state. The affinity of the blocking site for ACh is 1400 JIM (outside-out patches).

25 Pages·2005·2.73 MB·English

Journal ofPhysiology (1992), 454, pp. 129-153 129
With 11 figures
Printed in GreatBritain
A STUDY OF THE BOVINE ADRENAL CHROMAFFIN NICOTINIC
RECEPTOR USING PATCH CLAMP AND CONCENTRATION-JUMP
TECHNIQUES
BY D. J. MACONOCHIE* AND D. E. KNIGHT
From the Biomedical Sciences Division, King's College, London W8 7AH
(Received 14 September 1990)
SUMMARY
1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells
in the outside-out and whole-cell configurations, in response to step changes of
acetylcholine (ACh) concentration. The concentrations used ranged from 50 nm to
20 mM.
2. At high acetylcholine concentrations, the activation and desensitization
kinetics of the nicotinic receptor, as observed in outside-out patches, may be
described by a model incorporating a single, fast agonist binding step, and relatively
slow isomerization to the open state. The affinity of the closed receptor for ACh is
310 aM, the channel opening rate constant is 460 s-1, and the closing rate constant
is 29 sol.
3. Single channel events, observed when nanomolar ACh concentrations are
applied to whole cells, have two distinct channel lifetimes: 0(6 ms and 11-15 ms. The
variation ofthe frequencies ofthe events with ACh concentration, suggests that the
short lifetimes are openings of a singly liganded receptor and the longer lifetimes
are openings of a doubly liganded receptor.
4. Only a single exponential associated with receptor desensitization is seen with
outside-out patches, but two are seen with whole cells. It is postulated that there are
two nicotinic receptor types present on adrenal chromaffin cells.
5. The rate ofdesensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast
enough to be significant in determining the open channel lifetime.
6. A sudden increase in current (rebound) is observed when a high concentration
ofACh is abruptly removed from outside-out patches. This is evidence for a blocked
state. The affinity of the blocking site for ACh is 1400 JIM (outside-out patches).
7. The total number ofactivatable nicotinic channels per whole cell is estimated
to be 2600.
INTRODUCTION
As a modified autonomic ganglion, the adrenal medulla would be expected to
express neuronal-type nicotinic receptors. It has been known for some time that the
nicotinic receptors ofmuscle endplate and autonomic ganglia are different (Paton &
Zaimis, 1951; Chiappinelli, 1985), although recent work suggests that they share the
* To whom reprint requests should be addressed.
'N5 88(1
130 D. J. MACONOCHIE AND D. E. KNIGHT
samepentameric structure andinclude twoasubunits (Cooper, Couturier &Ballivet,
1991).
The kinetics ofthe muscle-type receptor have been characterized most effectively
by single channel analysis ofpatch clamp records in the cell-attached configuration.
Adrenal cells do not lend themselves particularly well to this sort ofanalysis for two
reasons. They have a low receptor density (calculated here to be of the order of
10 um-2) compared to the receptor density at the endplate of 10000gtm-2 (Saltpeter
& Eldefrau, 1973). Neuronal nicotinic receptors are observed here to desensitize
quickly. As a consequence, analysis ofthe kinetics ofthe neuronal type ofreceptor
has been restricted (Kuba & Nishi, 1979; MacDermott, Connor, Dionne & Parsons,
1980; Rang, 1982; Gurney & Rang, 1984; Ogden, Gray, Colquhoun & Rang, 1984;
Oortgiesen & Vijverberg, 1989).
On the basis ofnoise analysis and equilibrium current measurements at the frog
neuromuscular junction, a basic model for the muscle type ofnicotinic receptor has
beenproposed inwhich two successive ligand bindings arefollowed byisomerization
of the receptor to an open form (Dionne, Steinbach & Stevens, 1978).
This kinetic model has been widely used as the basis for explaining the behaviour
of the nicotinic receptor/channel, additional complications such as desensitized
states, a blocked state and a singly liganded open state being added to account for
additional experimental observations.
The final isomerization to the open state, governed by the channel opening rate
constant / and the closing rate a, is common to a range ofmodels. Measurements of
a and have been made from both single channel records, and from measurements
,
ofpostsynaptic currents. The value of/3 has been the subject ofsome controversy,
since widely different values have been calculated (Colquhoun & Sakmann, 1985;
Sine & Steinbach, 1986a, b, 1987).
