Adams, P.R., Brown, D.a., and Constanti, A. (1982b) - Pharmacological Inhibition of The M-Current

J. Phy8iol. (1982), 332, pp.
223-262 223
With 27 text-figures
Printed in Great Britain
PHARMACOLOGICAL INHIBITION OF THE M-CURRENT

BY P. R. ADAMS*, D. A. BROWNt AND A. CONSTANTIt
From the Department of Physiology and Biophysics, University of Texas
Medical Branch, Galveston, TX 77550, U.S.A.
(Received 18 February 1982)
SUMMARY
1. The effects of muscarinic agonists, luteinizing hormone-releasing hormone
(LHRH) analogues, uridine triphosphate (UTP) and divalent cations on K+-currents
in voltage-clamped bullfrog sympathetic neurones have been studied.
2. Muscarine (1-10 /UM), D-ala6 LHRH (1-5 psM), UTP (50-100 /SM) and Ba2+
(1-4 mM) selectively depressed the M-current (IM), without appreciable effect on the
delayed rectifier, Ca2+-activated or transient outward currents (IK, IC or IA).
3. Im-inhibition was characterized by: (a) elimination of slow current relaxations
accompanying voltage jumps in the membrane potential range -30 to -60 mV; (b)
reduced voltage-dependent chord conductance over this range with no change in the
voltage-independent chord conductance at more negative membrane potentials; (c)
suppression of outward rectification in the steady-state current-voltage curve
between -70 and -25 mV; and (d) development of an inward current which
increased in amplitude between -70 and -20 mV in proportion to the decrease in
steady-state IM. The kinetics and voltage sensitivity of residual IM were unchanged.
4. The magnitude of the inward current produced by muscarine or LHRH could
be accounted for quantitatively by the reduction in steady-state IM. No increase in
leak current could be detected in the range -60 to - 30 mV. In two cells muscarine
(10 /SM) increased the leak current and conductance at -70 to -100 mV, but not
at more depolarized levels.
5. Im was not modified by removing extracellular Ca2+, adding a selective
Ca2+-channel blocker (Cd2+), adding 1 mM-dibutyryl cyclic AMP or 8'Br cyclic GMP,
or by intracellular ionophoresis of Ca2+, 8'Br cyclic GMP, dibutyryl cyclic AMP,
GTP-y-S or S-adenosylmethionine.
6. It is concluded that the principal effects of these agents in unclamped neurones -
depolarization, increased input resistance, reduced outward rectification and increased
excitability - are due entirely to a selective inhibition of IMP The intracellular
transduction mechanism for IM inhibition is unknown.
* Present address: Department of Neurobiology and Behaviour, State University of New York
at Stony Brook. Long Island NY 11794 U.S.A.
t Present address: Department of Pharmacology, School of Pharmacy, University of London,
29/39 Brunswick Square, London WC1N lAX U.K.
224 P. R. ADAMS, D. A. BROWN AND A. CONSTANTI
INTRODUCTION
We have previously reported the presence of a time and voltage-dependent

K+-current in bullfrog sympathetic neurones which we termed the M-current (IM:
Brown & Adams, 1980; Adams, Brown & Constanti, 1982 a). IM is a non-inactivating,
non-rectifying current, activated relatively slowly (rmin 150 msec) between -60
-
and -10 mV. It appears to exert a strong potential-clamping effect on the neuronal
membrane potential when the latter is perturbed from rest potential by (for example)
a change in leak conductance or by current injection, and induces pronounced
outward rectification in the steady-state current-voltage curve between -60 and
-25 mV.
Several compounds have previously been observed to produce a 'slow' depQlariz-
ation of amphibian sympathetic neurones, during which the input conductance of the
cell is reduced. These include muscarinic acetylcholine-receptor agonists (Nishi, Soeda
& Koketsu, 1969; Kuba & Koketsu, 1976a) luteinizing hormone-releasing hormone
(Jan, Jan & Kuffler, 1979, 1980) and uridine nucleotides (Siggins, Gruol, Padjen &
Forman, 1977; Gruol, Siggins, Padjen & Forman, 1981).
