School ofMedicine, Miami,.FL 33101, USA 2. The current-voltage (IV)

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

ACETYLCHOLINE-EVOKED CURRENTS IN CULTURED NEURONESDISSOCIATED FROM RAT PARASYMPATHETIC CARDIAC GANGLIA

BY LYNNE A. FIEBER* AND DAVID J. ADAMSFrom the Department of Molecular and Cellular Pharmacology, University ofMiami

School of Medicine, Miami,.FL 33101, USA

(Received 26 April 1990)

SUMMARY

1. The properties of acetylcholine (ACh)-activated ion channels of parasym-pathetic neurones from neonatal rat cardiac ganglia grown in tissue culture wereexamined using patch clamp recording techniques. Membrane currents evoked byACh were mimicked by nicotine, attenuated by neuronal bungarotoxin, andunaffected by atropine, suggesting that the ACh-induced currents are mediated bynicotinic receptor activation.

2. The current-voltage (I-V) relationship for whole-cell ACh-evoked currentsexhibited strong inward rectification and a reversal (zero current) potential of-3 mV (NaCl outside, CsCl inside). The rectification was not alleviated by changingthe main permeant cation or by removal of divalent cations from the intracellular orextracellular solutions. Unitary ACh-activated currents exhibited a linear I-Vrelationship with slope conductances of 32 pS in cell-attached membrane patchesand 38 pS in excised membrane patches with symmetrical CsCl solutions.

3. Acetylcholine-induced currents were reversibly inhibited in a dose-dependentmanner by the ganglionic antagonists, mecamylamine (Kd = 37 nM) and hex-amethonium (IC50 1 UtM), as well as by the neuromuscular relaxant, d-tubocurarine(Kd = 3 /M). Inhibition of ACh-evoked currents by hexamethonium could not bedescribed by a simple blocking model for drug-receptor interaction.

4. The amplitude of the ionic current through the open channel was dependent onthe extracellular Na+ concentration. The direction of the shift in reversal potentialupon replacement of NaCl by mannitol indicates that the neuronal nicotinic receptorchannel is cation selective and the magnitude suggests a high cation to anionpermeability ratio. The cation permeability (Px/PNa) followed the ionic selectivitysequence Cs+(1 06) > Na+(1P0) > Ca2+(093). Anion substitution experiments showeda relative anion permeability, PCi/pNa 1< 005.

5. The nicotinic ACh-activated channels described mediate the responses ofpostganglionic parasympathetic neurones of the mammalian heart to vagalstimulation.

* Present address: Department of Cell Biology and Physiology, Washington University, 660 S.Euclid Avenue, St Louis, MO 63108, USA.

MS 8455

L. A. FIEBER AND D. J. ADAMS

INTRODUCTION

The mammalian cardiac ganglion, located in the atrial subepicardium, mediatesthe vagal innervation of the heart and is proposed to play a role in the control of theheart beat (Moravec & Moravec, 1987; Gagliardi, Randall, Bieger, Wurster, Hopkins& Armour, 1988). To date detailed electrophysiological studies of postsynapticresponses in parasympathetic neurones of the heart have been confined to theamphibian 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 neuronesto the neurotransmitter, acetylcholine (ACh), released upon vagal stimulation isimportant for understanding how postsynaptic neuronal responses modulatecholinergic innervation of the heart.Although many structural similarities exist between neuronal and muscle nicotinic

receptors (Lindstrom, Schoepfer & Whiting, 1987; Steinbach & Ifune, 1989), there isconsiderable pharmacological evidence of functional differences between the twotypes. 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-bindingsubunits (/82, 83, /4) of the neuronal nicotinic receptor have been identified. It appearsthat a functional neuronal receptor channel consists of at least one ACh-binding andone non-ACh-binding subunit (Ballivet, Nef, Couturier, Rungger, Bader, Bertrand &Cooper, 1988; Papke, Boulter, Patrick & Heinemann, 1989). Electrophysiologicalstudies of membrane currents evoked in response to exogenous ACh in mammaliancultured central and peripheral neurones provide evidence of numerous types ofneuronal 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 ganglionicblocking agents on neurally evoked postsynaptic currents in rat parasympatheticsubmandibular ganglia (Ascher, Large & Rang, 1979; Rang, 1982) and amphibianparasympathetic cardiac ganglia (Hartzell et al. 1977; Lipscombe & Rang, 1988)have been described, neuronal nicotinic receptor function has not been investigatedextensively in isolated neurones. A foundation for quantitative comparison of thefunctional properties of neuronal and muscle nicotinic receptor channels is lacking.

