. 7. neurones. A, I-V curves for ACh-evoked currents observed in normal [Na+]. (A), 2 normal [Na']. (70 mM-NaCl, 140 mM-mannitol; *) 4 normal [Na+]O (35 mM-NaCl, 210 mmmannitol; E]), and double normal [Na+]I (290 mM-NaCl) (A). Arrows indicate the measured reversal potential for the ACh-evoked currents in each of these test solutions. B, plot of the reversal potential of ACh-evoked currents as a function of the external Na+ concentration. The continuous curve represents the relation predicted by the GHK equation assuming PCs/PNa = 1-06.

1 C) is consistent with this hypothesis. The relative cation permeability of the. AChactivated channel was investigated by substitution of external Na' with various cations and measurement of shifts in the reversal potential. The I-V relationship for

NEURONAL NICOTINIC RECEPTOR CHANNEL

229

ACh-evoked currents obtained in the presence of I normal [Na+]. (45 mM) showed a reversal potential shift of -310+0-9 mV (S.E.M., n = 3) (Fig. 7A). The shift in reversal potential predicted by the GHK equation, assuming that [Na]i = 0 and that only Na+ and Cs+ are permeant, is -30 mV. The shifts in reversal potential measured B

A

pA 20

pA 20

* -30 -20

10

-40

20

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30

-40 W

-30 . mV

-

20

10

m

40

Na+

Na+ °

-80~~~~~~V4. ~

-60

-60 Na+

-20

Na+

L ~-80

-8

8

Fig. 8. Current-voltage relationship for ACh-evoked currents obtained on replacement of external NaCl with either CsCl or CaCl2. A, I-V curve for ACh-evoked currents in isotonic NaCl (al) and CsCl (A) external solutions. B, I-V curve for ACh-evoked currents in PSS

(ElO) and in isotonic CaCl2 solution (*). Arrow indicates the reversal potential of the ACh-

induced current obtained in I normal

[Na+],.

in I normal [Na+]o and double normal [Na']. were -19 + 2-0 and + 11.5 + 1-7 mV (n = 3), respectively. The current-voltage relations obtained when external NaCl was replaced with mannitol exhibit inward rectification suggesting that rectification follows the shift in reversal potential. The reversal potential of the ACh-induced current as a function of [Na+]. is illustrated in Fig. 7B. Acetylcholine-evoked currents obtained in approximately symmetrical Cs+ solutions exhibited a reversal potential of - 10 +0'9 mV (S.E.M., n = 3; Fig. 8A). The shift in reversal potential between Na+- and Cs+-containing extracellular solutions corresponds to a calculated relative permeability (Pcs/PNa) of 1-06. The relative permeability of the nicotinic receptor channel to Ca2+ was examined by measurement of the reversal potential of ACh-evoked currents following isosmotic replacement of NaCl by CaCl2. The average reversal potential in isotonic CaCl2 (-5 4+0 9 mV; S.E.M., n = 5) was similar to that measured in normal saline (Fig. 8B). The absence of a significant shift of the reversal potential in the presence of isotonic Ca2+ may be compared to the marked shift observed in I normal [Na+]o where mannitol replaced Na+ (arrow). The relative permeability of the neuronal nicotinic receptor channel to Ca2+ calculated from the observed shift in reversal potential in isotonic Ca2+ using a GHK voltage equation derived for Ca2+ (see Methods) was, with respect to Na+, PCa/PNa = 0*93. Although there is variability in the measurement of reversal potential in isotonic Ca2 , the calculated relative

L. A. FIEBER AND D. J. ADAMS

230

permeability ratio indicates that Ca2+ is permeant through the neuronal nicotinic receptor channel. Further evidence in support of a high relative Ca2+ permeability was obtained from measurement of ACh-evoked current amplitude as a function of reciprocal changes c

b

a

J 200 pA 1s n 100

.E

-

a

,600-_

_t

: 40C

0

m cu

20-

a) 0 XL

0

10

133

20

50

75

120

80

50

100 [Ca2+] (mM) 0

[Na+] (mM)

