membrane potential, which is explained by the voltage-dependent gating ofIns, Ache

15 Pages·2005·1.69 MB·English

JournalofPhysiology (1990), 424, pp. 57-71 57
With8figures
PrintedinGreatBritain
EFFECT OF MEMBRANE POTENTIAL ON ACETYLCHOLINE-INDUCED
INWARD CURRENT IN GUINEA-PIG ILEUM
ByR. INOUE* AND G. ISENBERG
From the Department ofPhysiology, University ofCologne, Robert-Koch-Strasse 39,
D-5000K6ln41,FRG
(Received 7June 1989)
SUMMARY
1. The whole-cell patch clamp technique with caesium aspartate internal solution
was used with single isolated cells from the longitudinal muscle layer ofguinea-pig
ileum, toinvestigate the voltage-dependentgatingofACh-induced inwardcurrent.
2. In voltage clamp experiments, at holding potentials ranging from -80 to
-30 mV, ACh (300,tM) produced a slow sustained inward current in physiological
saltbathsolution (PSS). Themeasurements ofthereversalpotentialsonsubstituting
Na+ by other monovalent and divalent cations showed that this current is through
non-selective cation channels (Ins,ACh)-
3. During hyperpolarizations, Ins,ACh instantaneously increased in amplitude and
then relaxed to a new steady-state level. The I-Vrelationship ofthe instantaneous
peakwas linear with a reversal potential of0 mV, while that ofthe steady state was
bell-shaped. The time course ofrelaxation appeared to be monoexponential and its
time constants were reduced by stronger hyperpolarizations.
4. These results were not affected by the organic Ca2+ antagonists D600 or
nitrendipine (10,tM). Under this condition, maximal chord conductance ofIns Ach
which was observed at 0 mV was about 1-5 nS. The steady-state activation
relationship waswell fittedby Boltzmann's equationwith a half-maximal activation
(Vh) of -50 mV and a slope factor (k) of -15 mV at membrane potentials negative
to 0 mV, but over 0 mV the degree of activation was again decreased. The time
constantsforrelaxationalso appeared tofollow asigmoid curve.
5. In current clamp experiments, superfusion of ACh (300gM) depolarized the
membrane up to -10 to 0 mV. Inward current injection resulting in the moderate
hyperpolarization of the membrane (-70 to -80 mV) attenuated ACh-induced
depolarization and strongerhyperpolarization (<-80 mV) abolished it.
6. These results show that ACh-induced depolarization is controlled by the
membrane potential, whichisexplainedbythevoltage-dependent gating ofIns,Ache
INTRODUCTION
Acetylcholine is a major excitatory neurotransmitter not only in visceral smooth
muscle, but also in secretary cells, and causes contraction or secretion by its ability
* Present address: Department of Pharmacology, University of Oxford, South Parks Road,
OxfordOXI 3QT.
MS7734
58 R. INOUE AND G. ISENBERG
to raise the intracellular Ca2+ concentration. In the process of increasing the
intracellular Ca2+ concentrations, a number ofionic mechanisms have been proposed
and studied (Bolton, 1979; Petersen & Findlay, 1987). Recently in the intestine, it
hasbeenreportedthatnon-selectivecationchannels (Ins,ACh)differentfromtheCa2+_
dependent ones (Yellen, 1982; Ehara, Noma & Ono, 1988) are activated by
muscarinic receptors and the sustained depolarization can induce Ca2+ influx via
voltage-operated Ca2+ channels (Benham, Bolton & Lang, 1985; Inoue, Kitamura &
Kuriyama, 1987). The channels discriminate poorly between Na+ and K+, have a
single-channel conductance of 20-30pS, and fluctuate between open and closed
states under the influence of the membrane potential (Inoue et al. 1987). Voltage-
dependent gating of these channels has been suggested from the time-dependent
decay of the ACh-induced inward current (Benham et al. 1985). However, since
previous experiments were all carried out with long-lasting holding potentials and
under conditions which would also activate K+, Cl-, and Ca2+ currents, the detailed
featuresoftheACh-inducedcurrentsuchasthesteady-stateactivationvariablesand
the time constants of voltage-dependent relaxation are still unknown. The present
study was designed to clarify these points using short pulse protocols and caesium
aspartate pipette solution to isolate Ins,ACh. As a result ofthis work, we found that
the membrane potential is an effective regulator ofACh-induced depolarization and
its mechanism is similar to that described for other types of receptor-operated
currents (del Castillo & Katz, 1957; Trautwein, Osterreider & Noma, 1981).
