comparison nicotinic neurones

Journal of Physiology (1994), 481.1

Comparison of neuronal nicotinic receptors in rat sympatheticneurones with subunit pairs expressed in Xenopus oocytes

P. J. 0. Covernton *, H. Kojima*, L. G. Sivilotti, A. J. Gibb and D. Colquhoun

Department of Pharmacology, University College London, Gower Street,London WC1E 6BT, UK

1. The agonist sensitivity of nicotinic acetylcholine receptors in rat superior cervicalganglion (SCG) neurones was compared with that of cloned receptors expressed inXenopus oocytes by pairwise injections of a3-,B2 or a3-,B4 neuronal nicotinic subunitcombinations.

2. Agonist responses in rat SCG neurones indicated that cytisine was the most potentagonist and lobeline the least potent (rank order of potency: cytisine > dimethylphenyl-piperazinium iodide (DMPP) > nicotine > ACh > carbachol > lobeline).

3. Receptors expressed in oocytes by injection of a3 and ,82 subunits had a relatively highsensitivity to DMPP and low sensitivity to cytisine (rank order of potency:DMPP > ACh > lobeline > carbachol > nicotine > cytisine), whereas receptorscomposed of a3 and /64 subunits had a high sensitivity to cytisine and low sensitivity toDMPP (rank order of potency: cytisine > nicotine - ACh > DMPP > carbachol >lobeline).

4. With the exception of responses to DMPP, agonist sensitivity measurements suggestthat nicotinic receptors in the rat SCG are composed of a3 and /14 subunits. The resultsare discussed in terms of the receptor subunit mRNAs known to be expressed in the ratSCG and previous evidence of functional heterogeneity of rat SCG nicotinicacetylcholine receptors.

Several subunit types for neuronal nicotinic acetylcholinereceptors (neuronal AChRs) have now been cloned(reviewed by Deneris, Connolly, Rogers & Duvoisin, 1991)with a total of seven a- and three fl-subunits so far. ThemRNAs encoding these subunits are expressed selectivelyin different parts of the nervous system, but it is notknown to what extent this is related to functional diversityamong native nicotinic receptors.The subunit structure of the Torpedo nicotinic acetyl-

choline receptor is now well known, and the musclereceptor is probably the same. It is a pentamer of subunitstermed a, ,B, y and 8, with the stoichiometry 2:1:1:1. Thestructures of native neuronal AChRs are, however, stilllargely unknown. When expressed in oocytes, a-fl subunitpairs form functional receptors, although the subunitcomposition of native receptors in autonomic ganglia maybe more complex (e.g. Listerud, Brussard, Devay, Colman& Role, 1991; Vernallis, Conroy & Berg, 1993). However,not all neuronal subunits appear to be functional whenexpressed in this manner: e.g. a5 and /13 in allcombinations tested so far (see Deneris et al. 1991) and some(e.g. a7) form functional receptors homomerically (Seguela,

Wadiche, Dineley-Miller, Dani & Patrick, 1993). Ratsuperior cervical ganglion (SCG) neurones express mRNAsfor the a3, a7, /2 and /14 subunits of the nicotinic acetyl-choline receptor, with conflicting information concerningthe presence of a4-1 and a5 mRNAs (Rust, Burgunder,Lauterburg & Cachelin, 1994; Mandelzys, Pie, Deneris &Cooper, 1994), but it is not known which of these subunitsco-assemble to form the native functional receptors. Thesereceptors have been extensively studied and show anumber of functional as well as structural differences fromthose found in muscle (reviewed by Role, 1992).One way to characterize receptor subtypes is by

measuring the relative potencies of a series of agonists; forexample, cytisine is a potent agonist, relative toacetylcholine, for a3-,B4 receptors, but very weak fora3-/12 receptors (Luetje & Patrick, 1991; and presentwork). In order to cast light on the nature of the nativereceptors in rat sympathetic neurones, we have madeparallel measurements, under comparable conditions, ofagonist potency ratios on (a) neurones of the rat superiorcervical ganglion, and (b) AChRs expressed in Xenopuslaevis oocytes using a3-/12 or a3-,/4 subunit pairs. These

* Authors listed alphabetically.