In this paper we investigate the pharmacology and ionic permeability of theneuronal nicotinic receptor channel in cultured neurones dissociated from ratparasympathetic cardiac ganglia to permit comparison with neuronal nicotinicreceptor channels in amphibian and rat autonomic ganglia. The findings presentedprovide a basis for future studies of the molecular structure of the receptor channel.Preliminary reports ofsome ofthese results have appeared (Adams, Fieber & Konishi,1987; Fieber & Adams, 1988).

METHODS

Preparation and 8olutionsNeonatal 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 bydissecting out individual ganglia following enzyme treatment. Dissected atria containing ganglia

216

NEURONAL NICOTINVIC RECEPTOR CHANNEL 217

were incubated in collagenase (1 mg/ml, Worthington-Biomedical) for 1 h at 37 °C, transferred toa sterile culture dish containing culture medium (Dulbecco's Modified Eagle Medium with 10 mm-glucose, 10% (v/v) fetal calf serum, 100 U/ml penicillin and 0 1 mg/ml streptomycin), trituratedwith a fine-bore Pasteur pipette, then plated onto 18 mm glass cover-slips coated with laminin. Thedissociated cells were incubated at 37 °C in a 95 % air, 5% CO2 atmosphere. Electrophysiologicalrecordings were made from neurones maintained in tissue culture for 48-72 h. At the time ofexperiments, the glass cover-slip was transferred to a low-volume (0 5 ml) recording chamber andviewed 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 wasinvestigated by replacement of NaCl with an osmotically equivalent amount of the following testcompounds: N-methylglucamine chloride, CsCl, CaCl2 and mannitol. Mannitol was chosen oversalts of large positively charged ions such as, N-methylglucamine, as a Na+ substitute because theamplitude of the ACh-evoked current in the presence ofN-methylglucamine was less than predictedfor the extracellular Na' concentration ([Na+]O) (see Adams, Nonner, Dwyer & Hille, 1981;Sanchez, Dani, Siemen & Hille, 1986). To investigate anion selectivity, reversal potentials weremeasured in external solutions containing Cl-, gluconate or S042- as the major anion. Reversalpotential measurements were corrected for differences in junction potential between the bathsolution 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 weremade with respect to a reference electrode (saturated KCl, reverse sleeve junction; Corning X-EL47619). 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 HEPES-CsOH, pH 7-2 (adjusted after addition of ATP), and either 2 CS4BAPTA or 1 CS2EGTA. In a seriesof experiments the free internal Mg2+ concentration ([Mg2J]i) was reduced to negligible levels byreplacing MgATP with Na2ATP in the intracellular pipette solution containing EGTA. Agonist-induced responses were investigated using a pressure application device (Picospritzer II, GeneralValve 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. Agonistwas applied during continuous bath perfusion and exchange of the external solution in therecording chamber was complete within 10 s. The pressure ejection pipette was positionedapproximately 30,tm from the soma mem')rane to evoke maximal responses to agonist undercontrol 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 eliminatefast 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 antagonistconcentration at the cell surface during pressure application of agonist alone will be equal to orlower than that in the bulk solution. The apparent dose-response curves thus may be shiftedtowards higher antagonist concentrations.

All chemical reagents were of analytical grade. Acetylcholine chloride, atropine sulphate,ethyleneglycol-bis-(/-aminoethylether) NN,N',N'-tetraacetic acid (EGTA), hexamethoniumbromide, laminin, mecamylamine hydrochloride, (-)nicotine di-(+)tartrate and d-tubocurarinechloride were obtained from Sigma Chemical Co. (St Louis, MO, USA). 1,2-Bis(2-amino-phenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA, tetracaesium salt) was obtained fromMolecular Probes, Inc. (Eugene, OR, USA). K-Bungarotoxin, a fraction of the venom a-bungaro-toxin (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 recordingsAgonist-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 generatedan 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

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

an analog-to-digital recorder adaptor (PCM- 1; Medical Systems, NY, UTSA). The membranecurrent and voltage were monitored continuously on a digital oscilloscope (Nicolet 3091). The cellcapacitance (Cm) was determined for each cell from the compensation of the capacity transient inresponse 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 currentsevoked by agonist was - t nA, then the voltage error due to R. would be < 6 mV. WVhole-cell an(lsingle-channel currents and voltage were displayed using a chart recorder (Gould 2200S; DC-125 Hz bandwidth) for analysis.For single-channel recording, patch pipettes (thin-walled borosilicate glass, Clark Elec-