Previous work on frog sartorius endplate, in which the relationship of initial
current amplitude to acetylcholine concentration is investigated (Adams, 1975)
suggests two equivalent non-co-operative ligand binding steps. Single channel
analysis of frog endplate currents (Colquhoun & Ogden, 1988) supports this.
However, thereis alsoevidence thatthetwo agonist bindingsites mayhavedifferent
affinities foragonist. Theaffinity ofradio-labelled toxinandsmallligandsto Torpedo
and Electrophorus acetylcholine receptors has been measured (Neubig & Cohen,
1979), andtwoaffinitiesuncovered. Singlechannelmeasurementshavealsoindicated
two non-equivalent binding steps (Jackson, 1988; Sine, Claudio & Sigworth, 1990).
Perhaps the strongest arguments for the non-equivalence of the bindings sites are
structural, and are based on the affinity ofthe a subunit, and subunitpairs for small
ligands (Blount & Merlie, 1989). An alternative suggestion is that glycosylation with
differentoligosaccharidemoietiesconfersagonistaffinitydifferences (Conti-Tranconi,
Hunkapillar & Raftery, 1984).
Concentration-jump experiments promise to clarify the concentration dependence
of the nicotinic channel (Franke, Hatt & Dudel, 1991), and to measure ft directly.
We show here, in the form of single channel measurements at nanomolar ACh
concentrations, strong evidence for two non-equivalent agonist binding steps in the
activation of the bovine adrenal chromaffin nicotinic receptor in response to
acetylcholine.
ACETYLCHOLINE RECEPTOR KINETICS 131
Open channels are described as being 'blocked' by high agonist concentrations
(Adams & Colquhoun, 1983; Colquhoun & Ogden, 1984; Ogden & Colquhoun, 1985;
Colquhoun & Ogden, 1988), and a simple model ofopen channel block has been used
to account for the observed channel kinetics. The simple model of channel block
needs to be modified to account for the activation kinetics ofthe nicotinic receptor
observed here.
METHODS
Cell culture
The method ofpreparing a primary culture ofadrenal chromaffin cells is modified from Knight
& Baker (1983).
Bovine adrenalglandswererapidly cooledbytheinjectionof5-10 mlofice-coldsolutionviathe
adrenal vein. The solution contained 1 mg ml-' Protease (Sigma P5147) in Ca2l-free physiological
saline. Theglands were thentransported onicetothelaboratory, whereexternal connective tissue
and fat were stripped away, and cortical tissue removed from the cranial surface to expose the
partly digested medullary tissue. The medulla was sliced with stacked razor blades, and the tissue
slices (ofthickness0-5-1 mm)putintocoldCa2+-free salineandrinsedtoremoveredcellsandtissue
debris. The tissue was then enzymatically digested at 37 0C for 30-40 min in Ca2+-free saline
containing bovine serum albumin (SigmaA7906) 5 mg ml-', collagenase (SigmaC6885) 2 mgml-',
hyaluronidase (Sigma H3506) 2 mg ml-' and DNAse 0-1 mgml-' (Sigma D0876). The tissue was
gently disaggregated every 10 min by passing through the widened end of a plastic 5ml
pipette tip. Isolated cells were separated from undigested tissue debris by passing through four
layers ofgauze swab, and a 25,um nylon mesh. The cells were repeatedly centrifuged at 100Yfor
2 minandre-suspended inCa2+-free saline, beforebeingre-suspendedinculture mediumandplated
on glass coverslips. Cellswere cultured inDulbecco's modified Eagle's medium supplemented with
10% fetal calfserum and used between 3 h and 14 days after isolation.
Solutions
A Ca2+-free saline was used for isolation of adrenal cells and as the extracellular solution in
experiments. It contained (mM): NaCl, 140; KCl, 5-4; MgCl2, 2; HEPES, 10; pH adjusted to 7-4
with NaOH.
The intracellular (pipette filling) solution, used for both whole-cell and outside-out patch
experiments, contained (mM): CS2SO4, 83; MgCl2, 2; CaCl2, 01; EGTA, 1; dithiothreitol, 1; ATP,
2; tetraethylammonium chloride, 10; HEPES, 10; pH adjusted to 7-4 with NaOH.