This manuscript was accepted as a Short Paper for rapid publication.

MS 3557, pp. 27-34 27

P. J. 0. Covernton and others

two pairs would appear to be a good choice for comparisonwith the rat SCG as the a3 and /34 mRNAs have thehighest level of expression in this tissue (Rust et al. 1994;Mandelzys et al. 1994). Our results indicate that the relativepotencies of agonists in rat SCG neurones are inconsistentwith those for the a3-/2 subunit combination alone, andare similar to, but not the same as, those for the a3-/34receptor. Some of these results have appeared in abstractform (Kojima, Covernton, Sivilotti, Gibb & Colquhoun, 1994).

METHODSAll measurements were made at room temperature (20-25 °C),using the low concentration end of the dose-response curve tominimize desensitization. Nominally calcium-free extra-cellular solutions were used for both preparations. In the caseof the oocytes this helped to avoid calcium-activated chloridecurrents (Leonard & Kelso, 1990).

Preparation of oocytes for injection and recordingSolutions used for the preparation and injection of oocytes hadthe following compositions (mM). Nuclear injection buffer:NaCl, 88; KCl, 1; Hepes, 15; pH 7 0. Sterile Barth's medium:NaCl, 88; KCl, 1; MgSO4, 0f82; Ca(NO3)2, 0 33; CaCl2, 0f41;NaHCO3, 2-4; Tris-HCl, 15; with 50 U ml- penicillin and50 jug ml-' streptomycin; pH 7-4. Recordings were made usinga nominally calcium-free solution of the following composition(mM): NaCl, 115; MgCl2, 18; KCl, 2X5; Hepes, 10; and 500 nMatropine sulphate (Sigma) to block endogenous muscarinicreceptors; pH 7-2. Stock solutions of acetylcholine chloride(ACh), (-)-nicotine hydrogen tartrate (Nic), (-)-lobelinehydrochloride (Lob), dimethylphenylpiperazinium iodide(DMPP), (-)-cytisine (Cyt) and carbachol (CCh) (all Sigma)were kept frozen and diluted to the desired concentrationwhen needed. Cloned cDNAs suitable for direct nuclearinjection (pcDNAIneo vector; see Luetje & Patrick, 1991) werekindly supplied to us by Dr J. Patrick (Baylor College ofMedicine, Houston, TX, USA).

Mature Xenopus laevis were anaesthetized with 0-2%tricaine methanesulphonate (Sigma) and ovarian lobes wereremoved surgically (Methfessel, Witzemann, Takahashi,Mishina, Numa & Sakmann, 1986). Stage V-VI oocytes weremanually dissected and stored in sterile Barth's medium forone day, then used for injection. A sharp bevelled pipette (tipdiameter, 5-10 /sm) was inserted into the animal pole of theoocyte, and about 10 nl cDNA (041 jug ,F1-) was injected intothe nucleus. Two days after injection, oocytes were treatedwith 1 mg ml-' collagenase (type IA, Sigma) in Barth'smedium for 1 h at 19 °C, and the follicle cell layer removed asdescribed by Methfessel et al. (1986). Defolliculated oocyteswere maintained at 19 °C in Barth's medium. Three days afterinjection, oocytes were tested for expression of nicotinic AChRby bath application of 10/M cytisine or 20 /M DMPP for thea3-/l4 and a3-/l2 combinations, respectively. Oocytes givingresponses of more than 200 nA (at -60 mV) were used forsubsequent experiments.

Oocytes were superfused continuously (at about2-16 ml min-'; chamber volume, 300 1l) with the recordingsolution, and agonists were bath applied for up to 40 s at5 min intervals. Current responses to agonist application weremeasured using a two-electrode voltage clamp (Axoclamp 2A,Axon Instruments, Foster City, CA, USA), at a holdingpotential of -60 mV, and recorded on a chart recorder.

Microelectrodes were filled with 3 M KCl and had a resistanceof05-30 MQ.