Fig. 9. Whole-cell ACh-induced current amplitude as a function of [Ca2+].. Data from two series of experiments are shown; measurement of ACh-induced current amplitude with first, a constant [Na+]. while varying [Ca2+]o between 0 and 10 mm, and second, in extracellular solutions exchanging CaCl2 isotonically for NaCl. The Ca2+ concentration of PSS, 2-5 mm, was used as the 100% control current amplitude. The lower activity coefficient of CaCl2 compared to NaCl may account for the reduced current amplitude observed in isotonic CaCl2. Each data point represents the mean of three cells + S.E.M. The curve of best fit is according to the equation described in Methods. Representative records of ACh-induced currents obtained in 0 (a), 2-5 (b) and 50 mm (c) [Ca2+]0.

in extracellular Na+ and Ca2+ concentrations (Fig. 9). The amplitude of ACh-evoked currents increased as the [Ca2+]o was raised from 0 to 2-5 mm, but then decreased as [Ca2+]o was raised from 2-5 to 20 mm. At [Ca2+]o of 20-80 mm the current amplitude remained relatively constant, and the rate of decay of the ACh-evoked current was unchanged (see Fig. 9a-c). These data suggest that Ca2+ can substitute equally well for Na+ as a charge carrier through the open nicotinic receptor channel. The anion permeability of the nicotinic receptor channel was examined by replacing extracellular Cl- with gluconate or S042. Substitution of extracellular C1 by gluconate caused a shift in reversal potential of + 1-3+0-3 mV (S.E.M., n = 3), a small change which may be related to a change in activity coefficient of the Na+ salt for the two extracellular solutions. Similarly, substitution of extracellular Cl- by so42- shifted the reversal potential by + 2 mV (not shown). If the nicotinic receptor channel were equally permeable to Na+ and Cl-, but impermeable to gluconate then

NEtKROIA4L N-TCOTINIC RECEPTOR CHANNEL

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the shift in reversal potential predicted from the GilK equation would be + 20 mV. The small shifts in reversal potential observed for AC(h-evoked currenlts in 80 42- an(1 gluconate solutions set the upper limit for PCj/lPNa at 005 and suggests that the neuronal nicotinic receptor channel is cation selective like the nicotinic receptor channel in vertebrate skeletal muscle (Adams, Dwyer & Hille, 1980). DISCUSSION

The observed excitatory response of rat cultured cardiac neurones to a brief pulse of ACh mimics that occurring in neurones of rat cardiac ganglia in situ upon stimulation of the vagus nerve (Seabrook, Fieber & Adams, 1990). The depolarizing response and initiation of action potentials induced by ACh in rat cardiac neurones is analogous to observations on other neurones, such as vertebrate sympathetic neurones (O'Lague et al. 1978; Kuba, Tanaka, Kumamoto & Minota, 1989), chick ciliary ganglion cells (Ogden et al. 1984; Margiotta, Berg & Dionne, 1987), and cardiac ganglion cells of the frog and mudpuppy (Dennis et al. 1971; Roper, 1976; Hartzell et al. 1977). lonophoretic application of ACh to parasympathetic cardiac neurones of the mudpuppy evokes a rapid excitatory postsynaptic potential (EPSP) followed by a slow atropine-sensitive inhibitory postsynaptic potential (IPSP). An ACh-evoked IPSP was not observed in rat cultured parasympathetic neurones (cf. Allen & Burnstock, 1990); the absence of such a response may be due to uncoupling of musearinic receptors from the effector ion channels, as the patch pipette 'intracellular' solution may dilute a necessary second messenger (Horn & Marty, 1988). The inward current elicited in response to exogenously applied ACh, however, indicates that the nicotinic receptor channel in rat cultured neurones retains function in short-term tissue culture, without the addition of growth factors. The response to ACh is similar to that observed in autonomic ganglion cells either innervated (in situ) (Ascher et al. 1979) or denervated (dissociated or cultured) (Ogden et al. 1984; Margiotta et al. 1987; Mathie et al. 1987; Kuba et al. 1989). Activation within 10 ms of ACh application indicates that the nicotinic receptor and ion channel are closely coupled. Ionic currents evoked by ACh in rat cardiac neurones were insensitive to atropine, and were mimicked by brief pulses of nicotine to the neuronal soma. Therefore, ACh-induced currents in rat parasympathetic cardiac neurones are due to the activation of nicotinic receptor channels. The reversal potential for ACh-evoked whole-cell currents in rat cardiac neurones is similar to that reported for ACh responses in retinal ganglion cells (Lipton et al. 1987) and chromaffin cells (Fenwick, Marty & Neher, 1982). Inward rectification of the ACh-evoked whole-cell I-V curve was observed using either K+ or Cs' as the main intracellular cation, and in the presence or absence of intracellular Mg2' and extracellular Ca2+. The rectification appears to follow the shift in reversal potential of the ACh-induced current upon replacement of external NaCl with mannitol and is expressed in the outward direction independent of absolute voltage. The ohmic behaviour of the single-channel I-V relationship in excised patches suggests that the rectification of the whole-cell and cell-attached I-V curves may be attributed, in part, to the voltage dependence of the open-channel current (Mathie et al. 1987). Elimination of internal Mg2+ and external divalent cations fails to alleviate the