We now report that these compounds all selectivity inhibit the M-current and
provide evidence that this inhibition underlies their depolarizing action. Some of the
results have been reported in abbreviated form (Brown & Adams, 1979; 1980; Adams
& Brown, 1980; Brown, Constanti & Adams, 1981; Constanti, Adams & Brown,
1981 a).
METHODS
Experiments were performed on neurones in trypsin-digested isolated bullfrog lumbar sympathetic
ganglia under two-electrode voltage clamp as described previously (Adams et al. 1982a). In
addition, a few tests were made in non-trypsinized cells using a single micro-electrode coupled to
a high frequency sample-and-hold voltage-clamp amplifier (Dagan 8100), as described by Constanti
& Brown (1981) and Halliwell & Adams (1982). Initial tests showed that the sampled voltage
responses recorded through the Dagan amplifier in switched voltage-clamp mode correspond well
with those observed with an independent voltage-recording electrode. This indicates that the soma
was well clamped, at least for the small (£ 10 nA), slow M-currents. The same form of single
electrode clamp was also used to record the membrane currents induced by intracellular ionophoresis
of various substances (applied through a second intracellular electrode), as described by Adams,
Constanti, Brown & Clark (1982 b).
The normal perfusing solution was Tris-buffered Ringer solution with 10 mM-added MgCl2 at pH
7-2 and 22 °C (Adams et al. 1982 a). The effects of omitting Mg2+ or modifying Ca21 were tested and
are described in Results. Drugs were applied by rapid bath perfusion,*using electronic switching
valves. For brief perfusion these were activated for a pre-set time (1-5 sec) by a pulse generator.
The perfusion rate was sufficiently high (1 ml. sec-1) and the bath fluid volume and perfusion dead
space sufficiently small (£ 0-5 ml.) that substantial exchange occurred within 1 sec. In a few
experiments muscarine was applied to the outside of the cell by 0 5-3 sec ionophoresis from a
micropipette filled with 100 mM-muscarine, using positive currents of a few nanoamps. However,
the effect was no more rapid, and less consistent, than that obtained by fast bath perfusion. For
intracellular ionophoresis, micro-electrodes (10-15 MQ) were filled with 10-100 mm solution and
ionophoretic current was monitored by subtraction of Dagan continuous current output from total
clamp current-to-ground (see Adams et al. 1982b).
The LHRH-analogue, D-ala6 LHRH, was kindly provided by Drs J. Rivier and W. Vale of the
Salk Institute. Other compounds were obtained from Sigma: (± )-muscarine chloride, methacholine
chloride, pilocarpine hydrochloride, atropine sulphate, uridine triphosphate, uridine diphosphate,
PHARMACOLOGY OF M-CURRENT 225
uridine 5'-monophosphate, orotidine 5'-monophosphate, orotic acid, dibutryl cyclic adenosine
monophosphate (db cyclic AMP), 8'Br-cyclic guanosine monophosphate (8'Br cyclic GMP),
guanosine 5'O-3-thiotriphosphate (GTP-y-S), isomethylbutylxanthine (IBMX) and S-adenosyl-
methionine (SAM).
RESULTS
Muwcarinic agonists
Inhibition of IMP
Figs. 1 and 2 illustrate the experimental protocol usually adopted for detecting the
inhibition of IM by muscarinic agonists or by the other pharmacological agents tested
in these experiments. In Fig. 1 A, the cell was held under voltage clamp at a relatively
depolarized membrane potential (-30 mV) such that some 60 % or so of the
M-channels were in the open state, thereby generating a steady outward K+-current
(see Adams et al. 1982a). The cell was then subjected to hyperpolarizing commands
ofsufficient duration ( > 0.5 sec) and magnitude (to membrane potentials > -60 mV)
to close all (or nearly all) of the M-channels. One of these commands is shown at a
fast recorder speed on the left of Fig. 1 A. Two clearly separable components of inward
current are seen during the hyperpolarizing command. First, there is an 'instan-
taneous' current step: this is the current initially generated by the 30 mV voltage
step flowing through the membrane 'leak' channels and also through those M-channels
which were open at the holding potential and which do not have time to close during
the rapid ( < 2 msec) voltage step. Subsequently, there is a further, and much slower,
inward relaxation: this reflects the reduction in the steady outward current flowing
through the M-channels as the latter slowly close in response to the change in
membrane voltage.