Preparation of and recording from sympathetic neuronesA calcium-free Krebs solution was used for preparation,enzyme treatment and recording from SCG neurones. Itscomposition was (mM): NaCl, 125; KCl, 2-5; NaHCO3, 26;NaH2PO3, 1P25; MgCl2, 1; glucose, 25; 500 nm atropinesulphate and 100 nm tetrodotoxin (both Sigma); pH 7N4 whenbubbled with 95% 02-5% CO2.

Macroscopic currents were recorded using the whole-cellpatch clamp configuration with a pipette solution containing(mM): CsF, 135; CsCl, 5; Hepes, 10; EGTA, 5; Mg-ATP, 4;adjusted to pH 73 with NaOH. It has previously beendemonstrated that intracellular Cs+ does not influence theACh-evoked whole-cell currents (Mathie, Colquhoun & Cull-Candy, 1990). In some cells an ATP-regenerating system wasincluded in the pipette solution (containing 20 mm phospho-creatine, 50 U ml-' creatine phosphokinase and 25 U ml-'protein kinase A catalytic subunit) in order to avoid possibleloss of ATP due to spontaneous hydrolysis. Responses wereindistinguishable from those recorded with the standardpipette solution.

Whole SCG were obtained from 4-6-week-oldSprague-Dawley rats, killed by decapitation. Once removed,whole ganglia were kept in ice-cold Krebs solution and cleanedof connective tissue ('desheathed'). Ganglia were then treatedfor 15 min with 1 mg ml-l trypsin, followed by 45 min in1 mg ml-' collagenase (type IA), 1 mg ml-' trypsin inhibitor,and 041 mg ml-' thermolysin (all Sigma). This was thenfollowed by a 5 min treatment in 150 AM methanesulphonyl-fluoride (MSF; Aldrich Chemical Co., Gillingham, Dorset,UK). All enzyme treatments took place at 37 °C in 10 ml Krebssolution, bubbled with 95% 02-5% CO2.

Ganglia were held in place in the recording chamber using anylon-mesh grid (F. A. Edwards, personal communication).Healthy cells were identified as those having a smooth andshiny appearance, with the cell nucleus not visible. Severalsatellite cells on the surface of each neurone could be removedusing gentle suction and careful manipulation of a suitablysized cleaning pipette (- 5,m diameter). Attempts to cleanthe cells without enzyme treatment were not successful. Allrecordings were made on the day of cell preparation.

Drugs were applied to a particular cell using a gravity-feddouble-barrelled glass pipette (- 100 ,sm diameter for eachbarrel). Cells were exposed to control solution flowing from onebarrel and then to drug solution flowing from a second barrelby the lateral movement of the pipette using a stepper motor.Control solution continually flowed down one barrel, while thesolution flowing down the second barrel was switched betweena number of different drug solution reservoirs using an eight-way tap.

Cells were voltage clamped at -60 mV using an AxopatchlB amplifier (Axon Instruments). Patch electrodes were pulledfrom thin-walled borosilicate glass (GC150TF-7T5; ClarkElectromedical, Reading, UK), coated with Sylgard resin(Dow Corning 184), and fire polished to a final tip resistance of5-10 MQ. Cells were viewed under Nomarski optics (ZeissAxioskop, Oberkuchen, Germany) using a x 40 water-immersion objective lens (16 mm working distance).Macroscopic currents were stored on FM tape (Racal Store 4,DC - 5 kHz) after filtering at 10 kHz (-3 dB, 8-pole Besselresponse), as well as being plotted directly with a pen recorderafter being filtered at 1 Hz (8-pole Bessel filter, Barr & Stroud,East Kilbride, UK).

28 J. Physiol. 481.1

Native and cloned neuronal nicotinic ACh receptors

Amplitudes of the whole-cell currents were measured fromthe pen recorder traces. Current amplitude increased withtime in the majority of recordings, and never decreased. Theextent of any whole-cell 'run-up' was calculated for eachrecording from response versus time plots for a drug standard(10 #M ACh) which constituted every third drug application.Current amplitudes were corrected by linear interpolation.

