University College London, Gower Street, London WC1E 6BT current evoked by ACh in whole-cell ...

34 Pages·2005·3.68 MB·English

Journal ofPhysiology (1991), 439, pp. 717-750 717
With 15figures
Printed in GreatBritain
CONDUCTANCE AND KINETIC PROPERTIES OF SINGLE NICOTINIC
ACETYLCHOLINE RECEPTOR CHANNELS IN RAT SYMPATHETIC
NEURONES
BY ALISTAIR MATHIE*, STUART G. CULL-CANDY AND
DAVID COLQUHOUN
From the MRC Receptor Mechanisms Group, Department ofPharmacology,
University College London, Gower Street, London WC1E 6BT
(Received 9 October 1990)
SUMMARY
1. The unitary conductance ofnicotinic acetylcholine (ACh) receptor channels in
rat sympathetic neurones has been studied. Conductance estimates varied from
26-48 pS with a mean of 36-8 pS in 1 mM-Ca2+. The main conductance level varied
from patch to patch and the presence (or absence) ofadditional conductance levels
also varied.
2. The channels showed large open channel noise and experiments with 300 mM-
NaCl in the patch pipette substantially increased the open channel noise. The
appearance of detectable step-like transitions within this noise strongly suggested
the existence of closely spaced discrete levels.
3. Removal of divalent cations from the external solution increased the unitary
channel conductance. Altering the main permeant ion in divalent-free solutions gave
the following conductance sequence: K+ (93 pS) > Cs' (61 pS) > Na+ (51 pS) >
Li+(23 pS).
4. Replacement of Na+ by Cs' in the external solution considerably reduced the
current evoked by ACh in whole-cell recordings and the channel-opening frequency
in outside-out patches.
5. The kinetic properties of channels activated by ACh and 1,1-dimethyl-4-
phenylpiperazinium iodide (DMPP) were also studied. At low concentrations of
ACh and DMPP the gap distributions were complex and best fitted by the sum of
four exponential components. Individual activations (bursts) were interrupted by
the two shortest closed periods the briefer ofwhich had time constants of36,ts for
ACh and 67 ,ts for DMPP.
6. The distribution of burst lengths had two components for each agonist, each
component making up about 50% ofthe total area under the distribution. For ACh,
the time constant ofthe longer component (12-2 ms) was similar to the decay time
constant ofexcitatory postsynaptic potentials (EPSCs) at similar temperature and
potential. For DMPP the time constant ofthe longer component was 17-6 ms.
* Present address: Department of Pharmacology, Royal Free Hospital School of Medicine,
Rowland Hill Street, London NW3 2PF.
MS8856
718 A. MATHIE, S. G. CULL-CANDY AND D. COLQUHOUN
7. The relative number ofbriefgaps per long burst was much largerforACh than
for DMPP. Therefore the corrected mean open time for ACh (0-86 ms) was much
shorter than that for DMPP (2-3 ms).
8. In terms ofreceptor mechanism, the values ofthe channel opening equilibrium
constant (,/oc) estimated from these numbers (ACh, 23; DMPP, 25) suggest that
both agonists are efficaceous.
9. DMPP is a potent blocker of the channel with an equilibrium dissociation
constant (KB) ofaround 50/tM and blockage gaps ofaround 1 ms duration. ACh also
blocks the channel but with a higher KB of around 470,UM.
10. The equilibrium concentration-response relationship was estimated for a
range of ACh concentrations by measuring the proportion of time a channel stays
open (po) during the clusters ofopenings that occur on escape from desensitization.
The p0 values for clusters at a given ACh concentration were found to be very
variable, however, division ofclusters into 'lowpo' and 'normal' allowed a plausible
activation mechanism to be fitted to the data.
11. Arandomization test suggested that theexistence ofsuchheterogeneity isdue
to more than one population of clusters made up of channels with quite different
open and closed times. It isnot known, however, whether such differences are due to
the existence of more than one channel type or to variation in the behaviour of a
single channel type with time.