METHODS
Cell isolation. Guinea-pigs weighing between 500 and 1000g were anaesthetized by intra-
peritoneal injection of pentobarbitone (50-100mgkg-'). They were exsanguinated by surgical
bleeding. Within 5cm from the ileocaecal valve, the terminal part ofthe ileum was resected and
thentransferredintoadissectingchamberfilledwith PSS (physiologicalsaltsolution). Themucosa
was removed under a binocular microscope and small pieces (2-5mm) ofthe longitudinal muscle
layer ofileum were prepared. These were transferred into nominal Ca2l-free solution containing
0-5-1-0mgml-' collagenase (Boehringer, Mannheim) and 0-5-1-0mgml-' pronase (Serva, Heidel-
berg) and incubated at 36TC stirred at a rate of 2-5 Hz for 15-20min. The supernatant was
discarded and softened chunks were gently agitated for several minutes until spindle-shaped cells
were obtained. After diluting the cell suspending medium 1:1 with Kraft Briihe (KB) medium
(Isenberg& Kl6ckner, 1982) theisolated cellswerestoredat7'C. Theexperimentswereperformed
at room temperature (20-22 °C), usually within 6h ofcell isolation.
Solutions. The composition ofthe solutions isgiven in mmol 1-1. PSS: NaCl, 130; KCI, 5; CaCl2,
2; MgCI2, 2; glucose, 10; HEPES/Tris, 10 (pH 7.4). Calcium-free solution forcell isolation: NaCl,
120; KCl, 12; MgCl2, 1-2; glucose, 20;taurine, 20; HEPES, 10;pyruvicacid, 5. KBmedium; KCI,
85; K2HPO4, 30; Na2ATP, 2; MgCl2, 5; pyruvic acid, 5; creatine, 5; taurine, 20; glucose, 10;
succinic acid, 5; EGTA, 1; fatty acid-freealbumin, 1 g-. KCIinternal solution for current clamp
experiments: KCl, 130; MgCl2, 2; creatine, 5; succinic acid, 5; oxalacetic acid, 5; EGTA, 20,am;
HEPES/KOH,5 (pH 7-2).Caesiumaspartateinternalsolutionforvoltageclampexperiments;Cs+,
130;Cl-, 24; asparticacid, 110; Mg2+2; EGTA, 10uM;HEPES/Tris5(pH7 2).
Experimental set-up. After the cells had settled on the bottom of the experimental chamber
(0-1 ml in volume), they were continuously superfused with PSS at a constant flow rate of
0-05-0-1 ml s-i. The time needed to complete a solution change was 5s-1, and thiswasestimated
fromtheeffectsofinorganicCa2+antagonists (La3+, Ni2+,etc.) ontheleakcurrent.Therecordings
were performed in the whole-cell configuration. The electrodes used had a series resistance of
2-3MQinPSS.Aftertheintracellulardialysiswiththeinternalsolutions,thecellsshowedaninput
resistance of 2-10GQ1 before and > 0-25GQ during the application ofACh. Assuming that the
series resistance ofelectrode would not increase more than 2 or 3 times after the rupture ofthe
VOLTAGE-DEPENDENCE OF ACH-INDUCED CURRENT 59
membrane, the voltage error introduced by the series resistance would be less than 5%, and was
notcompensated.
Data recording and evaluation. Theelectrodes were connected to the head stageofahome-made
patch clamp amplifier with the facility ofcurrent clamp. For monitoring the membrane currents
or the membrane potentials in real time, a Brush pen-recorder (50Hz frequency response) was
Ii~~~~~~~~~~~~I
A li~~~~~~~~~~~~~~11111IiW ==
I ~~~~~IIl
ACh, 300 mM
Vh -30 mV
=
B
Fig.1.Netmembranecurrentsinresponseto300/LM-AChrecordedfromacaesiumaspartate
(10,sM-EGTA)-loaded smooth muscle cell. On-linerecording byapen recorder. Fromthe
holding potential of -30mV, 450ms long hyperpolarizations to -40, -50, -60, -70,
-80, -90, -120, -140, -160 and -180mV were applied at a frequency of04Hz.