Analysis of potency ratiosPotency ratios were estimated from all the results on one cell,separately for each cell. Initial attempts to normalize theresponses from different cells via the response to a standarddose of agonist were not very effective. Extensivedesensitization at high agonist concentration made fulldose-response curves misleading, so the aim was to find thelow-concentration limit of the relative potency of pairs ofagonists. All the curves from a single cell were fittedsimultaneously by weighted least-squares with Hill equationsthat were constrained to be parallel (i.e. had the same Hillslope). The parameters to be estimated were the horizontalposition (nominal EC50) for one of the curves (the 'standard'),nH (the common Hill coefficient) and potency ratios for each ofthe other curves relative to the 'standard'. The maximumresponse was fixed at a value much larger than any observedresponse, and the fit was weighted by assuming that thecoefficient of variation was the same for each response. Thisprocedure amounts to fitting curves that are linear,constrained to be parallel and have constant errors whenplotted on double-logarithmic co-ordinates (see Fig. 2). Theadequacy of the parallel simultaneous fit was judged by eye,and by performing a variance ratio test on the decrease in thesum of squared deviations when each curve was fittedseparately with its own nH value. Potency ratios so estimatedwere averaged and converted to a common standard (ACh).Errors for the ratios were estimated by Fieller's theorem(e.g. Colquhoun, 1971).

RESULTS

Whole-cell current responses in oocytesinjected with cDNA pairsFigure IA shows typical inward current responses of asingle oocyte injected with the a3-/32 cDNA combinationto the nicotinic agonists ACh, DMPP and cytisine. Theaverage peak response to 1 UM DMPP in a3-/12-injectedoocytes clamped at -60 mV was 246 + 3.4 nA (n = 9).Figure lB shows typical responses to the same threeagonists of a single oocyte injected with an a3-/14 cDNApair. The average peak response to 1 /M cytisine in a3-/14-injected oocytes clamped at -60 mV was 256+ 92 nA(n = 8). As previously reported by Cachelin & Jaggi (1991),responses in oocytes injected with the a3-/12 combinationoften show desensitization, which was not apparent ina3-fl4-injected oocytes.

Whole-cell current responses in SCG neuronesFigure 1C shows typical inward current responses of asingle SCG neurone to the agonists ACh, DMPP andcytisine. The average peak response to 10/M ACh in SCG

neurones clamped at -60 mV was 113 + 24 pA (n = 12).This value applies to the first application of 10/1M ACh assubsequent applications tended to result in larger currentresponses due to the effects of 'run-up'. The highestconcentration of ACh used on SCG neurones was 10/SM;this was usually the concentration at which desensitizationbecame apparent. No obvious desensitization was observedwith any of the other agonists up to the highestconcentrations used, even though the magnitude of theresponse may have been larger than that to 10/uM ACh.The time to reach peak response was shortest for ACh(< I s at 10/M) and longest for nicotine (up to 20 s at 5/uM).Solution exchange, measured as the junction potentialchange at the end of an open patch pipette in response tochanging between dilute and normal Krebs solutions, wascomplete in around 5 ms.

Dose-response curvesFigure 2A shows a typical set of data using nicotinicagonist responses from a single oocyte injected with thea3-/12 cDNA pair. The data has been fitted with the Hillequation (see Methods) to obtain a set of dose-responsecurves constrained to be parallel. For the particularagonists applied to this oocyte a clear rank order ofpotency can be seen: DMPP > ACh > Nic - CCh > Cyt. Inthis example the fitted potency ratios (relative to ACh)are: DMPP, 1P54; Nic, 0-025; CCh, 0-024; and Cyt, 0 005.Figure 2B shows a similar set of data from a single oocyteinjected with an a3-/4 cDNA pair. In this example therank order of potency is: Cyt > ACh - Nic > DMPP. Thepotency ratios (relative to ACh) are: Cyt, 5412; Nic, 1-01;and DMPP, 0 45.