When the cell is repolarized to -30 mV the same sequence of instantaneous and
slow outward currents is observed. The instantaneous current step is clearly much
smaller than that at the beginning of the command pulse, since it only reflects current
flow through the leak channels and not through M-channels (the latter having been
closed). The slow outward relaxation is due to the slow re-opening of the M-channels
at -30 mV. It is larger than the initial inward relaxation because the driving force
for the M-current (reversal potential about -90 mV) is twice as great at -30 mV
than at -60 mV.
After recording several such current relaxations at a reduced recorder speed the
perfusion fluid was switched to one containing 10 /tM-( ± )muscarine. This compound
is a potent and selective muscarinic receptor agonist on ganglion cells (Ambache,
Perry & Robertson, 1956; Konzett & Waser, 1956; Brown, Fatherazi, Garthwaite
& White, 1980), and was used as the preferred muscarinic agonist for most of our
experiments. The effect of muscarine is to close those M-channels previously open at
the holding potential. Three manifestations of this effect can be discerned in Fig. 1 A.
Firstly, the steady outward current at the holding potential is reduced, giving a net
inward current. In contrast, the base line current level attained at -60 mV at the
end of the command pulse remains the same - i.e. muscarine does not produce an
inward current at voltages where the M-channels are normally shut. (This point is
amplified later.) Secondly, the inward current relaxation during the command pulse
is suppressed: the M-channels, having been already closed by muscarine, cannot be
8 PR'Y 332
226
B
A
j ~ ~ ~ ~ mV
P. R. ADAMS, D. A. BROWN AND A. CONSTANTI
TfflI[W
Muscarine
I111111111111
10 pM
XI30mv
Time (sec)
]2nA
-60
]12nA
Fig. 1. Effect of 10/M-(±) muscarine on a bullfrog sympathetic neurone recorded (A)
under voltage clamp and (B) when the clamp was released. In A the cell was clamped at
a holding potential of -30 mV and subjected to 0-58 see voltage jumps to -60 mV at
5 see intervals. The expanded records show clamp-currents (lower trace) generated by
these voltage jumps (middle trace) immediately before and just after starting the
muscarine superfusion; between these records the recording speed was slowed x 100, to
show the development ofthe muscarine-induced inward current. Just prior to and just after
the two clamp-pulses shown in A, the clamp was released and the voltage deflexions
produced by single 0-58 see current injections (-16 nA) were recorded ('current clamp'
records in B). The resting potential was about -42 mV before muscarine and -22 mV
in the presence of muscarine. Interrupted lines show initial holding current and membrane
potential levels. (Note: these commands were succeeded by an extensive series of voltage-
and current-pulses, to generate the current-voltage curves shown in Fig. 2. Hence,
muscarine perfusion was prolonged and no attempt at reversal was made: reversal after
short applications is shown in Figs. 6 and 10.) The brief downward deflexions ofthe current
records in A at the end of each voltage command are due to anode-break spikes generated
in the axon.
further closed by hyperpolarization. (The very small and slow residual outward
relaxation in the presence of muscarine following repolarization, which subsides
within a second or so, may reflect a just suprathreshold, slowly inactivating delayed
rectifier current which is not sensitive to muscarine: see below. The small outward-
current 'blip' immediately after repolarizing is the transient A-current, which is also
insensitive to muscarine: see Fig. 11 below.) Thirdly, the instantaneous inward
current step at the onset of the hyperpolarizing pulse is now very much smaller, and
indeed is no larger than that accompanying the repolarizing step. This is because both
instantaneous current steps now reflect current flow only through membrane leak
channels, and the leak conductance does not change with membrane potential over
this voltage range. The reduction of the initial instantaneous current step shows that
muscarine has reduced the membrane chord conductance at the holding potential (see
Fig. 3 below).