INTRODUCTION
Although nicotinic receptors in mammalian autonomic ganglia serve the same
function in synaptic transmission as those at the endplate, neuronal receptors show
a number ofstructural and functional differences from those in muscle.
Structurally, muscle nicotinic receptors are composed offour distinct subunits, ac,
/3, y (or e) and 8, arranged as an asymmetric pentamer with two a-subunits per
molecule (Conti-Tronconi & Raftery, 1982; Mishina, Takai, Imota, Noda, Takahashi,
Numa, Methfessel & Sakmann, 1986). It is thought that neuronal nicotinic receptors
which occur centrally and peripherally contain only two subunit types (ac and ,3),
with a stoichiometry as yet unknown (Boulter, Connolly, Deneris, Goldman,
Heinemann & Patrick, 1987; Lindstrom, Schoepfer & Whiting, 1987) with at least
five (for c) and four (for fl) subtypes ofeach subunit (see Papke, Boulter, Patrick &
Heinemann, 1989; Boulter, O'Shea-Greenfield, Duvoisin, Connolly, Wada, Jensen,
Gardner, Ballivet, Deneris, McKinnon & Patrick, 1990). The messenger-ribonucleic
acids (mRNAs) for these subtypes are located throughout the nervous system, with
different combinations predominating indifferentregions (see Wada, Wada, Boulter,
Deneris, Heinemann, Patrick & Swanson, 1989).
Functionally, neuronal nicotinicreceptors have beenknownforsometime todiffer
from muscle nicotinic receptors in their sensitivity to many blocking drugs and
toxins (e.g. Paton & Zaimis, 1951; Ascher, Large & Rang, 1979). More recently the
functional characteristics of the responses to agonists have been shown to differ
(Kuba & Nishi, 1979; Rang, 1981; Derkach, Selyanko & Skok, 1983), results which
have been confirmed by initial studies at the single-channel level (Cull-Candy &
Mathie, 1986; Derkach, North, Selyanko & Skok, 1987; Mathie, Cull-Candy &
Colquhoun, 1987). Some studies suggest that heterogeneity exists between nicotinic
ACh CHANNELS IN SYMPATHETIC NEURONES 719
receptors within a single population ofneuronal cells (Bormann & Matthaei, 1983),
during the development of particular neurone types (Margiotta & Gurantz, 1989;
Moss, Schuetze & Role, 1989) or when comparing data from one neurone to another
(see Colquhoun, Ogden & Mathie, 1987; Steinbach & Ifune, 1989; see also Mulle &
Changeux, 1990).
In the present paper we have examined the properties of the neuronal nicotinic
receptors of rat sympathetic ganglion cells with criteria and methods previously
established forstudying the conductance (Sakmann, Methfessel, Mishina, Takahashi,
Takai, Kurasaki, Fukuda & Numa, 1985) or kinetic properties (Colquhoun &
Sakmann, 1985; Sine & Steinbach 1986; Colquhoun & Ogden, 1988) of muscle
nicotinic receptors. This has allowed us to assess the degree ofheterogeneity within
apresumed uniform population ofnicotinic receptors, and should provide abasis for
comparison with single-channel studies of neuronal nicotinic receptor subunits
expressed in Xenopus oocytes or in cell lines (e.g. Ballivet, Nef, Couturier, Rungger,
Bader, Bertrand & Cooper, 1988; Papke et al. 1989). Apreliminary report ofsome of
these results has appeared (Mathie, Cull-Candy & Colquhoun, 1989).
METHODS
Preparation ofcells
Experiments were on sympathetic neurones obtained from the sympathetic chain and superior
cervical ganglion ofyoung female Sprague-Dawley rats (17-21 days after birth). Rats were killed
with chloroform. The method of cell dissociation has been described previously (Cull-Candy,
Magnus & Mathie, 1986).