Figure 2C and D show two typical sets of data obtainedfrom two separate SCG neurones. For the cell in Fig. 3Cthe rank order of potency is: Cyt > DMPP > ACh, and thepotency ratios (relative to ACh) are: Cyt, 6f66; and DMPP,2-66. For the cell in Fig. 2D the rank order of potency is:ACh > CCh > Lob, and the potency ratios (relative toACh) are: CCh, 0-32; and Lob, 0-068. In these twoexamples the separate fits for each agonist (fits that are notconstrained to be parallel) are shown as dashed lines.

It is apparent from Fig. 2C that DMPP gives a steeperHill slope estimate than the other two agonists - in thisparticular case nH for ACh = 1-84, nH for Cyt = 1-83 and nHfor DMPP = 2-69. It was a consistent observation thatDMPP gave a steeper Hill slope estimate than any othernicotinic agonists applied to the same SCG neurone, but nocorresponding difference was observed in oocytes injectedwith either the a3-fl2 or a3-,B4 cDNA pairs.

Potency ratiosTable 1 shows the mean potency ratios for both the nativeSCG receptors and cloned receptors. It is clear that in ratSCG neurones cytisine is the most potent of the agoniststested, being almost five times as potent as ACh. DMPPand nicotine are also more potent than ACh. The rank

J. Physmiol. 481.1 29


Table 1. Potency ratios for nicotinic agonists in oocytes and rat SCG neurones

SCG neurones (n = 3-4)Oocytes: a3-fl4 (n = 4-7)Oocytes: a3-fl2 (n = 7-21)

Cytisine

4-76 + 0705-06 + 076

0-020 + 0-012

DMPP

250 + 0240 43 + 0-081P62 + 0 49

Nicotine

1P98 + 0.13110 + 0-31

0-038 + 0-012

Carbachol

0f361 + 0-0220-25 + 004

0-068 + 0-028

Lobeline

0-066 + 0-014018 + 004

0-207 + 0-081

The relative potency ratios calculated individually from each oocyte and neurone were pooled andexpressed as the mean potency ratio relative to ACh (± S.E.M.).

order of potency is Cyt > DMPP > Nic > ACh > CCh > Lob.For oocytes injected with a3-/54 cDNA pairs the rankorder of potency is Cyt > Nic z ACh > DMPP > CCh > Lob.Similarly for oocytes injected with a3-fl2 cDNA pairs therank order of potency is DMPP > ACh > Lob > CCh > Nic> Cyt. There is clearly a marked difference between therank orders of potency obtained with these two cDNApairs, the most striking being cytisine, which is the mostpotent agonist with a3-/4 cDNA pairs but the weakest

A Oocyte: a3-#23 yM ACh

B Oocyte: a3-/45 #M ACh

50 nA

20 s

C SCG neurone

10 FM ACh

3/uM DMPP

agonist with a3-/2 cDNA pairs, as also found by Luetje &Patrick (1991).

Thus the rank order of the potency ratios in SCGneurones does not exactly match those obtained witheither of the cDNA pairs, but it is close to that of thea3-/14 cDNA, pair with only one agonist in a differentposition (DMPP is more potent than ACh with SCGneurones, but less potent with the a3-fl4 cDNA pair).Closer inspection of the actual values of the potency ratios

50 #M Cyt

10 nA

10 s

10 FM DMPP

3 FM DMPP

1 FM Cyt

3 FM Cyt

100 pA

20 s

Figure 1. Examples of whole-cell responses in oocytes and rat SCG neuronesInward current responses to bath applications of the nicotinic agonists ACh, DMPP and cytisine.A, a single oocyte with an a3-fl2 cDNA pair (DMPP is clearly the most potent agonist). B, a singleoocyte injected with an a3-,84 cDNA pair (cytisine is clearly the most potent agonist). Both oocyteswere voltage clamped at -60 mV, and the traces were obtained directly from the chart recorder.C, inward current responses to stepped flow-pipe applications of the same agonists in a singleneurone from an undissociated rat SCG; cytisine is clearly the most potent agonist. The neuronewas voltage clamped at -60 mV. Traces were replayed from FM tape, filtered at 3 Hz and sampledat 200 Hz.