Thereafter, 300,gM-ACh was superfused for 10s which induced a net inward current.
When the inward current was steady, a series ofthe hyperpolarizing clamp steps were
applied again. Note: the large current deflections result mostly from the capacitive
current. A, current trace; B, voltage trace.
used. Step andramp command signalsweregenerated by a PDP23 minicomputerwhich was also
used to digitize the currents (1024 points of 12-bit resolution). The time course ofrelaxation was
fitted byaleast-squares routine. Forthe measurement ofthe reversal potentials, ramppulses (1-9
or 2-0V sol, 100ms, in duration) were applied both before and during the application of ACh
(usually 300/SM) and corresponding ramp currents were recorded in the presence oforganic Ca2+
antagonists (nitrendipine orD600, 10 eM). TheACh-sensitive currentwasobtainedbysubtracting
the control ramp current (before) from the test one (on the application ofACh) and the reversal
potential was defined as the potential at which the ACh-sensitive current becomes zero (see Fig.
2B).
RESULTS
Ins,AChmeasured incaesiumaspartate-loaded cellbathed innormalPSS
In order to analyseIns,ACh, the other superimposed ionic currents, e.g. K+ and C1-
currents were suppressed by loading the cells via the patch electrode with high
caesium aspartate solution (substitution of intracellular K+ and Cl-, respectively).
When the membrane potential was clamped to negative potentials (-80 to 30 mV),
superfusion of ACh induced net inward currents. Figure 1 shows, an example at a
holding potential of -30 mV, where the inward current peaked within 10 s to about
-100 pA, and then slowly decayed. This decay most likely results from at least two
effects, i.e. simple wash-out of ACh and desensitization. Desensitization was
indicated bythefactthattheresponse to asecondapplication ofACh 1 minlaterwas
almost absent, and the responses recovered gradually as the interval between
applications became longer. Complete recovery from desensitization was usually
observed after 5 min, but in some cases, was not obtained, probably because some
|~~~~A
60 R. INOUE AND G. ISENBERG
kind of 'run-down' decreases the response to ACh time dependently with a suction
electrode (Horn & Marty, 1988). Thus, the period ofstable whole-cell recording was
normally limited to within 30 min.
In order to analyse the current sensitive to ACh, InsACh was defined as the
A
1.0 0
/
0~~~~~~
0.
*0
z-o
-7 -6 -5 -4 -3
0.5~~~~~~~log~[~AC~hJ(~M)~
B
50
-1 00 -50 0~~~~~10
i' ~~~-50m
iig. 2. Concentration response curve and instantaneous I-V relationship of 'nsACh* A,
concentrationresponse curve; normalized Ins ACh is plotted versus the log[ACh]. Data
from three different cells are shown by different symbols. The curve is drawn using the
Michaelis-Menten equationIn~ACh = 1/{1+Km/[ACh]8}, wherethedissociation constant
(Kin) is 10/SM and the Hill coefficient (n) is 10. B, I-V curve of the instantaneous ACh
(300,SM)-sensitive currentsevaluated with quick ramppulses (100 ins, +80to-120 mV,
see inset) from a holding potential of-30 mV in the presence of nitrendipine (10O4UM).
Note that I-V curve shows rectification at potentials~positive to the reversal potential
(+6mV).