tromedical, UK) of 1-2 MQ resistance were made and coated with Sylgard (Dow Corning Corp.,Midland, MI, USA) to within 100lOm of the tip to reduce extraneous electrical noise due tocapacitative 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-HEPES-CsOH, pH 7-2. To obtain an excised membrane patch, a cell-attached patch was pulled slowly awayfrom the soma membrane, carried briefly through the solution-air interface and the bathingsolution exchanged for one corresponding to the pipette solution. Experiments were performed inpairs, the first of each pair in the absence of agonist and the second with the same patch pipettesolution, but containing 10 /tM-ACh. It is possible that single-channel currents obtained from thelatter patches were subject to desensitization. When single-channel activity was observed in a cell-attached membrane patch, the patch was evaluated based on the dependence of single-channelevents on the presence of ACh in the patch pipette. A small conductance channel (<7 pS) wasobserved in approximately one-third of membrane patches in the absence of ACh in symmetricalCsCl solutions, but was not studied. Single-channel currents were analysed to obtain histogramsof current amplitude by either direct measurement using the digital storage oscilloscope and/orby sampling videotape records at 20 kHz (Tecmar Labmaster DMA interface) for analysis on aPC 80286 computer using the pCLAMP programs (Axon Instruments Inc., CA, USA).

Analysis of receptor-antagonist interactionsFor experiments investigating actions of antagonists of the neuronal nicotinic receptor, it was

assumed that the receptor-channel interaction obeyed the law ofmass action. It was also assumedthat the agonist (or blocker)-receptor interaction equilibrates rapidly and that the observedphysiological response resulting from agonist-receptor binding is proportional to receptoroccupancy:

k+1A(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), andsolving for p gives

KJ[A] k+1 1p= where Ka= Kd(1 +Ka[A]) k 1 Kd

Since p is proportional to the observed response,I

control

on (t1oA]dwhere C is a constant. In the case of competitive antagonists, [A] becomes [B], the concentrationof blocker, and the equation is

I 1 | C xd1B

'control = ( +K-l[B

This equation allows calculation of the half-inhibitory concentration of drug, IC50, or for thispurpose referred to as the dissociation constant Kd, by plotting '/Icontrol versus log[B] in the

218

.NEURONAL NICOTINIC RECEPTOR CHANNEL

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 andend-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 permeabilitie8Relative 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 shiftsin 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'] arethe 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 thesalts 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 = 4Pa V([ ]2)= 4Pa V(( ;] r)Ca Ca I-exp (2V) Ca (I1+exp(V) (1-exp(V')

iNa =Na Vle;p()

and ics = PC. V [1 ex (V)I-ep (V)

where V =FRT

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 + PNa[Na ]O+ PNa[NaJ]OX P5[Cs]]iX PCs[1]X= 0.

Solving for PCaNa

PCa (PC/ PNa) [CsJ]X(1 +X)-[Naa ] (1 +X)PNa 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)}_N2PNa 4[Ca ]

219

L. A. FIEBER AiND D. J. ADAMS

RESULTS

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

Acetylcholine-evoked currents in intracardiac neuronesAcetylcholine applied from a pressure ejection pipette evoked either a single action

potential or a train of action potentials in the neurone when recorded in currentclamp mode (Fig. 1A), whereas pressure ejection of PSS (without ACh) produced noresponse. Under voltage clamp conditions, ACh evoked a transient inward currentin cultured cardiac neurones, with the amplitude dependent on the membranepotential (Fig. 1B). The amplitude of the ACh-induced current at a holding potentialof -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 amplitudebefore the cessation of the pulse. The half-time of decay of the ACh-induced currentwas 10+0 13 s (S.E.M., n = 20 cells) at -70 mV and was independent of membranep6tential over the range -150 to + 50 mV. Desensitization of ACh-evoked currentswas apparent from the marked reduction of current amplitude observed in responseto 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 conditionsaveraged -3+0-1 mV (S.E.M., n = 25). ACh-evoked currents obtained at negativeholding potentials were larger than at the corresponding positive potentials. Inwardrectification occurred with either Cs' or K+ as the major intracellular cation and inthe absence of external Ca2+. In six cells dialysed with an intracellular pipettesolution containing no added Mg2+ (and 10 mM-EGTA), inward rectification of theI-V relationship also was observed (Fig. 1D).