Native and cloned neuronal nicotinic ACh receptors

Table 2. Hill slope estimates for dose-response curves from oocytes and rat SCG neurones

SCG neuronesOocytes: a3-fl4Oocytes: a3-#2

ACh

1P62 + 0'06 (12)1P70 + 0-08 (6)1P13+0-07(5)

Cytisine1P75 + 008 (3)1P97 + 0-21 (4)

0.95 (1)

DMPP

2'65 + 0 04(3)1P33+0-16(4)1P25+0'09(21)

Mean Hill slope + S.E.M. (the number of cells used to calculate each mean is given in parentheses).The number of cells is lower for the very weak agonists as it was not always possible to test enoughconcentrations to get a good estimate. In SCG neurones a steeper Hill slope estimate wasconsistently obtained with DMPP.

shows that the values for cytisine are almost identical forSCG neurones and oocytes injected with a3-/4 cDNApairs (4-76 + 0 70 and 5-06 + 0-76, respectively, but only0-020 + 0-012 with the a3-fl2 pair). Similarly the value fornicotine in SCG neurones is close to that obtained with thea3-/14 cDNA pair (1P98 + 0413 and 1-10 + 0-31, respectively,but only 0-038 + 0-012 with the a3-/J2 pair). This is alsothe case with carbachol (0-361 + 0-022 and 0-25 + 0'04,

A

a

a

0 10'aC.

respectively, but only 0-068 + 0-028 with the a3-fl2 pair).However, the value for DMPP in SCG neurones is similarto that in oocytes injected with a3-/2 cDNA pairs(2-50 + 0-24 and 1P62 + 0 49, respectively, but only0 43 + 0-08 with the a3-/l4 pair). Lobeline gives similarvalues for both a3-f4 and a3-/32 cDNA pairs (i.e.0-18 + 0 04 and 0-207 + 0-081 respectively), but has a lowerpotency in SCG neurones (0-066 + 0-014).

C

100

10

0-1000

B

Cyt DlSCG neurone I

I

I

,,I" ,,,X,.. I.

MPP ACh

10. I

10

DACh CCh

100

10

1 10

Concentration (,uM)100 1 10

Concentration (uM)

Figure 2. Examples of fitted dose-response curves for sets of whole-cell responses from singleoocytes and rat SCG neurones

Data sets containing the whole-cell responses from each individual cell were fitted separately toform a set of parallel dose-response curves (see Methods) from which the relative potency ratioswere obtained. Oocyte responses: whole-cell responses from (A) a single oocyte injected with an

a3-fl2 cDNA pair, and (B) a single oocyte injected with an a3-,84 cDNA pair. C and D, SCGresponses, showing two fitted dose-response curves each using data obtained from a single SCGneurone (the dashed lines show the fitted curves obtained when the fits were not constrained to beparallel).

Nicotine

1P55+0'12(4)1'48 + 0 09 (4)0-82 + 013 (5)

Carbachol

1P85 + 0-08 (3)1P66 + 002 (4)0-87 + 0'14 (5)

Lobeline

1P37 + 0-20 (5)1P59 + 0-28 (7)

CL0L-

CuC

3:c

100

J. Physiol. 481.1 31

.1 1


Hill slope estimatesThe steeper Hill slope estimates encountered with DMPPin the SCG neurones (see Table 2) do cast some doubt on

the accuracy of the DMPP potency ratio value whenestimated from parallel fits, but this would probably notbe sufficient to account for the discrepancy between thea3-/34 subunit combination in oocytes and the rat SCGresponses. The reasons for the steeper Hill slope withDMPP are unclear; factors such as desensitization or

receptor heterogeneity will tend to decrease the Hill slope.DMPP-evoked neurotransmitter release via a presynapticreceptor is unlikely to be involved due to the presence ofTTX and the absence of external Ca2+.