Current recordings
Membrane currents were recorded in the outside-out and whole-cell tight-seal recording modes
(Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The currentswere amplified and converted to
avoltage signal with apatch clamp amplifier (List EPC7; ListMedical, Darmstadt, Germany) and
recorded on an FM tape-recorder (Racal store 4DS, Racal Recorders, Southampton, UK) before
being filtered (purpose-built 5-pole Bessel filter) and digitized by an analog-to-digital interface
(CED 1401, Cambridge Electronic Design, Cambridge, UK). Consecutive traces were averaged
using a signal averaging program also supplied by Cambridge Electronic Design.
Solution changes
The method of making fast solution changes is described elsewhere (Maconochie, 1987;
Maconochie & Knight, 1989). Essentially, an excised membrane patch is positioned in the cross-
stream oftwo glass capillary perfusion pipes. Solutions are fed first from one pipe and then the
other, causing a sharp interface between the two solutions to move across the patch.
The time taken to make a change of solution was measured by following the liquid junction
potential at an open patch pipette tip while switching between solutions of different ionic
compositions. At the relatively low (100 mm s-1) perfusion speeds used for outside-out patch
experiments, junction potential 5-95% rise-time was routinely 0-2 ms or better. Although faster
perfusion speeds (400 mm s-') allowed faster solution changes with a 10-90% rise-time of005 ms
132 D. J. MACONOCIIE AND D. E. KNIGHT
(Maconochie & Knight, 1989), the higher perfusion speeds were not used, since 02 ms was more
than adequate to follow the kinetics ofthe recorded current, and a higher perfusion speed led to
a shorter patch lifetime. The slower perfusion speeds needed for perfusing whole cells resulted in
a longer solution change time of3 ms.
Fitting exponentials to current traces
Averaged data were fitted with exponentials by a novel method, DELP, (differential equation
legendre polynomial) described elsewhere (Maconochie & Martin, 1989; D. J.Maconochie, J. L.
Martin & D. E. Knight, in preparation).
Estimation ofthe error associated withfitting exponentials to noisy data
Theerrorinvolved infittingexponentials arisesfromthepresence ofrandomfluctuationsofthe
recorded current. Theprinciple noise component isrelatedtotheopen-channel lifetime (ofaround
10ms for the adrenal nicotinic receptor).
Traces were synthesized using the reaction scheme below:
k+XA P kD
state 4 . state 3 state 2-state 1 (1)
k- a
State 4 is the inactive state, state 3 the active state, state 2 the open state and state 1 a
desensitized state; x;A is the agonist concentration, kA/k+ the affinity for agonist, f and a the
openingandclosingrateconstantsandkDtherateofdesensitization. Byvaryingthereactionrates,
and the total number ofchannels involved, the amplitude and rate constants ofthe exponential
relaxations following a concentration jump were varied while maintaining the open-channel
lifetime. An example of the current passing through a single channel is given in Fig. 1A. The
response ofa number ofreceptors to a step change ofagonist concentration (xA) is shown in Fig.
1B, C and D. These traces were computer synthesized using a direct Monte-Carlo approach
(Hammersly & Handscomb, 1968). As the model used had three kinetically distinguishable states
(the agonist binding step is assumed to be fast), the response to a step change of agonist
concentration was a current with two exponential components (A2 and A1), relating to activation
and desensitization ofthe receptor.
The synthesized traces were fitted by the same program usedtofitexperimental data. Twenty
tracesweresynthesized, usingthesamemodelwiththesamenumberofreceptors;theywerefitted,
andestimatesofthemeanandstandarddeviationoftheexponential rate constantsobtained. The
standard deviation ofthe exponential rate constants in thesample population oftwenty istaken
tobeanestimateoftheerrorassociatedwithfittingasingle synthesized trace. Itisfoundthatthe
errorassociatedwithmeasurement oftheexponentialrateconstantsisdependentonthesizeofthe
peakcurrent (andthereforeonthenumberofchannelsmodelled),andonthesizeoftheexponential
rate constants themselves. This relationship is shown in Fig. 1E. Each point on the graph
represents a standard deviation oftwenty measurements.
In order to obtain an estimate ofthe error associated with fitting a real data trace, the peak
currentismeasured,andavalueofthepercentageerroroftheexponentialrateconstantsreadfrom
the graph ofFig. IE. Thisfigure is referred to subsequently asthe errornomogram. Experimental
traces were used for analysis ifthe sum ofthe errors in the fitted exponential rates constants, as
read from the error nomogram, was less than 25%.
This method oferrorestimation is only suitable fortraces withthe type ofnoise described, and
with only two closely spaced exponential components.