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L. A. FIEBER AND D. J. ADAMS

rectification, in contrast with rectification induced by intracellular Mg2+ block of inwardly rectifying K+ channels (Matsuda, Saigusa & Irisawa, 1987). The I-V curve for ACh-evoked whole-cell currents is similar to that reported in rat cultured sympathetic neurones (Mathie et al. 1987), mouse parasympathetic neurones (Yawo, 1989) and porcine cultured hypophyseal intermediate lobe cells (Zhang & Feltz, 1990). The mechanism underlying the non-linearity of the whole-cell I-V relationship remains to be resolved but may result from a voltage dependence of the kinetics of the ACh-activated channel whereby a population of the channels enter an inactive conformation at potentials positive to the reversal potential. The linear I-V relationship for ACh-activated single channels in excised membrane patches is consistent with that observed for ACh-induced unitary currents in other autonomic neurones (Margiotta et al. 1987; Mathie et al. 1987), but contrasts with the inwardly rectifying single-channel I-V curve observed in the presence of intracellular Mg2+ in outside-out patches from PC12 cells (Ifune & Steinbach, 1990). The conductance of ACh-activated channels in rat cardiac neurones is 32 pS in cellattached membrane patches and in excised patches is 38 pS with symmetrical CsCl solutions. Single-channel conductances of 20-50 pS have been determined for other nicotinic receptor channels in mammalian central and peripheral neurones (Aracava et al. 1987; Derkach, North, Selyanko & Skok, 1987; Lipton et al. 1987; Mathie et al. 1987). The amplitude ratio of whole-cell current to single-channel current suggests that there are > 103 functional nicotinic receptor channels per cardiac neurone and, for an estimated surface area of 2000 ,Um2, the average channel density is 0 5 channels per /tm2. K-Bungarotoxin, an antagonist of nicotinic responses in central and peripheral neurones insensitive to the crude snake venom a-BTX (Brown & Fumagalli, 1977, Lipton et al. 1987; Schulz & Zigmond, 1989), blocked ACh-evoked currents in rat cardiac neurones. This sensitivity suggests that K-BTX may be a more reliable marker for the functional neuronal nicotinic receptor channel in mammalian neurones than oc-BTX. The nicotinic ganglionic blocking drugs, mecamylamine and hexamethonium, inhibited ACh-evoked currents in rat cardiac neurones in a dose-dependent manner and exhibited voltage-dependent block, whereby ACh-evoked current amplitude was reduced to a greater extent at hyperpolarized than at depolarized membrane potentials. Potentiation of current amplitude at low concentrations of mecamylamine similar to that seen here was observed for currents evoked by a brief ionophoretic pulse of ACh in the presence of low concentrations of d-TC in rat submandibular ganglion cells (Ascher et al. 1979). The anomalous potentiation of the ACh-induced current in the presence of d-TC was suggested to be due to antagonist interaction with receptor binding sites already occupied by agonist molecules, thus retarding loss of agonist and subsequent closure of channels. Block of ACh-induced currents in cardiac neurones by high concentrations of mecamylamine was voltage dependent, and when considered with a Hill slope, nH < 1 for the dose-response curve suggests that mecamylamine may have multiple sites of action with the nicotinic receptor channel. Hexamethonium produced 50% block of ACh-induced responses in rat cardiac neurones at a concentration of - 1,UM, though this value may represent an -