Voltage dependence of ACh-induced single-channel currentsTo investigate the mechanism(s) underlying the inward rectification of the

macroscopic I-V curve for ACh-evoked currents, the I-V relationship for single ACh-induced channels was determined in cell-attached and excised membrane patches.Typical records of ACh-induced single-channel currents from cell-attached andinside-out membrane patches are shown in Fig. 2A and B respectively. Thecorresponding single-channel I-V relationships are shown in Fig. 2 C and D. Unitary

220

NEURONAL NVICOTINIC RECEPTOR CHANNEL 221

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

A

AI

B

-J 20 mV

1 s

AChV

+30 mV

+10

0

-10

-30

C

-120 -90 -60 -30

pA/pF D pA/pFr 10 rF 1o

-10

-20

-30

-40

-50

-60

*-70

Fig. 1. Excitatory response of rat cultured cardiac neurones to exogenous ACh. A, voltagerecord 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 membranepotentials 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. Eachdata 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.

j 100 pA

1 s

50mV


ACell attached

+100 mV -c

+60 . -c

-60 -c

@ jl3 ~~1 pA200 ms

BInside-out

+60 mV9 -c

0 -c

-20 f -c

-60 -c

I 2 pA

100 msFig. 2. For legend see facing page.

patches with symmetrical CsCl solutions. In symmetrical BaCl2 solutions, the ACh-induced single-channel I-V relationship also was linear and unitary currentsexhibited two distinct conductance levels of 20 and 66 pS (n = 3 patches; L. A.Fieber & D. J. Adams, unpublished observations). Since the conductance of thesingle ACh-gated channel in excised patches appears voltage independent, thenumber of channels open (NPo, where N is the number of functional channels and POis 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 potentialindicates that an e-fold decrease in NPo occurs with approximately 100 mVdepolarization.

222

NEURONAL N\ICOTINIC RECEPTOR CHANNEL

C pA D pA2 3

2

-100 -50 50 100 -100 -50 50 100, X 1 ,mV ,..., 'mV

-1 ~~~~~~~~~~~~~~~-1

-2

-2-3

-3 -4

Fig. 2. ACh-activated single-channel currents obtained in cell-attached and excisedmembrane patches from rat cardiac neurones. A, ACh-induced single-channel currentsobtained from a cell-attached patch at the potentials indicated applied across themembrane patch. The resting membrane potential was -40 mV. The closed state of thechannel is indicated (c). Temperature 23 'C. B, ACh-induced single-channel currentsobtained in symmetrical CsCl solution from an inside-out membrane patch at themembrane potentials indicated. C, single-channel current amplitudes obtained from acell-attached patch plotted as a function of the membrane potential. Each data pointrepresents the average from > sixty events+S.E.M. These data suggest rectification atpositive membrane potentials and a slope conductance of 32 pS was obtained for thelinear portion of the I-V curve. D, single-channel current amplitudes from excised patchesplotted as a function of the membrane potential. Each data point represents the averagecurrent arnplitude from ) twenty events from each of three different patches + S.E.M. TheI-V relationship was fitted by linear regression (P < 001) with a slope conductance of38 pS.

Acetylcholine activates neuronal nicotinic receptorsThe 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 %, andthis inhibition was maintained during the 20 min wash-out that followed exposure toK-BTX. Cultured neurones in the same dish, examined 30-60 min later, responded toACh by producing currents of normal magnitude, suggesting that inhibition of ACh-evoked currents by K-BTX is slowly reversible. Pre-treatment of cultured neuroneswith K-BTX prior to agonist application inhibited the response to ACh, suggestingthat block of ACh-evoked currents by K-BTX did not require the binding of ACh andopening 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 ofthe ACh-induced response in rat cardiac neurones. The time course and peakamplitude of ACh-evoked currents were unchanged during bath application of PSScontaining 1 ,aM-atropine (Fig. 3B). Furthermore, the I-V relationship for whole-cellcurrents evoked by ACh was similar in the absence and presence of atropine (Fig.

223


3C). The ACh-evoked inward current also could be mimicked by a brief pulse ofnicotine (100 1am) applied to the soma (not shown). These results suggest thatnicotinic receptors alone mediate the excitatory response to ACh.

Antagonists of the nicotinic receptor channelThe pharmacological properties of the nicotinic receptor channel were investigated

to characterize the neuronal nicotinic receptor and to permit a comparison with

A ACh

VControl

0.2 ,uM-K-BTX--

200 pA1 s

BControl

+10 mV0

-10

-30 _it,

C

-120 -80 -40

Atropine

nA- 0.5

40

*-0.5

--1.0

--1.5

- -2.0

J 200 pA1 s

Fig. 3. Effects of K-bungarotoxin (K-BTX) and atropine on ACh-evoked whole-cellcurrents. 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-inducedcurrents shown in B obtained in the absence (@) and presence (A) of 1 /IM-atropine.

nicotinic receptor channels of peripheral autonomic neurones and the vertebratemotor endplate. The actions of the ganglionic blocking agents mecamylamine andhexamethonium (C6). and the neuromuscular relaxant, d-tubocurarine (d-TC), onACh-evoked currents in parasympathetic neurones were examined.