Fitting the concentration dependence ofthe A
Asimplekinetic modelwasusedtodescribethedatafromoutside-outpatches;thisisdescribed
by the reaction scheme (1) above. The agonist binding step is assumed to be fast. The
desensitization step is assumed to be irreversible, since the equilibrium current recorded in the
presenceof100gM-AChisidenticaltothecurrentintheabsenceofACh.TheplotsofAagainstACh
concentration were fitted using a Marquardt least-squares process (Enzfitter, Elsevier-Biosoft,
Cambridge,UK).ThevariationoftheexponentialrateconstantA1,associatedwithdesensitization
ofthereceptor, withACh concentration wasinsufficiently dependentonaandf forafitofall the
variables (a,fi, kDandagonistaffinity) to befullydeterminate. Soaand werekeptconstantand
,
ACETYLCHOLIVE RECEPTOR KINETICS 133
A B
''.'.h .
I;i 'p !,*
C D
E
201
6c.n
a)
X 10
480
CaL, 420
280
5
*90
30 60
50 100 200 500 1000
Peak number of open channels
Fig. 1. The use ofsynthesized current traces to estimate the error associated with fitting
experimental data. Trace A represents a typical single channel current from a nicotinic
receptor in the presence of 100/tM-ACh, filtered at 3 kHz (-3 dB 5 pole Bessel). Traces
B, CandDrepresent typical responses of 20, 50 and 100 receptors, similarly filtered, and
all lasting 100ms. All four traces have been synthesized using direct Monte-Carlo
techniquesfromamodelconsistingofasinglefastagonist-bindingstep,slowisomerization
totheopenstate, and irreversible desensitization. Gaussian noise hasbeen added tomore
closely simulate real currents. Traces B-D have been overlaid with double exponential
fits. From groups oftwenty such traces, estimates ofthe mean and standard deviation of
the fitted exponential rate constants have been derived as functions of peak current.
These are the bases of the points used in part E. Graph E shows the percentage
error of the exponential rate constants fitted to a current trace, plotted against peak
current amplitude (expressed asthe number ofsingle channels open), fordifferent values
ofthe exponential rate constant.
kD and the agonist affinity allowed to vary. Similarly, the exponential rate constant A2 was fitted
byvariationofaand, only. Theestimatesofaand,wereusedinfittingA,again. Thisprocesswas
repeated until further change in the fitted parameters was less than 10% ofthe estimated error.
134 D. J. MACONOCHIE AND D. K KNIGHT
Theratioofslowtofastdesensitizingreceptorswasdeterminedasfollows.Firstthereactionrate
constants and agonist affinityforeach receptortypewere calculated using the methods described
above. Next, these values were usedtogenerate the amplitudes oftheexponential rate constants
A. and A2 as functions ofconcentration using the methods ofColquhoun & Hawkes (1977). The
ratio ofthe two receptor types was adjusted until the function A1/A2 most closely matched the
graph ofA1/A2.
RESULTS
Concentration-jump experiments with patches and whole cells
Concentration-jump experiments were performed with both outside-out patches
and whole cells. Some examples oftypical traces at a variety ofacetylcholine (ACh)
concentrations are shown in Fig. 2 (patches) and Fig. 3 (whole cells). The traces
A B
C D
E F
20 pA
10 ms
Fig. 2. The response of outside-out patches of adrenal chromaffin cell membrane to
different concentrations ofACh. These traces ofmembrane current were recorded from
outside-out patches excised from cultured bovine adrenal chromaffin cells. The current
baseline isnearto zero. The downward displacement indicates an inward current due to
openingofnicotinic cholinergic ion channelsinresponsetopulsesofACh; 2 mm, tracesA
and B; 100uM, traces C and D; and 10/SM, traces E and F. The presence of ACh is
indicatedbythefilledbar. Thetracesinthefirst column areresponses toasinglestepin
theAChconcentration; thesecondcolumn showstheresultofaveraging between 60and
120 successive responses. The averaged traces are smaller than the single traces because
themagnitude oftheresponsetoACh declineswithtime. Therepetitionratewas01 Hz.
Themembrane potentialwas -80mV. Thesignalswerefilteredat 10kHz (5poleBessel
-3dB), digitized at 32kHz and then filtered again digitally at 2kHz (8 pole Bessel
-3dB).
ACETYLCHOLINE RECEPTOR KINETICS 135
follow a double exponential time course, the current first activating, and then
desensitizing.