233 NEURONAL NICOTINIC RECEPTOR CHANNEL underestimate due to a lower antagonist concentration at the cell surface during the pulse of ACh. In contrast, the IC50 for hexamethonium block of ACh responses in rat skeletal muscle is approximately one-hundredfold higher (Rang & Rylett, 1984). The potency of the methonium compounds in block of neuronal ACh-activated channels has been examined previously in rat submandibular ganglia (Gurney & Rang, 1984). The short-chain methonium (C5-C8) compounds exhibited use-dependent block and apparently could be trapped in the closed channel. The ability of the short-chain methonium compounds to enter the open channel and partially occlude the pore may underlie the shape of the dose-response curve for C6 in cardiac neurones and suggests that C6 acts within the membrane electric field. The neuromuscular relaxant, d-TC, produced a dose-dependent block of the AChevoked current in rat cardiac neurones with a Kd for inhibition of 3 /tM. This value is higher than that reported for d-TC block of endplate currents in rat skeletal muscle (IC50 = 1 /,M; Gibb & Marshall, 1984). At low concentrations of d-TC (< 3/tM) inhibition of ACh-induced currents in rat parasympathetic neurones was not voltage dependent, in contrast with observations at the frog neuromuscular junction (Manalis, 1977; Colquhoun, Dreyer & Sheridan, 1979). Incomplete block at high concentrations may be due to a population of nicotinic receptor channels either insensitive to d-TC or partially blocked by d-TC, such that even when bound to the receptor some current flow persists through the 'blocked' channel. A comprehensive study of the actions of nicotinic receptor antagonists has been carried out on neurally evoked and ACh-evoked currents in parasympathetic neurones of the rat submandibular ganglion (Ascher et at. 1979; Rang, 1982). The Kds for inhibition of AChevoked postsynaptic currents in submandibular ganglion cells by C6, d-TC and mecamylamine were similar to those obtained in cultured cardiac neurones suggesting that the pharmacological profile of the nicotinic receptor channel is similar in different mammalian parasympathetic ganglia. The dose-response and kinetic effects of d-TC and C6 on excitatory postsynaptic currents (EPSCs) in frog cardiac ganglia, however, resemble more closely those observed at nicotinic receptors in skeletal muscle (Lipscombe & Rang, 1988). Although d-TC and C6 blockade of ACh-induced responses in rat cardiac neurones exhibited a voltage dependence similar to that observed in frog cardiac neurones, d-TC and C6 increased the rate of decay of ACh-evoked currents. It is not possible to extrapolate the pharmacological properties of neuronal nicotinic receptor channels in amphibian parasympathetic ganglia, which share similarities with muscle nicotinic receptors, to neuronal nicotinic receptors in mammalian parasympathetic ganglia. One explanation for this is that neuronal nicotinic receptor channels composed of different combinations of a- and non-a-subunits have different functional properties (Papke et al. 1989). Although the ionic dependence of the postsynaptic current evoked by ACh in neurones of autonomic ganglia on extracellular cations has been described (Connor, Neel & Parsons, 1985; Lipton et al. 1987; Mathie et al. 1987), there has been no quantitative study of the ionic selectivity or relative permeabilities of the neuronal nicotinic receptor channel to monovalent and divalent cations. The cation permeability of the receptor channel was demonstrated by substitution of extracellular Na+ with Cs+, Ca2+ and mannitol. The measurement of the reversal potential of ACh-induced current as a function of [Na+]o followed that predicted by