Whole-cell ACh-evoked currents obtained in the absence and in the presence of 0 1and I 0 /tM-mecamylamine are shown in Fig. 4A. Mecamylamine (0 1yaM) reduced theamplitude 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

224

NEURONAL NICOTINIC RECEPTOR CHANNEL 225

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

AControl 0.1 jiM-mecamylamine

Control 1 .0 gM-mecamylamine

J 500 pAB 1s

n 120-

E 100-

, 80-

' 60-

o 40-

m 20-

0a,

-9 -8 -7 -6 -5[Mecamylamine] (log M)

Fig. 4. Dose-response relationship for inhibition of ACh-evoked currents by mec-amylamine. A, representative records of ACh-evoked currents obtained at - 120 mV atthe concentrations of mecamylamine indicated. Arrow-head indicates a 100 ms pulse ofACh (< 100 UM). B, dose-response relationship for inhibition of ACh-evoked inwardcurrents by mecamylamine. Each data point represents the mean current ampli-tude + S.E.M. measured at - 120 mV in three different neurones. The curve of best fit tothe 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 nm-mecamylamine in the dose-response curve. A Kd of 37 nm and Hill coefficient of -0 7were calculated from the fit of the dose-response relationship (continuous line, Fig.4B). Complete block of the ACh-evoked current was not achieved, even atconcentrations far exceeding the Kd for mecamylamine inhibition.

Whole-cell currents evoked by ACh under control conditions and following bathapplication of either 3- or 100 jtM-d-TC are shown in Fig. 5A. Low concentrations ofd-TC (< 3 aUm) reversibly depressed the peak current amplitude without affecting thetime course of ACh-evoked responses, and the current was inhibited to the same

PHY 434

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

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 ofACh-induced currents was enhanced. For example, the half-time of decay of the ACh-evoked current was reduced by an average of 30% (n = 3 cells) in the presence of

AControl

Control

3 pM-d-TC

100 IM-d-TC

J 200 pA1 s

-6 -5

[d-TC] (log M)

-4 -3

Fig. 5. Dose-response relationship for inhibition of ACh-evoked currents by d-tubocurarine (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-TCinhibition of ACh-evoked currents. Each point represents the mean relative currentamplitude + S.E.M. determined in at least five neurones at - 120 mV. The fitted curve hada Kd = 3 /M and nH = -1.

100 JtM-d-TC (see Fig. 5A). The dose-response relationship was best fitted assuminga 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 currentswas increased by approximately 55% in the presence of 0l tM-C6. The blockade ofACh-evoked currents by C6 exhibited a marked voltage dependence whereby the

B-o 100-,-.

E 80-

CDt 60-0

° 40-C)00

a) 20-CU)Ca)C. 0a) -0L -7

NEURONAL NICOTINIC RECEPTOR CHANNEL

degree of inhibition increased with membrane hyperpolarization (Fig. 6B). Thedose-response curve for block of ACh-induced currents by C6 was not fitted by amodel 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 inthe micromolar range.

BAControl 10 ,UM-C6

-30 mV

-70 --

-120 7

j 200 pA

1 s

C

V

a.)

-0

0

0)

CDa)0C

100 -

80 -

60 -

40 -

20 -

n_-

-8

10 PM-C6

Control

pAr 100

-100

-200

-300

-400

-500

0

C

T

C

-7 -6[C61 (log M)

-5 -4

Fig. 6. Inhibition of ACh-evoked currents by hexamethonium (C6). A, whole-cell ACh-evoked currents obtained in the absence and presence of external 1O0UM-C6. B, I-Vrelationship for inhibition of ACh-evoked currents in the absence (@) and presence ofeither 10/tM-C6 (V) or 30 /tM-C6 (V). Recovery upon wash-out of hexamethonium isindicated by 0. C, dose-response relationship for inhibition of ACh-evoked currents byhexamethonium. 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 ACh-evoked currents were not well described by a simple model for receptor-antagonistinteraction (see Methods).

8-2

227


Ionic selectivity of the nicotinic receptor channelThe neuronal nicotinic receptor channel has been assumed to be a non-selective

cation channel analogous to the nicotinic receptor channel in skeletal muscle. Thereversal potential (-3 mV) of the ACh-activated current obtained in PSS (see Fig.

ApA

r 100

' Na+4

2 Na+

2 Na+i -200

B20 -

10"

-. 0-

c0ao -10

X' -20-a)) -30.

.

0 100 200 300[Na+l (mM)

Fig. 7. Effects of [Na']0 substitution on whole-cell ACh-induced currents in rat cardiacneurones. 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 mm-mannitol; E]), and double normal [Na+]I (290 mM-NaCl) (A). Arrows indicate themeasured 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 GHKequation assuming PCs/PNa = 1-06.