Whole-cell records are well fitted by a sum ofthree exponential: one activating,
and two desensitizing. If more than three exponentials are fitted, the extra
exponentials are oflow amplitude, and have variable (and frequently positive) rate
constants. Figure 4 shows how closely the response of a whole adrenal cell to a
sustained application of ACh may be fitted with three exponential. Outside-out
patches are, in contrast, best fitted by a sum of two exponential components.
In the continued presence ofhigh concentrations ofagonist (10/M to 1 mM), the
currentsrecordedfrombothwholecellsandfromoutside-outpatchesdecaytoalevel
that is indistinguishable from the current baseline. This is confirmed in truncated
traces (responses to shorter applications ofagonist), the value ofthe constant term
included in the multiple exponential fit is close to the value ofthe current prior to
the application ofagonist.
Afterisolation, andrepeated applications ofACh, the size ofthe currentsrecorded
from outside-out patches declines and eventually disappears. Thetime course ofthis
patch 'run-down' is variable, and can result in anywhere between 20 and 120 single
responses being collected at 10 s intervals, from one patch. This phenomenon is not
observed withwhole cells. The amplitude ofthe response to ACh may be maintained
over periods of 1 h or more.
The exponential rate constants are not stable with time. In experiments with
outside-out patches, the rate of desensitization can increase by up to 30% over
30 min. However, most data were collected from outside-out patches in 5-10 min.
Theresponse ofwhole cells also varieswith time. Responses to, forexample, 100gm-
ACh, are on average 20% faster after 20 min. These time variations add to the error
involved in measuring exponential rate constants.
Simultaneous measurement ofvoltage-jump and concentration-jump responses
It has been shown (Eigen & de Maeyer, 1963; Colquhoun & Hawkes, 1977) that
a given reaction scheme, constructed of first-order reaction steps, will relax
exponentially following a perturbation. If the Markov hypothesis applies to the
behaviour ofthe bovine nicotinic receptor, then given the same external conditions,
the exponential relaxation rate constants following a perturbation will be
independent ofthe nature ofthe perturbation.
Two simple ways of perturbing a receptor/channel are to step the agonist
concentration (as described above) or to step the membrane potential (and modify
voltage-sensitive reaction rates). Both of these manoeuvres are performed on the
same cell in Fig. 5. The cell is held at -80 mV. A step change ofagonist is applied
(100/lm-ACh); section a-d ofFig. 5. After a short time, during which the nicotinic
receptors have activated and are beginning to desensitize, the membrane potential
is stepped to +40 mV for 5 ms and then returned to -80 mV. At this point, the
current begins to rise again, before desensitizing further; section c-d.
Sincethe sections oftrace a-b and c-d areshort, onlytwoexponential components
were fitted to the data (the third exponential component appears in the constant
term). The results offitting exponentials to section a-b and c-d are shown in Table
1. Although the amplitudes A are different, as would be expected since the initial
136 D. J. IACO4NOCHIE AND D. E. KNIGHT
conditions are different, the exponential rate constants are nearly identical. This is
as predicted by the Markov hypothesis. and opens the way for the analysis of the
results by constructing reaction schemes with constant reaction rates to model the
data.
2
A B
C D
' 9'~~-0~
E F
500
ms
Fig. 3. The response of voltage-clamped adrenal chromaffin cells to different con-
centrations ofACh. Thesetraces ofmembrane current were recorded from asingle bovine
adrenal chromaffin cell in the 'whole-cell' recording configuration. The current baseline
isveryclosetozero. Thecurrent isduetotheopeningofnicotinic ion channels in response
toACh: 100puM. traceA 20yIM, trace (1; 5#AI, trace E. TracesB.DandFshow theresult
of averaging fifteen to thirty single responses. The repetition rate was 0067 Hz. The
signals were filtered at 2 kHz (5 pole Bessel -3 dB), (ligitize(1 at 5 kHz, and filtered
digitally at 600 Hz (8 pole Bessel -3dB).
The variation ofthe exponential rate constants with agonist concentration
A total of ninety experiments were performed on outside-out patches, and two
exponential fitted to each trace. For each trace, an estimate ofthe error involved in
fitting exponential to the traces was made, using the procedure described in the
Methods section. If the sum of the error associated with each exponential rate
constant for a particular trace was greater than 25%, then that trace was
ACETYLCHOLINE RECEPTOR KINETICS 137
discarded. A total offorty-two traces were left, representing experiments with ACh
concentrations of 10 aM to 2 mm.