difference current obtained by the subtraction method (see Methods and Figs 2B and
3). 'ns,ACh recorded with caesium aspartate electrodes showed a similar ACh
concentration-response relationship to that described previously (KCl electrodes;
Inoue et al. 1987). The curve was ofthe Michaelis-Menten type with a Hill coefficient
of 0 8-1 0. The half-maximal activation required about 101sM-ACh (Fig. 2A, these
values are similar to those evaluated by binding studies using radioisotopes; Birdsall
et al. 1979). Atropine (1 /tM) completely blocked Ins,ACh' thus suggesting that the
receptor is ofthe muscarinic type. In ACh was reduced in amplitude in parallel with
a reduction ofthe extracellular Na+ concentration and was almost zero when NaCl
VOLTAGE-DEPENDENCE OF ACH-INDUCED CURRENT 61
was isosmotically replaced by Tris-Cl in PSS. The reversal potential shifted from
+1P6+ 4-2 mV (Fig. 2B) to -32-6+ 3-7 mV by reducing the extracellular Na+ from
130 to 30 mm (n = 3). The reversal potential of Ins,ACh did not however change
significantly whentheextracellularNa+wassubstituted byequimolarLi+, K+orCs+
(±+1-5+ 23 mV, -1.75+2-4 mV, -1-4± 2-6 mV, respectively, n = 2-4). Therefore,
it is concluded that Na+, LiW, Cs+ and K+ are almost equally permeable to this
channel. Substitution of 130 mM-NaCl by 110 mM-CaCl2 (10 mM-EGTA in the
pipette) still produced an inward current in response to ACh (0-57±0-16nS: a
steady-state chord conductance at -60 mV, n = 4) but the reversal potential was
shifted to +199+95 mV (n = 3). This finding strongly suggests that Ca2+ can
permeate the ACh-activated non-selective cation channel comparably or more
potently than Na+. Nearly identical values and the same conclusion were obtained
when 110 mM-Ba2+ was present in bath instead of Ca2+ (0-44±0-28 nS at -60 mV,
22-7+2-6 mV, n = 3). These properties indicate thatIns,ACh measured in the present
contextwith caesium aspartate electrodes isthe same astheIns,ACh currentrecorded
with KCl internal solution (Inoue etal. 1987).
Influence ofmembranepotentialonIns,ACh
Hyperpolarization to more negative potentials caused a relaxation ofIns ACh (Fig.
3). Forexample, at -140 mV,In~s ACh jumped to an instantaneous peak of -250 pA
and then rapidly decayed (Fig. 3D). The time course of this relaxation followed a
single-exponential curve. The rate and degree of relaxation changed voltage
dependently, i.e. at -140 mV, Ins ACh fell quickly (short r) to almost zero, while at
-80 mV, it fell more slowly to a more negative current level. The rate ofrelaxation
is more clearly seen in Fig. 4A. The time course of relaxation during hyper-
polarization waswell fitted byanequation:
I(Vm,t) = a+bexp(-t/r),
where I(Vm,t), (a+b), a and r denote the current amplitudes at a time t, of the
instantaneouspeakandthesteady state, andatimeconstantofrelaxationatagiven
membrane potential (Vm) for Ins ACh respectively. Obviously, the time constant r
decreased as the membrane washyperpolarized (Fig. 4B).
Upon repolarization to the holding potential of -30 mV, tail currents were
observed (Fig. 3D). The tail currents started from an initial peak with little delay
and converged to almost the same amplitude regardless ofthe size ofthe preceding
hyperpolarization. The amplitude ofthe initial peak ofthe tail current depended on
the preceding testpulses, i.e. at -140 mVitwas almostzero, butincreased to about
-20 pA at -60 mV. Nevertheless, the time constants forthetail currentswere very
similar (in this case, 150-200 ms). These results suggest that the voltage-dependent
gatingoftheACh-activated channels inthispreparation can be describedasasimple
two-state model (open-closed).
I-Vcurvesandsteady-stateactivationcurveofIns,ACh
Figure 5A shows the voltage dependence ofthe I-Vrelationships evaluated from
the experiment shown in Fig. 3. The instantaneous I-V relationship (0) is nearly
linear. This curve extrapolates to a reversal potential of +4 mV and gives a chord
62 R. INOUE AND G. ISENBERG
conductance of 1P7 nS at the holding potential ofthis experiment (-30 mV). In five
cells, the extrapolated reversal potential was -02+3-9 mV. These results are in
good agreement to that obtained by ramp pulses (see above). The I-V relationship
ofthe steady state is indicated by the filled circles and the dashed line in Fig. 5A. As
A Vt Vh=-30 mV
450 ms -140
-60
450ms -140 mV
B It 200 ms
a 0
im . i P i R
-250
pA
C
0
--250
pA
DI mv -140 mV
-8o
-4 ...1 0
-80 mV
-60 mv
-40 mV -40 mV I -100
-60 mV
-200
-140 mV
II
pA
Fig. 3. Evaluation of the ACh-sensitive current InsACh Computer print-outs of the
experimentdescribedinFig. 1. Theschematicofthepulseprotocol (A) isshown together
withthenetcurrentsbefore(B)andinthepresenceofACh(C).Note:thetransientinward
andoutwarddeflectionsaremostlyduetothecapacitiveartifacts. InthepresenceofACh
the current is more inward and tracings cross each other. D, In,, ACh' defined as the
differencecurrent (C-B). Notetheenlargedcalibrationofthesetracings.