Solutions
The normal external and internal solutions had the following composition (mM): external
solution: NaCl, 150; KCl, 2-8; MgCl2, 2; CaCl2, 1 and HEPES (Na), 10 (pH to 7-2 with NaOH);
internal solution: CsCl, 140; CaCl2, 1; EGTA (K), 10 and HEPES (K), 10 (pH to 7-2 with KOH).
Different solutions were used for some of the conductance experiments. For the relative
conductance of each ion in outside-out patches, both external and internal solutions comprised
(mM): XCl, 150; where X = Cs, K, Li or Na; HEPES, 10 and EGTA, 10. For the high Na+ cell-
attached patches, thepatchpipette contained thenormalexternal solutionwiththeexceptionthat
NaCl was increased to 300 mM.
Two agonists were used, acetylcholine chloride (ACh) and 1,1-dimethyl-4-phenylpiperazinium
iodide (DMPP), both from Sigma. Agonists were applied to whole cells or outside-out patches by
bath perfusion via the manual switching of a two-way Hamilton tap or added to the pipette
solution for cell-attached patches.
Recording
Experimentsusedthreevariantsofthepatch-clamp technique (whole-cell recording, outside-out
patchesandcell-attachedpatches) (Hamill, Marty, Neher, Sakmann &Sigworth, 1981). Recordings
were made with a List EPC-7 or an Axopatch 1B patch-clamp amplifier (capacitance range
10-17 pF). Small-diameter cells with noobviousprocesses were chosen forexperiments. Electrodes
werepulledfromthin- orthick-walled filament-containing glass (Clark EMI). The resistance ofthe
electrodes was 2-5 MCIforwhole-cell recording and 5-10 MQ2forpatches. Series resistance wasnot
compensated and no corrections were made for liquid junction potentials. Experiments were at
room temperature 20-23 'C.
Whole-cell records
Signals were stored on magnetic tape. (Racal store 4; tape speed 71, 15 or 30 in/s. Whole-cell
records were replayed from the tape onto a Tektronix digital oscilloscope and sent from there to
a Gould plotter. The amplitudes ofwhole-cell currents were measured from these plots.
720 A. MATHIE, S. G. CULL-CANDY AND D. COLQUHOUN
Single-channel records
Single-channel datawere filtered at 1-5-4kHz (-3dB, 8-pole filter with Bessel characteristics)
then digitized at sampling rates ranging from 18-48 kHz. (For higher rates this was achieved by
reducing the tape playback speed by a factor oftwo).
Amplitudes
Amplitude distributions were obtained by two methods. The first, during time-course fitting (see
below), was to superimpose a cursor on the record when a channel was open and measure the
difference in amplitude from the measured baseline when the channel was closed. By this method
each individual opening contributes one point to the amplitude distribution ofthe whole record.
The second method was to obtain the amplitude distribution ofall the individual digitized data
points, apoint amplitude histogram. Thiswasdetermined separately forsections ofdatainwhich
thechannelwasdeemedopenorclosedbutnotintransitionbetweenthetwo. Bythismethodeach
opening contributes anumberofpoints,proportionaltothelengthoftheopening,totheamplitude
distribution ofthewhole record. Both typesofhistogram were fittedwithappropriate probability
density functions representing the sum ofone or more Gaussian components using the method of
maximum likelihood (Colquhoun & Sigworth, 1983).
Sublevel detection plots
In addition, the sublevel detection method ofPatlak (1988) was used. Short stretches ofdata
when the channel is open, that have a low variance, represent sojourns at a fairly constant
amplitude level. Therefore the variance and mean amplitude were calculated, during channel
openings, forsections oftendatapoints, successive sections beingtakenbyadvancingthedigitized
record by one point at a time. All ofthese sections for which the standard deviation was greater
thanthebaselineroot-mean-squarenoisewereeliminatedandhistogramswereconstructedfromthe
mean amplitude ofthe sections remaining. For comparison, a similar analysis was carried out on
sections ofthe data when no channels were open.