Fig. 1 B shows the behaviour of the same neurone before and after muscarine-
addition when the voltage clamp was removed and the voltage commands were
replaced by constant negative current commands ('current clamp'). As expected, the
inward current induced by muscarine under voltage clamp generates a sustained
membrane depolarization, from -42 to -22 mV. Further, the steady-state voltage
deflexions produced by the current injection are increased in amplitude, i.e. the
apparent 'input conductance' is reduced. This accords with earlier descriptions of
the effects of muscarinic agonists on ganglion cells under current clamp (see for
example, Kobayashi & Libet, 1970; Kuba & Koketsu, 1976a; Brown & Constanti,
1980). The conductance drop does not appear as large as that observed under voltage
clamp; this is because the input conductances before and after muscarine are now
measured at different membrane potentials (see Discussion).
There is also a very striking change in the shape of the voltage response on adding
muscarine. Under normal conditions the hyperpolarization produced by the negative
current injection itself induces M-channel closure resulting in a slow inward current
like that seen under voltage clamp in Fig. 1 A. This inward current in turn generates
a secondary depolarizing 'sag' in the voltage trajectory in Fig. 1 B. In the presence
of muscarine this closure-current is lost and the voltage response now becomes flat.
The significance of these voltage trajectories is considered further in Discussion.
Current-voltage relations. The traces in Fig. 1 formed part of an experiment in which
several series of voltage and current pulses of differing amplitude were applied before
and during muscarine application. A further sample of some of the responses to these
commands, recorded under voltage clamp and current clamp respectively, are
illustrated in Fig. 2A and B. In voltage-clamp mode, as the voltage command carries
the membrane potential through the reversal potential for IM (VM, = -90 mV), so
the current relaxation generated by M-channel closure changes from a slow inward
current (panel a) to a flat trajectory at VM (panel b) and then inverts to a fast outward
relaxation (panel c). The acceleration of the current relaxation at hyperpolarized
commands reflects the voltage sensitivity of the time constant for M-channel closure
described previously (Adams et al. 1982a). (Following this inverted IM relaxation
there is quite a separate small, slow inward current which is manifest at very
hyperpolarized potentials.) Correspondingly, the secondary depolarizing sag to the
voltage trajectory recorded under current clamp which is induced by M-channel
closure is eliminated when the current injection is sufficiently great to drive the
membrane potential to VM (Fig. 2B, panel b), though the above-mentioned extra
inward current at hyperpolarized potentials induces an additional sag with even
larger current pulses (Fig. 2B, panel c).
In the presence of muscarine both inward and outward IM relaxations recorded
under voltage clamp are eliminated, and - apart from residual slow inward currents
and A-currents - the current responses become square and increase ohmically with
increasing voltage jumps: Likewise, the voltage response to current pulses take on
a 'squarer' appearance.
By plotting the final current level at the end of the command pulse against the
clamp command potential, the 'steady-state' current-voltage (I/V) curves in Fig.
2 C were obtained. The I/ V curve obtained under current clamp superimposed quite
well on that obtained under voltage clamp when appropriately offset by the amount
8-2
A B
Control Muscarine Control Muscarine

VH -30 Em -43
-30
-61
-60 3.4L --~

b
-9m -100
-4.4
m9015 R E V
nA 5nA
c
%.-I
I---
---7
5 nA 150 mV
-124 0-5sec
nA
+8
mV
V
V -6
Fig. 2. Current-voltage relations of the cell illustrated in Fig. 1 recorded before (Control)
and during (Muscarine) perfusion with 10 SM-( ±) muscarine. Records in A show sample
current responses to hyperpolarizing voltage jumps of increasing amplitude (30, 60 and
90 mV) applied from a holding potential (VH) of -30 mV, recorded under voltage clamp.