expected from the voltage-dependent relaxation ofIns,ACh, the curve is bell-shaped
with a maximum at -30 mV declining toward the voltage axis at very negative
potentials. This strong voltage dependence is more clearly observed when the ratio
ofthevalueatthesteadystate tothatoftheinstantaneous current isplotted against
the membrane potential (0 in Fig. 5B). The steady-state activation curve rises as
the membrane is depolarized and shows an e-fold change for about 35 mV at the
membrane potentials between -80 and -30 mV. The curve is also calculated from
the amplitude ofthe initial peak ofthe tail currents upon repolarization (O in Fig.
5B). Using adequate calibration (right ordinate), both curves superimpose.
VOLTAGE-DEPENDENCE OF ACH-INDUCED CURRENT 63
Voltagedependence of1ns,ACh inthepresence ofCa2+ antagonists
The concentrations ofACh used in the present experiments (041-1000/tM) affected
othermembraneconductanceaswellasIns,ACh i.e. thevoltage-operatedCa2+ current
diminishedduringtheapplication ofACh. Furthermore,Ins, ACh can bemodulated by
A
B
0
irn
0
E
1= I
0 is--
-180 -140 -100 -60
Membrane potential (mV)
Fig. 4. Time course and voltage dependence of relaxation of I.,ACh during hyper-
polarization from the experiment ofFig. 3D. A, normalized time course ofrelaxation of
InsACh at -140, -90, -70 mV. For better comparison, peak amplitude of InsACh is
normalizedto 10foreachmembranepotential. Smoothcurvesaredrawnbyleast-squares
fitting using anequationI(Vm,t) = a+bexp(-tIT) (seetext). Thevaluesaand T are 0X02
and 28ms, 013 and 78 ms, 0-23 and 95 ms at -140, -90 and -70mV, respectively. B,
time constants (-r) are plotted against the membrane potential.
the intracellular Ca2+ concentration via Ca2+ influx through voltage-operated Ca2+
channels. (For both observations, see Inoue & Isenberg, 1990). Such effects could
introduce an error when obtaining the purely voltage-dependent properties of
Ins,ACh. A similarproblem is discussed in the case ofatrial cells (Noma & Trautwein,
1978). Therefore, the experiments were repeated in the presence of organic Ca2+
antagonists (10,tM-nitrendipine or D600) over a wide range of the membrane
potential (- 160 to +40 mV), and the results obtained were compared with those in
the absence ofCa2+ antagonists.
Figure 6A shows an example of records of Ins ACh during jumps to various
potentials. At potentials negative to the holding potential, the rate and degree of
64 R. INOUE AND G. ISENBERG
relaxation are very similar to those in the absence ofCa2+ antagonist (compare with
Fig. 3D). The most prominent thing is that at potentials positive to the holding
potential currents increased in amplitude time dependently and their corresponding
tail currents showed relaxation instead of reactivation. The maximal activation is
A
mv
-300 pA
B
100
0.
75 '
as 1.0
0.
50 X
-5101 n.- l0L0i-
25 m
E
0 <
-160 -120 -80 -40 0
Membrane potential (mV)
Fig. 5. I-Vcurves ofIs,,AChevaluated fromexperiment ofFig. 3D. A, instantaneous (0)
and steady-state current (@) plotted versus the potential ofthe hyperpolarizing clamp
step. Connecting lines were drawn by eye. B, steady-state activation curve ofIng,ACh'
Duringthepotentialclampstep,theratioofthesteady-statecurrenttotheinstantaneous
current is marked by filled circles and calibrated on the left. Repolarization back to
-30mV evoked a tail current. The amplitude of the instantaneous tail current (the
amplitude at its initial peak measured from the zero current level) follows the same
activationcurve. Itismarkedbyopencirclesandcalibratedontherightordinate.
seen near 0 mV where InS,ACh reverses the polarity, and the amplitude of the tail
current increased to about 3-5-fold of that at -60 mV. Interestingly, at more
positive potentials than 0 mV, the initial peak of tail current decreased again, to
about 80% ofthat at 0 mV in this case. The maximal chord conductance ofIns,ACh
appearing at 0 mV is calculated to be 1-54+0-59 nS (n = 5) in the presence ofCa2+
antagonist.