Kinetics
Opening and closing transitions were measured byfitting the time course ofthesignal using the
measured-step response function of the recording system as described previously (Colquhoun &
Sigworth, 1983; Colquhoun & Sakmann, 1985). The resolution of the system using this method
varied from patch to patch but was usually between 60-100,s for openings and 50-80/is for
closings. Theidealizedrecord obtainedwasthenusedtoconstruct histograms ofopentimes, closed
times, burst lengths etc. which were then fitted with appropriate probability-density functions
representing the sum of several exponential or geometric terms, again using the method of
maximum likelihood.
In some experiments using high agonist concentrations, it was considered unnecessary to have
a very high resolution so single-channel transitions were detected by a 50% threshold crossing
program. Although much fasterthan time-course fitting, the resolution imposed ontheresultswas
much lower (120-200Ius).
The critical gap length, Te, for bursts, below which a gap was classified as a gap within a burst
was calculated bythe method ofColquhoun & Sakmann (1985) to minimize theproportion ofgaps
misclassified as gaps within or between bursts. Values obtained from the open-time distributions
were corrected to take account ofmissed gaps as described before (Colquhoun & Sakmann, 1985;
Cull-Candy, Mathie & Powis, 1988). The probability of being open, p0, for clusters at high ACh
concentrations was calculated by numerical integration ofthe digitized record (see Colquhoun &
Ogden, 1988).
Randomization test
Clusters of channel openings evoked by high ACh concentrations were compared by a
randomization test (Patlak, Ortiz & Horn, 1986) inorder todetermine the degree ofheterogeneity
betweentheclusters. ThistestwasusedtocomparethevaluesofpO,meanopentimeandmeanshut
time for clusters at the same ACh concentration, each with at least twenty openings.
ACh CHANNELS IN SYMPATHETIC NEURONES 721
The observed scatter (Sobs) was calculated according to:
N
SOS= z ly-)
f-i
whereNis the number ofclusters compared, n, is the number ofopenings in the ith cluster, g, is
the mean open time (orp0 orshut time) fortheith cluster andyisthe overall meanopentime (or
p0 orshuttime). Histograms ofrandomized scatter (Sran) were obtained underthenull hypothesis
that all clusters are homogenous by generating 1000 artificial sets of clusters. These artificial
clusters were generated by random selection ofopen and shut times from the total population of
observed intervals. Avalue ofSran was calculated asforSobs bythe equation above foreach setof
Nclusters generated inthis way. IftheSran values rarelyorneverexceedSob. this implies that the
open time (orpo or shut time) genuinely differs between clusters.
RESULTS
Two agonists have beenused throughout this study: ACh, thephysiological ligand
for the receptor, and DMPP a commonly used agonist at neuronal nicotinic
receptors.
Conductance properties
The aim of the first part of this study was to consider the single-channel
conductance properties ofnicotinic receptors in rat sympathetic neurones. We have
previously described these channels as having a unitary conductance of 35 pS in
normal external solution, this increased to 51 pS on removal of external divalent
cations (Mathie et al. 1987; Mathie, Colquhoun & Cull-Candy, 1990). However, the
conductance values showed substantial variation both from patch to patch, and
within patches when individual amplitudes were compared (Mathie et al. 1987).
The number ofconductance levels. The variability ofthe conductance is illustrated
by the single-channel current records in apatch shown in Fig. IA. In addition, some
openings areclearlynoisierthanothers (comparethelowertwotracesinFig. IA, and
see Fig. 3). Figure 1B and C shows point amplitude histograms, obtained in the
presence ofthenicotinic agonists ACh and DMPP, in twofurtherpatches. Bothopen
pointandshutpointamplitudes canbefittedwellbyasingleGaussiandistribution in
this case, but the standard deviation ofthe Gaussian is considerably largerwhen the
channels are open. In the examples illustrated the standard deviation of the noise
during openings exceeds that ofthe baseline by 2-2-fold for open ACh channels and
by 284-fold for DMPP channels. We have previously considered the possibility that
thislarge standard deviation mayreflecttheexistence ofdiscrete, butcloselyspaced,
multiple-conductance states ofthe neuronal ACh channel. To some extent this was
borne out when amplitudes were measured by the time-course fitting method
(Colquhoun & Sigworth, 1983); this gave distributions ofamplitudes that suggested
the presence ofmultiple-conductance levels (see Mathie et al. 1987).