The two exponential rate constants, A, associated with desensitization and A2
associated with activation ofthe receptor, are plotted against ACh concentration in
A
500 pA
100 Ms
B C
100 Ms
Fig. 4. A triple exponential fitted to whole-cell currents. The average ofsix responses of
abovine adrenal cell toalong (2s) pulse of100gm-ACh. The trace is overlaid with afit
ofthree exponential components, withrate constants -33, -18-1 and -131 s-1. Trace
Ashowsthewholeoftheresponse,overlaidwiththefittedcurve. TraceBshowstherising
phase in more detail, with the fitted curve overlaid in white. The fit starts -3ms after
the start ofthe pulse ofACh. Trace C shows the difference between the recorded signal
and the fitted curve of trace B. The current baseline is near to zero, and the trace
approaches asymptotically the current baseline. The membrane potential was -80mV.
The signal was filtered at 2kHz (5 pole Bessel -3 dB) and digitized at 6kHz.
TABLE 1. A comparison ofconcentration-jump and voltage-jump responses
Amplitudes and rate constants
Al A1 A2 A2
Part oftrace (pAi) (srl) (pA) (($-1)
Following concentration jump -1819 -5-1 1394 -108
Following voltage jump -1209 -4-3 6806 -102
After agonist removal -18 -9-72 -310 -76
Sections ofthe trace in Fig. 5A were fitted by two exponential. The amplitudes A and rate
constantsAaregiven. Theexponential componentofpositiveamplitudeandfastexponential time
constant relates to activation ofthe receptor. The similarity of the exponential rate constants
following thevoltagejump and the concentration jump ispredicted fromthe Markov hypothesis.
Fig. 6. It is apparent that there is a wide variation in the values of the two
exponential rate constants, much wider than the estimated error ofmeasurement.
The curves drawn through the graphs of Al and A2 are the results of fitting the
138 D. J. MACONOCHIE AND D. E. KNIGHT
A B
a bc d
100-0ppA I
50ms
:I
_
+40 mV- -
-80mV- -
Fig. 5. Anexaminationofavoltage-jump response andaconcentration-jumpresponsein
thesamecell. Theresponseofabovineadrenalchromaffin celltoa50mspulseof100fiM-
ACh. The membrane potential was -80mV. After 10ms the membrane potential was
stepped to +40mV for 5ms. The capacitive transient cancellation circuitry ofthe List
EPC7 amplifier was used to balance the transients dueto the impedance ofthe cell and
the electrode. A part ofthe fast transient remains uncancelled. TraceA is anaverage of
twenty single records. TraceB shows the initial rising phase oftraceA on an expanded
timescale,withthepartofthetraceimmediatelyfollowingthevoltagestepsuperimposed
(partoftheuncancelledcapacitivetransientisevidentatthestartofthissectionoftrace).
Theinitialdownwarddisplacementisduetotheopeningofnicotinicchannelsonbinding
withACh. Thevoltage pulseproduces ashiftinthe currentbaseline of240pA. Afterthe
potential step, the current rises again with a similar time course to the initial
displacement. All the traces were filtered at 10kHz (5 pole Bessel -3 dB), digitized at
30kHz, and filtered digitally at 3kHz (8 pole Bessel -3dB). ACh is applied at a, a-b
shows the activation and partial desensitization ofthe receptors at -80mV. At b, the
membrane potential is stepped to +40mV for 5ms. At c, the membrane potential is
returned to -80mV. The section oftrace c-d shows the continuation ofactivation and
desensitizationofthereceptorsat -80mV. Atd,theAChisremoved, sofromdonwards,
the current decays in the absence ofagonist.
model reaction scheme (1) tothe data, using the approach described in theMethods.
The following values were found:
a =29+12r1,
A= 460+60 s-11
kD = 24+2 s-l
k-lk+ = 310+100#M.
The three components fitted to currents recorded from whole cells are shown in
Fig. 7A,B and C (Al, A2 and A. respectively). The two exponential rate constants
associated with desensitization are Al andA2. The third exponential rate constant A3
is associated with activation. In the majority ofexperiments performed with whole
cells, the currents obtained were of the order of 100 times the amplitude of the
response in a similar experiment with outside-out patches. These peak amplitudes
were out ofrange ofthe nomogram constructed for error estimation. Moreover, the
errornomogram ofFig. 1 appliestodataconsisting oftwoexponential withopposite