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

228


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

A BpA pA

20 20

* ' W . mV-40 -30 -20 - 10 20mV m

-30 -20 10 20 30 40

Na+ ° Na+ --80~~~~~~V4.~-40

-60-60

Na+ -8Na+ L ~-80

8

Fig. 8. Current-voltage relationship for ACh-evoked currents obtained on replacement ofexternal NaCl with either CsCl or CaCl2. A, I-V curve for ACh-evoked currents in isotonicNaCl (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 wasreplaced with mannitol exhibit inward rectification suggesting that rectificationfollows the shift in reversal potential. The reversal potential of the ACh-inducedcurrent 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 extracellularsolutions 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 ofACh-evoked currents following isosmoticreplacement 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 ofisotonic Ca2+ may be compared to the marked shift observed in I normal [Na+]owhere mannitol replaced Na+ (arrow). The relative permeability of the neuronalnicotinic receptor channel to Ca2+ calculated from the observed shift in reversalpotential in isotonic Ca2+ using a GHK voltage equation derived for Ca2+ (seeMethods) was, with respect to Na+, PCa/PNa = 0*93. Although there is variability inthe measurement of reversal potential in isotonic Ca2 , the calculated relative

229


permeability ratio indicates that Ca2+ is permeant through the neuronal nicotinicreceptor channel.

Further evidence in support of a high relative Ca2+ permeability was obtained frommeasurement of ACh-evoked current amplitude as a function of reciprocal changes

a b c

J 200 pA

1 s

n 100

E. - a

,600-_ _t

: 40-C0

m 20-cua)

0XL 0 10 20 50 75 100 [Ca2+] (mM)

133 120 80 50 0 [Na+] (mM)Fig. 9. Whole-cell ACh-induced current amplitude as a function of [Ca2+].. Data from twoseries of experiments are shown; measurement of ACh-induced current amplitude withfirst, a constant [Na+]. while varying [Ca2+]o between 0 and 10 mm, and second, inextracellular solutions exchanging CaCl2 isotonically for NaCl. The Ca2+ concentration ofPSS, 2-5 mm, was used as the 100% control current amplitude. The lower activitycoefficient of CaCl2 compared to NaCl may account for the reduced current amplitudeobserved in isotonic CaCl2. Each data point represents the mean of three cells+ S.E.M. Thecurve of best fit is according to the equation described in Methods. Representative recordsof 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-evokedcurrents 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 amplituderemained relatively constant, and the rate of decay of the ACh-evoked current wasunchanged (see Fig. 9a-c). These data suggest that Ca2+ can substitute equally wellfor 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 C1by gluconate caused a shift in reversal potential of + 1-3+0-3 mV (S.E.M., n = 3), asmall change which may be related to a change in activity coefficient of the Na+ saltfor the two extracellular solutions. Similarly, substitution of extracellular Cl- byso42- shifted the reversal potential by + 2 mV (not shown). If the nicotinic receptorchannel were equally permeable to Na+ and Cl-, but impermeable to gluconate then

NEtKROIA4L N-TCOTINIC RECEPTOR CHANNEL

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 8042- an(1gluconate solutions set the upper limit for PCj/lPNa at 005 and suggests that theneuronal nicotinic receptor channel is cation selective like the nicotinic receptorchannel in vertebrate skeletal muscle (Adams, Dwyer & Hille, 1980).

DISCUSSION

The observed excitatory response of rat cultured cardiac neurones to a brief pulseof ACh mimics that occurring in neurones of rat cardiac ganglia in situ uponstimulation of the vagus nerve (Seabrook, Fieber & Adams, 1990). The depolarizingresponse and initiation of action potentials induced by ACh in rat cardiac neuronesis analogous to observations on other neurones, such as vertebrate sympatheticneurones (O'Lague et al. 1978; Kuba, Tanaka, Kumamoto & Minota, 1989), chickciliary ganglion cells (Ogden et al. 1984; Margiotta, Berg & Dionne, 1987), andcardiac ganglion cells of the frog and mudpuppy (Dennis et al. 1971; Roper, 1976;Hartzell et al. 1977). lonophoretic application of ACh to parasympathetic cardiacneurones of the mudpuppy evokes a rapid excitatory postsynaptic potential (EPSP)followed by a slow atropine-sensitive inhibitory postsynaptic potential (IPSP). AnACh-evoked IPSP was not observed in rat cultured parasympathetic neurones (cf.Allen & Burnstock, 1990); the absence of such a response may be due to uncouplingof 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 functionin short-term tissue culture, without the addition of growth factors. The response toACh 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 msof ACh application indicates that the nicotinic receptor and ion channel are closelycoupled. Ionic currents evoked by ACh in rat cardiac neurones were insensitive toatropine, and were mimicked by brief pulses of nicotine to the neuronal soma.Therefore, ACh-induced currents in rat parasympathetic cardiac neurones are due tothe 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 ofthe ACh-evoked whole-cell I-V curve was observed using either K+ or Cs' as the mainintracellular cation, and in the presence or absence of intracellular Mg2' andextracellular Ca2+. The rectification appears to follow the shift in reversal potentialof the ACh-induced current upon replacement of external NaCl with mannitol and isexpressed in the outward direction independent of absolute voltage. The ohmicbehaviour of the single-channel I-V relationship in excised patches suggests that therectification of the whole-cell and cell-attached I-V curves may be attributed, inpart, 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