Two methods were used to try to clarify the reasons for the noisiness ofthe open-
channel current. One was to attempt to fit discrete levels during channel openings
with cursors on an X-Y oscilloscope display of the data, the second was to use a
subleveldetection method. Thelatterapproach isillustrated inFig. 2, whichisbased
on the data illustrated in Fig. 1A. A subconductance level is defined as a period of
722 A. MATHIE, S. G. CULL-CANDY AND D. COLQUHOUN
A ACh
70 ms 40 ms
100 ms 50 ms
B ACh
¢., 1-00_
0-00
x
0.7
0.50
G)
0.2-1-0 00 1-7-530 4-
C.)
U-o
-1.-0 0.0 1.0 2.0 3.0 4.0 5.0
Pointamplitude (pA)
C DMPP
2.5
2.0
0
'1.0
0.5
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
Point amplitude (pA)
Fig. 1. For legend see facing page.
ACh CHANNELS IN SYMPATHETIC NEURONES 723
time (of specified duration) during which the variance ofthe current is low (e.g. the
same as when the channel is shut). The mean amplitudes ofsuch stretches ofdata are
displayed as a histogram (see Methods, and Patlak, 1988, for further details). The
open point amplitude histogram for this patch is clearly more complex than a single
Gaussian, although it is difficult to define more than one distinct peak. Figure 2B
shows a sublevel detection plot; there are distinct peaks in the distribution thatwere
not previously detected. In particular, a clear level occurs with a mean current of
around 2 pA, in addition to the main peak at about 3-8 pA (as in Fig. 2A). There also
appear to be otherpeaks in thehistogram, butit isnot cleartowhatextentthisarises
merely from sampling error. While in some patches the distinction between different
subconductance levels was better than in Fig. 2B, in most it was even less clear.
It is easy to imagine that at least some ofthe noise in open channels results from
transitions between closely spaced discrete conductance levels (asalready illustrated
by Mathie et al. 1987, Fig. 2). In an attempt to increase the resolution of such
putative transitions, experiments were done with 300 mM-NaCl in the patch pipette
(in place ofthe normal 150 mM-NaCl), and ACh-activated channels were recorded in
cell-attached mode. The results, illustrated in Fig. 3A, show that the open-channel
noise is even more prominent under these conditions (in which the mean current
amplitude is substantially increased). The record shows, in many places, the
appearance of small step-like transitions. The open point amplitude histogram for
these results, in Fig. 3C, is quite obviously not Gaussian, though no more than two
Gaussian components could be fitted to it with any degree of confidence. Three
examples ofsingle-channel currents (in normal Na+) are shown, on an expanded time
scale in Fig. 3B. The central trace looks quiet, but the noisy appearances ofthe top
and bottom traces are more typical. The continuous line superimposed on the data
shows time-course fits, in which anything that could be interpreted as a 'square'
transition was so fitted. The amplitudes were specified by eye, and the times of the
transitions were then fitted (by least squares). Many subjective and arbitrary
decisions had to be made during this fittingprocess. Nevertheless the distribution (in
Fig. 3D) ofthe amplitudes fitted totheresults illustrated in Fig. 3A, appears to show
clear multiple peaks. A good fit could be obtained with five closely spaced Gaussian
components, with amplitudes separated by about 05 pA (though the fitting process
was stable onlywhen the standard deviations ofthe components were constrained to
be equal; the common estimate of this standard deviation was 0-15 pA which is
comparable to, though slightly less than, the standard deviation ofthe shut-channel
noise, namely 0-22 pA). Some, or perhaps all, ofthe multiple peaks in Fig. 3D could
Fig. 1.A,examplesofsinglechannelswhichcontributedtothedistrubituonshowninFig.