The filled circles in Fig. 6B show the steady-state activation curve in the presence
of nitrendipine or D600, which was obtained from the ratio of the steady-state
VOLTAGE-DEPENDENCE OF ACH-INDUCED CURRENT 65
A
t= -140, -80, -60, 0, 40 mV
Vh=-0 mV
- - ,z~~~/0' -80
\ 4° ~40-_w
-140 0 j 40 pA
100 Ms
B
1.0
-0-
.~05
E
Z
0
-160 -120 -80 -40 0 40
Membrane potential (mV)
C
200
E
o100
E
0
-160 -120 -80 -40 0
Membrane potential (mV)
Fig. 6. Voltage dependence of I.,ACh in the presence of organic Ca2+ antagonists. A,
currenttraces ofIn.ACh recorded during steppulses tovariouspotentials in thepresence
ofCa2 antagonists (either 10/tM-D600 or 10ItM-nitrendipine). B, steady-state activation
curves in the absence (0) and in the presence (@) ofCa2+ antagonists. In the potential
range between -160 and 0mV data were fitted by a Boltzmann curve
t/{1+exp[(V-Vh)/k]}, wherethepotential ofhalf-maximal activation Vhis -50 mVand
slope factor k is -15 mv. C, voltage dependence ofrelaxation time constants in various
conditions; in the absence (at two different holding potentials of -30 mV (0) and
-80mV (A)), and in the presence ofCa2+ antagonist (A). The asterisk means that the
time constant was evaluated by an envelope oftail currents which is normally used to
studythetimeconstantsforactivationofvoltage-operatedCa2+currents (seeforexample
Fig. 7DinInoue& Isenberg, 1990).BandCshowthemean+s.E.M. (n = 4-12).
3 PHY424
66 R. INOUE AND G. ISENBERG
current to the instantaneous current or from the tail currents (mean+S.E.M., n =
6-10). Forbetter comparison, each valuewasnormalized tomake the maximal value
(i.e. at 0 mV) I 0. Negative to -30 mV, the values compare to those obtained in the
absence ofCa2+ antagonists (O), i.e. these drugs are virtually ineffective on Ins,ACh
A
-[-- 0 y
a-50 mVy
ACh, 300 mM 10s
b
50 mV
ACh, 300 mM 10 S
B
a b
25 m 25 mV
--------- --- -70 mV
5s 5s
Fig. 7. Effect ofmembrane hyperpolarization on ACh-induced depolarizations recorded
with a current clamp mode. The pipette contained KCl internal solution (20,/M-EGTA).
A, ACh (300liM) was added between filled circles to the PSS. a, inward currents of
-10pA (4s) were injected at the bars. b, inward currents of -10pA was constantly
injected to hold the membrane potential near -70 mV but cancelled by short injections
of outward currents (+10pA, 4s) at the bars which allowed the production of
spontaneous action potentials. Ba and b, the expanded records from Aa and b,
respectively. *,theonsetofapplicationofACh.
at this concentration (100gM-D600, however, depressed InsACh severely). Up to
0 mV, the steady-state activation relationship is sigmoid-shaped and adequately
expressed byaBoltzmann distribution:
normalized us,ACh = 1I0/{1 +exp[(V-Vh)/k}
setting the potential for half-maximal activation Vhto -50 mV and slopefactor kto
-15 mV. This suggests thatnearlythehalfofIns,ACh channelsareinactivated atthe
resting membrane potential (-55 to -60 mV, Suzuki & Kuriyama, 1975) and small
changesinmembranepotential cangreatlyaffecttheactionofAChonthemembrane
conductance. At positive potentials, the curve falls again, thus indicating that
activation ofIns,ACh at +40 mVis only 35% ofthe maximum. Someexplanation for
this fall are presented in the Discussion.
Figure 6C compares the time constants of relaxation process in three different
conditions. When a preceding holding potential was changed (-30 mV (0) versus