231


rectification, in contrast with rectification induced by intracellular Mg2+ block ofinwardly rectifying K+ channels (Matsuda, Saigusa & Irisawa, 1987). The I-V curvefor ACh-evoked whole-cell currents is similar to that reported in rat culturedsympathetic 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 relationshipremains to be resolved but may result from a voltage dependence of the kinetics ofthe ACh-activated channel whereby a population of the channels enter an inactiveconformation at potentials positive to the reversal potential. The linear I-Vrelationship for ACh-activated single channels in excised membrane patches isconsistent with that observed for ACh-induced unitary currents in other autonomicneurones (Margiotta et al. 1987; Mathie et al. 1987), but contrasts with the inwardlyrectifying single-channel I-V curve observed in the presence of intracellular Mg2+ inoutside-out patches from PC12 cells (Ifune & Steinbach, 1990).The conductance of ACh-activated channels in rat cardiac neurones is 32 pS in cell-

attached membrane patches and in excised patches is 38 pS with symmetrical CsClsolutions. Single-channel conductances of 20-50 pS have been determined for othernicotinic receptor channels in mammalian central and peripheral neurones (Aracavaet 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 suggeststhat 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 5channels per /tm2.

K-Bungarotoxin, an antagonist of nicotinic responses in central and peripheralneurones insensitive to the crude snake venom a-BTX (Brown & Fumagalli, 1977,Lipton et al. 1987; Schulz & Zigmond, 1989), blocked ACh-evoked currents in ratcardiac neurones. This sensitivity suggests that K-BTX may be a more reliablemarker for the functional neuronal nicotinic receptor channel in mammalianneurones than oc-BTX.The nicotinic ganglionic blocking drugs, mecamylamine and hexamethonium,

inhibited ACh-evoked currents in rat cardiac neurones in a dose-dependent mannerand exhibited voltage-dependent block, whereby ACh-evoked current amplitude wasreduced to a greater extent at hyperpolarized than at depolarized membranepotentials. Potentiation of current amplitude at low concentrations of meca-mylamine similar to that seen here was observed for currents evoked by a briefionophoretic pulse of ACh in the presence of low concentrations of d-TC in ratsubmandibular ganglion cells (Ascher et al. 1979). The anomalous potentiation of theACh-induced current in the presence of d-TC was suggested to be due to antagonistinteraction with receptor binding sites already occupied by agonist molecules, thusretarding loss of agonist and subsequent closure of channels. Block of ACh-inducedcurrents in cardiac neurones by high concentrations of mecamylamine was voltagedependent, and when considered with a Hill slope, nH < 1 for the dose-responsecurve suggests that mecamylamine may have multiple sites of action with thenicotinic 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

232


underestimate due to a lower antagonist concentration at the cell surface during thepulse of ACh. In contrast, the IC50 for hexamethonium block of ACh responses in ratskeletal muscle is approximately one-hundredfold higher (Rang & Rylett, 1984). Thepotency of the methonium compounds in block of neuronal ACh-activated channelshas been examined previously in rat submandibular ganglia (Gurney & Rang, 1984).The short-chain methonium (C5-C8) compounds exhibited use-dependent block andapparently could be trapped in the closed channel. The ability of the short-chainmethonium compounds to enter the open channel and partially occlude the pore mayunderlie the shape of the dose-response curve for C6 in cardiac neurones and suggeststhat C6 acts within the membrane electric field.The neuromuscular relaxant, d-TC, produced a dose-dependent block of the ACh-