2A andB. Notethevariation in amplitude andpossible fluctuations between amplitudes
in some cases. Records filtered of 2 kHz (-3dB). Calibrations as indicated. B, point
amplitude histogram for a single cell-attached patch, membrane potential Vm= Vrest
-30mV, where Vrestisthe resting potential ofthe cell. Channels were activated by 3 LM-
ACh. Open point and closed point histograms were constructed separately and each was
fitted with a single Gaussian distribution. Closed level = 0-0003+0-17 pA (mean+S.D);
open level = 3-11+0-38pA. C, point amplitude histogram for another cell-attached
patch Vm = Vrest -70mV. In this patch, channels were activated by 5,UM-DMPP. Open
points and closed points are each fitted by a single gaussian distribution, closed level =
0009+0-19pA; open level = 4-54+0-46pA.
724 A. MATHIE, S. G. CULL-CANDY AND D. COLQUHOUN
result from samplingerror, but theseresults, togetherwiththe appearance oftheraw
data in Fig. 3A, suggest that atleastpart ofthe largeexcess open-channel noise does
result from the existence of closely spaced discrete levels. Although the fitting
process, illustrated in Fig. 3B, is highly subjective, it is very unlikely that the person
A B
? 1.50 500
-
x 1.25
1.00~~~~~~~
6 0.75 2-025
0.50~~~~~~~~~~~~~~~~0
>.
(D0.2
>. 0.50 0
-1.0 0.0 1.0 2.0 3-0 4-0 5-0 6-0 -1-0 0-.0 1-.0 2-0 3-0 4-0 5-0 6-0
Point amplitude (pA) Mean low-variance amplitude (pA)
Fig. 2. A, point amplitude histogram for a single cell-attached patch V. = Vr,.t-40mV.
Channels were activated by 5/,lm-ACh. Closed points were fitted by a single Gaussian
distribution =-0-64x 10-4+0-20pA. Theopenpoints have adistribution more complex
thansingleGaussianandhavebeenleftinunfitted.B,subleveldetectionplotforthesame
dataasmadeupthehistogramin(A)(seetextfordetails). Notetheappearanceofdistinct
components in the distribution ofopen levels. Ten data points or more were required at
aparticularlow-varianceamplitudebeforethelevelcontributed tothetotaldistribution.
analysing the experiment could (unconsciously) memorize, with sufficient accuracy,
the positions ofthese rather closely spaced levels in such a way that substantial bias
arose in choosing where to put the cursor.
Weanalysed twenty-twopatches withnormal external sodium (eleven outside-out
andeleven cell-attached) in some detail, andthe conductance values obtained forthe
main state and putative additional conductance states are given in Table 1. Two
features are apparent from the data. Firstly, there is a wide variability in
conductance estimates between patches. This ranged from 26-48 pS. The main level
varied from patch to patch, and the presence (or absence) ofadditional conductance
levels also differed. Secondly, there was no discernible pattern to the data, the
numbers donot seem tofall into discrete groups. The mean value ofall conductances
obtained was 36-8 pS which is similar to the value of 35 pS that we previously
obtained in outside-out patches with less rigorous amplitude criteria.
The effect on conductance of changing the permeant ion. Despite the uncertainty
concerning the existence of subconductance levels and their absolute values, it is
clear that removal ofdivalent cations from the external solution caused an increase
in unitary single-channel conductance. When Na+ was the mainpermeant cation the
meanconductancevalueroseto51 pS. Figure4showssingle-channel current-voltage
relationshipsforfiveoutside-outpatchesbathedin 150 mm-XCI, where X = K, Cs, Li
and Na, each with 10 mm-HEPES and 10 mm-EGTA, so the solutions are effectively
free of divalent cations. All channel openings were elicited by lO/um-ACh. The
ACh CHANNELS IN SYMPATHETIC NEURONES 725
conductance sequence obtained was K+ (93 pS) > Cs' (61 pS) > Na+ (51 pS) > Li+
(23 pS). The sequence, and indeed the absolute conductance values, are similar to
those found previously for fetal or adult calf muscle or Torpedo nicotinic receptor
expressed by mRNA injection inXenopus oocytes (Sakmann et al., 1985; Mishina et
al., 1986).