234

L. A. FIEBE1R ANI) D. J. ADAMS

the GEIK equation, indicating that Na' is the major permeant cation in the normal extracellular solution. The deviation of the experimentally determined reversal potential at double normal [Na+]0 from the GHK prediction was also observed for the ACh receptor channel at the amphibian neuromuscular junction (Lewis, 1979; Takeda, Barry & Gage, 1982), and may be due to effects of ionic strength of the extracellular solution and/or ion saturation of the channel. The small change in reversal potential observed when Na' was replaced isotonically by either Cs' or Ca2+ suggests that these cations also can permeate the open channel. The selectivity for monovalent and divalent cations is weak with a sequence Cs+ > Na+ > Ca2+, and permeability ratios (PX/PNa) of 1P06, 10 and 0 93. The relative permeability of the neuronal nicotinic receptor channel to monovalent and divalent cations may be compared with that of the endplate channel in frog skeletal muscle where the following sequence and relative permeabilities were obtained: Cs+ (14) > Na+ (10) > Ca2+ (0 2) (Adams et al. 1980). Although the Ca2+ permeability of the neuronal nicotinic receptor channel is lower than that reported for the N-methyl-D-aspartate (NMDA) receptor channel in cultured central neurones (PCa/,-Na = 10 6; Mayer & Westbrook, 1987), nevertheless, the ACh-induced Ca21 influx may have significant implications for the control of second messenger pathways and neuronal function in parasympathetic ganglia. The anion permeability of the nicotinic receptor channel is low, as indicated by the direction and magnitude of the shift of the reversal potential when NaCl was replaced by mannitol and by the small shifts observed in the presence of either gluconate or S042. The amplitude of ACh-evoked currents in rat cardiac neurones increased as the [Ca2+]o was raised from 0 to 2 5 mm. A similar phenomenon was described in bull-frog sympathetic neurones (Connor et al. 1985) where a sixfold increase in [Ca2+]. (09-54 mM) caused a fivefold increase in ACh-evoked current amplitude. The increase in current amplitude upon raising [Ca2+]. is inconsistent with Ca2+ block of the neuronal nicotinic receptor channel and suggests an effect of Ca2+ on channel gating as proposed in bull-frog neurones (Connor et al. 1985). Despite the relatively high Ca2+ permeability of the channel in rat cardiac neurones, the reduced amplitude of ACh-evoked currents with [Ca2+]. concentrations > 2 5 mm suggests that the rate of Ca2+ movement through the open ACh-activated channel is less than for Na+ and that saturation may occur at high [Ca2+]0. It is apparent that Ca2+ can substitute for Na+ as a charge carrier through the open channel and that the decrease in current amplitude at higher [Ca2+]o is unlikely to be due to Ca2+-dependent gating because the rates of ACh-induced current decay were similar. These effects of [Ca2+]0 may be compared to those at the muscle endplate where raising [Ca2+] reduces endplate current (EPC) amplitude and increases the rate of EPC decay (Magleby & Weinstock, 1980). The nicotinic receptor channels in rat cardiac neurones mediate postganglionic neuronal responses following electrical stimulation of the vagus nerve. In this study, we have described some of the pharmacological and ionic permeability properties of the neuronal nicotinic receptor channel and conclude that there are significant functional differences between the nicotinic receptors in mammalian neurones and those found in amphibian ganglia and in skeletal muscle.

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We thank Dr Shiro Konishi for participation in prelimninary experiments and Drs Karl Magleby and Wolfgang Nonner for helpful comments. This work was supported by National Institute of Health grant HL 35422 to D. J. Adams. L. A. Fieber was supported by the Lucille P. Markey Foundation and NIH Trainin1g grant HL 07188.

REFERENCES

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