Our Hill slope estimates in oocytes (see Table 2) are

somewhat lower than those reported by other authors. ForL3-,fl2 we find nH for ACh = 1-13 and nH for DMPP = 1P25;and for a3-fl4, nH for ACh = 1P70 and nH for DMPP = 1P33.For example, Cachelin & Jaggi (1991) report for rat a3-fl2,nH for ACh = 1P4 and nH for DMPP = 1P7; and for rat z3-,B4,nH for ACh = 1-8 and nH for DMPP = 1-8. The lower theagonist concentrations, the better the estimate of the Hillslope due to minimal desensitization, and any factor thatenhances the response of the oocyte to a nicotinic agonistwill allow the use of lower concentrations. Steeper Hillslope estimates could therefore be due to higher levels ofreceptor expression or due to the presence of Ca2+ in theextracellular medium, as used by most authors. ExtracellularCa2+ increases agonist responses of neuronal nicotinicAChRs (with 1-8 mm Ca2+ almost doubling the response

recorded in 0-18 mm Ca2+; Vernino, Amador, Luetje,Patrick & Dani, 1992).

Another problem encountered when using extracellularCa2+ is that neuronal nicotinic AChRs have a significantCa2+ permeability (Vernino et al. 1992). In oocytes Ca2+ influxwill activate a transient chloride current, which is inwardat this holding potential (Leonard & Kelso, 1990), and willtherefore amplify the response to nicotinic agonists,resulting in an increased Hill slope. We have usednominally Ca2+-free extracellular media in order to allow a

direct comparison between oocytes and SCG neurones.

DISCUSSION

This study shows that the agonist profile of nicotinicreceptors in rat SCG neurones is similar to that of thea3-fl4 receptor subunit combination (see especially thehigh potencies of cytisine and, to a lesser extent, nicotine).However, the relatively high potency of DMPP in the SCGneurones, which is characteristic of the a3-fl2 combinationin oocytes, suggests that SCG receptors are not simply a

combination of a3 and /14 subunits. The oocyte datapresented here are largely consistent with those publishedpreviously by other authors with fewer agonists underdifferent conditions (e.g. Luetje & Patrick, 1991; Cachelin &Jaggi, 1991), although no comparable estimates of potency

ratios are available from these studies. The agonist profilein rat SCG neurones does not appear to match anyobserved in central neurones such as the rat habenular andinterpeduncular nucleus (Mulle, Vidal, Benoit & Changeux,1991), or those observed in oocytes for other functionalpairs of rat subunits (Luetje & Patrick, 1991) or for thehomomeric a7 subunit combination (Seguela et al. 1993).One possible explanation for the high potency of both

DMPP and cytisine is heterogeneity in the rat SCGreceptors, as suggested by single channel studies (Mathie,Cull-Candy & Colquhoun, 1991), so it is not entirelyunexpected that the whole-cell SCG agonist profile doesnot resemble that of a single a-3 pair. In oocytes neuronalbungarotoxin (NBT) has been shown to block a number ofa-4 subunit pairs depending on the manner in which it isapplied (Papke, Duvoisin & Heinemann, 1993); e.g. a3-/2receptors show a long-lasting inhibition after pre-incubation with NBT, whereas a3-f4, a4-fl2 and a4-/J4receptors (and several other combinations including themuscle al/lly8) are at least partly blocked by co-application of NBT with the agonist. Co-application withNBT has been found to block completely the whole-cellresponses to ACh in rat sympathetic neurones (Mathie,Cull-Candy & Colquhoun, 1988); this result resembles mostclosely that reported for a3-/34 receptors (if the receptorsactually contain only two subunit types). It should benoted that most patch recordings are made from the cellsoma, whereas most synaptic contacts in rat SCG neuronesare found on the proximal dendritic processes where theAChR density is high (as obtained with a- and neuronalbungarotoxin labelling; Loring, Sah, Landis & Zigmond,1988). Many of these dendritic processes may be lost whenSCG neurones are dissociated and the nature of thenicotinic AChRs present on new dendritic growth inducedduring the culturing process is uncertain, asmeasurements of receptor turnover in chick sympatheticganglia in culture suggest that new receptors are formedwithin 24 h (Listerud et al. 1991). The use of freshundissociated SCG neurones from mature rats in thisstudy will remove any such uncertainty that may beencountered when recording whole-cell currents fromdissociated and cultured neurones.