evoked current in rat cardiac neurones with a Kd for inhibition of 3 /tM. This valueis 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 voltagedependent, in contrast with observations at the frog neuromuscular junction(Manalis, 1977; Colquhoun, Dreyer & Sheridan, 1979). Incomplete block at highconcentrations may be due to a population of nicotinic receptor channels eitherinsensitive to d-TC or partially blocked by d-TC, such that even when bound to thereceptor some current flow persists through the 'blocked' channel. A comprehensivestudy of the actions of nicotinic receptor antagonists has been carried out on neurallyevoked and ACh-evoked currents in parasympathetic neurones of the rat sub-mandibular ganglion (Ascher et at. 1979; Rang, 1982). The Kds for inhibition of ACh-evoked postsynaptic currents in submandibular ganglion cells by C6, d-TC andmecamylamine were similar to those obtained in cultured cardiac neuronessuggesting that the pharmacological profile of the nicotinic receptor channel issimilar in different mammalian parasympathetic ganglia. The dose-response andkinetic effects of d-TC and C6 on excitatory postsynaptic currents (EPSCs) in frogcardiac ganglia, however, resemble more closely those observed at nicotinic receptorsin skeletal muscle (Lipscombe & Rang, 1988). Although d-TC and C6 blockade ofACh-induced responses in rat cardiac neurones exhibited a voltage dependencesimilar to that observed in frog cardiac neurones, d-TC and C6 increased the rate ofdecay of ACh-evoked currents. It is not possible to extrapolate the pharmacologicalproperties of neuronal nicotinic receptor channels in amphibian parasympatheticganglia, which share similarities with muscle nicotinic receptors, to neuronalnicotinic receptors in mammalian parasympathetic ganglia. One explanation for thisis that neuronal nicotinic receptor channels composed of different combinations ofa- 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 noquantitative study of the ionic selectivity or relative permeabilities of the neuronalnicotinic receptor channel to monovalent and divalent cations. The cationpermeability of the receptor channel was demonstrated by substitution ofextracellular Na+ with Cs+, Ca2+ and mannitol. The measurement of the reversalpotential of ACh-induced current as a function of [Na+]o followed that predicted by

233

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

the GEIK equation, indicating that Na' is the major permeant cation in the normalextracellular solution. The deviation of the experimentally determined reversalpotential at double normal [Na+]0 from the GHK prediction was also observed forthe ACh receptor channel at the amphibian neuromuscular junction (Lewis, 1979;Takeda, Barry & Gage, 1982), and may be due to effects of ionic strength of theextracellular solution and/or ion saturation of the channel. The small change inreversal potential observed when Na' was replaced isotonically by either Cs' or Ca2+suggests that these cations also can permeate the open channel. The selectivity formonovalent and divalent cations is weak with a sequence Cs+ > Na+ > Ca2+, andpermeability ratios (PX/PNa) of 1P06, 10 and 0 93. The relative permeability of theneuronal nicotinic receptor channel to monovalent and divalent cations may becompared with that of the endplate channel in frog skeletal muscle where the followingsequence and relative permeabilities were obtained: Cs+ (14) > Na+ (10) > Ca2+(0 2) (Adams et al. 1980). Although the Ca2+ permeability of the neuronal nicotinicreceptor 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 implicationsfor the control of second messenger pathways and neuronal function in par-asympathetic ganglia. The anion permeability of the nicotinic receptor channel islow, as indicated by the direction and magnitude of the shift of the reversal potentialwhen NaCl was replaced by mannitol and by the small shifts observed in the presenceof 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-frogsympathetic neurones (Connor et al. 1985) where a sixfold increase in [Ca2+].(09-54 mM) caused a fivefold increase in ACh-evoked current amplitude. Theincrease in current amplitude upon raising [Ca2+]. is inconsistent with Ca2+ block ofthe neuronal nicotinic receptor channel and suggests an effect of Ca2+ on channelgating as proposed in bull-frog neurones (Connor et al. 1985). Despite the relativelyhigh Ca2+ permeability of the channel in rat cardiac neurones, the reduced amplitudeof ACh-evoked currents with [Ca2+]. concentrations > 2 5 mm suggests that the rateof Ca2+ movement through the open ACh-activated channel is less than for Na+ andthat saturation may occur at high [Ca2+]0. It is apparent that Ca2+ can substitute forNa+ as a charge carrier through the open channel and that the decrease in currentamplitude at higher [Ca2+]o is unlikely to be due to Ca2+-dependent gating becausethe rates of ACh-induced current decay were similar. These effects of [Ca2+]0 may becompared to those at the muscle endplate where raising [Ca2+] reduces endplatecurrent (EPC) amplitude and increases the rate ofEPC 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 ofthe neuronal nicotinic receptor channel and conclude that there are significantfunctional differences between the nicotinic receptors in mammalian neurones andthose found in amphibian ganglia and in skeletal muscle.

234

lNEUROlNAL NICCOTINIC RECEPTOR CHANNEL 235

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

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School ofMedicine, Miami,.FL 33101, USA 2. The current-voltage (IV)