B v
A
0.
5 ms
0. r k-PW V,A
i--w-
Re
7 ms
5 ms
5 ms
C D
1.25 a 20.0
1.00
o
a1)I
CL0. 0.75
0 o a 10.0
uc 0 0.50- 0
C
0.25
a)
0.0-01.0 0.0. 1.0 2..0..3.04-0. I5...0...6...0.7I..,0 8.0 9.0 LL. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
Point amplitude (pA) Amplitude (pA)
Fig. 3. A, single-channel currents activated by 5 /sM-ACh in a cell-attached patch
containing 300mM-NaCl, Vm Vr.., -90mV. Note the large increase in noise during
=
channel openings. B, examples ofsingle-channel currents; shown on an expanded time
scale;activatedby5luM-AChinacell-attachedpatch, Vm = Vr..t-50 mV. Thecontinuous
linesshowtheidealizedrecordsobtainedfromtheseopeningsusingthetime-coursefitting
method. Note the apparent presence ofdiscrete amplitudes during an opening. C, point
amplitude histogram for a single cell-attached patch in high Na+ (same cell as A) Vrest
-90 mV. Each distribution is fitted by a single Gaussian distribution: closed level =
000+0-22pA, open level = 6-1+095pA. D, the distribution ofamplitudes (same patch
as C) was estimated by superimposing a cursor on openings in the same record. The
continuous line shows the fit by five Gaussian distributions, estimated S.D =0-15pA
(constrained to be the same for each component). The values ofthe amplitudes ofthe
five Gaussians are: 5-21+0-15pA (area=27%); 5-71pA (25%); 6-16pA (11%);
6-61 (18%) and 7-19 (19%).
726 A. MATHIE, S. G. CULL-CANDY AND D. COLQUHOUN
Membrane potential (mV)
-40
-2
a
X
-4 c;
-4
0
Na+
-6 :n
C
0)
s
-8 C
cn
._
-10
K+94pS
Fig. 4. Single-channel currents elicited by 10/LM-ACh in outside-out patches where the
solutiononbothsidesofthemembranewas 150mM-XCl (whereX = Na+,Cs+, K+orLi')
10mM-HEPES and 10mM-EGTA buffered to pH 7-2 with NaOH or KOH. Single ACh
channelsweredifficult toobtainwhen 150mM-CsClwasintheexternal solution (seetext
andFig. 5) so thedata foroutward currents carried by Cs+ on the intracellular (pipette)
sidewasobtainedwith 150mM-NaCl ontheextracellular (bath) side. The single-channel
conductance for the main permeant ion in each case was: K+ = 94pS (3 patches);
Cs+ 61 pS (4 patches); Na+ 51 pS (5 patches) and Li+ 21 pS (4 patches).
= = =
TABLE 1. Values for the main state and putative additional conductance states
Patch Agonist Single channel Patch Agonist Single channel
no. conc. (,UM) conductance (pS) no. conc. (,UM) conductance (pS)
22 5 34 37 3 43
43
23 5 28 35 38 3 32
24 39 39 3 32 36
10
28 3 39 48 40 3 37
30 3 37 41 3 35 37
31 30 28 42 1 36 41
32 5 33 62 1 34 41
33 5 44 63 1 31 38
34 5 37 64 6 31 41
35 5 35 41 65 6 26 32 44
36 5 48 75 50 34 39
Examples ofthe variability in estimated unitary channel conductance from patch to patch for
eleven outside-out patches and eleven cell-attached patches. In some cases the patches contained
channelswith only one clearamplitude level. Otherpatches contained channelswith two orthree
distinctamplitudes. Valueswereobtainedatbetweenthreeandfourpotentialsforeachpatch. The
external solution contained 150mM-NaCl, mM-CaCl2, and 2mM-MgCl2
1