If the native functional nicotinic AChRs do not consistof a simple a3-/12 or a3-/14 subunit pair, then what couldtheir subunit composition be? The data presented herewould be consistent with a situation in which both typeswere present, but the high potencies obtained withcytisine and nicotine (and block by NBT) would suggestthat an a3-/14 population was dominant, whereas the highpotency obtained with DMPP would suggest that ana3-/12 population was dominant. Immunoprecipitationstudies in chick ciliary ganglia reveal a population ofreceptors composed of three different subunits, a3, a5 andfl4 (Vernallis et al. 1993), and other studies suggest theincorporation of a7 into receptors containing the a3subunit (Listerud et al. 1991). While a5 subunit mRNA


J. Physiol. 481. Native and cloned neuronal nicotinic ACh receptors 33

may not be found in rat SCG (Rust et al. 1994), othersubunit combinations are possible. It is therefore notunlikely that a similar situation to that in the chickganglia might exist in the rat SCG, with receptorscontaining more than one type of a- and/or fl-subunit.

az-Bungarotoxin binds with high affinity (equilibriumconstant, Ka 6 x 10-1 M) to rat SCG neurones (Fumagalliet al. 1976), and also blocks the homomeric oc7 receptorin oocytes (Seguela et al. 1993). However, a-bungarotoxinhas no effect on either synaptic transmission orresponses to nicotinic agonists in the rat SCG (e.g.Brown & Fumagalli, 1977), although such an effect hasnow been demonstrated in the embryonic chick ciliaryganglion (Zhang, Vijayaraghavan & Berg, 1994). Therank order of potency (Seguela et al. 1993) for rathomomeric a7 receptors (Nic - DMPP > Cyt > ACh, atlow concentrations) is quite different from that of therat SCG. There is, therefore, no evidence to suggest thepresence of functional homomeric a7 receptors in ratSCG neurones, although this does not exclude thepossibility of incorporation of the a7 subunit intoreceptors with multiple subunits.

Interpreting the results of the functional comparativeapproach used in this study requires caution. It is possiblethat there may be additional regulatory and structuralproteins associated with the nicotinic receptor in theganglion that have not been expressed in the oocyte, andany post-translational modification may be different. Inaddition, other subunit combinations so far not investigatedcould have identical agonist profiles to those investigatedhere and in previous studies. There is therefore a need forother corroborative information to back up the findingspresented here, such as immunoprecipitation studies withsubunit-specific monoclonal antibodies (e.g. Vernallis et al.1993), single channel data and the daunting prospect,considering the number of possibilities, of furtherfunctional studies with more complex subunit combinations.

In conclusion, this study demonstrates that agonistpotency ratios may provide a quite subtle means ofdistinguishing receptor subunit composition. In the case ofreceptors in rat SCG neurones, the results are consistentwith that of an a3-,B4 subunit combination, except for thepotency of DMPP, which is approximately 6-fold morepotent in the SCG than expected from the a3-fl4combination in oocytes. This single result thereforehighlights the need for other corroborative functional andbiochemical studies to be undertaken before a firm conclusioncan be reached regarding the subunit composition ofnicotinic ACh receptors in the rat SCG.

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AcknowledgementsWe would like to thank the MRC and the Wellcome Trust forsupporting this work. We would also like to thank thefollowing: the Sandoz Institute (especially John Wood andMike Webb) for housing the Xenopus; Jim Patrick andDaNong Chen (Baylor College of Medicine' for supplying thecDNA clones; David Osborne for construction of the steppermotor apparatus, and Liz Ensor for technical assistance.P. J. 0. C. was supported by an MRC studentship.

Received 1 July 1994; accepted 5 September 1994.

J. Physiol 481.134

comparison nicotinic neurones

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