(An anode-break spike artifact is present in record a during the outward current
of holding current at the holding potential of -30 mV. The most noticeable feature
of the I/ V curves before addition of muscarine is the strong outward rectification
between -70 and -20 mV. This is due to the outward current generated by the
progressive opening of M-channels over this voltage range since the experimental
points accord well with the amplitude of IM predicted from our previously determined
kinetic scheme for IM (continuous curve: cf. Adams et al. 1982 a). In the presence of
muscarine, this rectification is abolished, so that the I/ V curve remains linear up to
that point (between -25 and -20 mV) where the muscarine-insensitive delayed
rectifier current becomes prominent.
This profound effect of muscarine on I/ V rectification accords with previous
observations in unclamped neurones (Kuba & Koketsu, 1976 a: 'type 2' neurones;
Brown & Constanti, 1980) but is much more dramatic under voltage clamp, primarily
because of the greater ease with which the cell can be driven to more positive
potentials. Equally significantly, and in complete contrast, neither the position nor
the slope of the linear part of the I/ V curve beyond -70 mV were changed, i.e. there
was no inward current or change in slope conductance outside the M-current range.
Although illustrated for convenience with reference to one neurone, the effects
shown in Figs. 1 and 2 were replicated in full in a very large number of neurones.
Membrane conductance. The instantaneous current-steps accompanying the voltage-
steps in Figs. 1 and 2 are linearly related to the amplitude of the voltage jump and
provide a measure of the membrane chord conductance 0ch (see Adams et al. 1982 a).
As shown in Fig. 3 (control) 0ch normally increased steeply as the membrane was
depolarized between -60 and -30 mV: this reflects the progressive opening of
M-channels over this voltage range. Muscarine reduced this voltage-dependent
increase in 0ch, without normally affecting the voltage-independent 'leak' conduc-
tances at membrane potentials more negative than -60 mV (see Fig. 3) (Fig. 10 below
shaws an exception to this latter statement). In consequence the apparent reduction
in total chord conductance varied with membrane potential, in confirmation of
relaxation.) Records in B show sample voltage deflexions produced by injecting increasing
negative currents (1-5, 3-4 and 4-4 nA) recorded under 'current clamp', starting from a
resting potential (Em) of -43 mV. The graphs in C show the clamp-currents (filled
symbols: *, control; V, + muscarine) and voltage deflexions (open symbols: A, control;
V, + muscarine) measured at the end of the command pulses, plotted against the
amplitude of the command pulses. Axes are drawn through the initial resting potential
(= zero current) - i.e. a positive current of +4-6 nA is registered at the clamp holding
potential (VH) of -30 mV before adding muscarine and a negative current of -0-8 nA
after adding muscarine. The continuous curve is drawn according to the equivalent circuit
for the resting membrane currents described by Adams et al. (1982a), comprising a
voltage-independent membrane leak current and a voltage-dependent M-current, and
using the following constants: leak conductance 0L = 48 nS; leak reversal potential
VL = 10 mV: M-current reversal potential VM = -90 mV. The M-conductance GM is given
by GM =M { epT(V°V)}
where e = + 25, V0 =-35 mV and V is the membrane potential. The maximum M-

conductance GM was set at 150 nS at rest and 0 nS in muscarine solution. Note that this
formulation described the current-voltage curves reasonably well over the range -100
to -25 mV. (This cell was unusually large and values for GL and OM accordingly exceed
the average values given in Adams et al. 1982a.)
previous inferences from slope conductance measurements (see Kuba & Koketsu,
1976 a; Brown & Constanti, 1980). However, in experiments where rather less intense
effects of muscarine were recorded, the fraction by which the voltage-sensitive
component of GCh was reduced was constant over the range -60 to -30 mV. This
implies that the fraction of M-channels closed by muscarine was not itself strongly
voltage-dependent, and that the voltage dependence of muscarine's effect on total
Gch stemmed simply from the different proportion of M-channels open at different
membrane potentials.
nS
80
_~~ VH
V
V
/ign(VH)
60 -
/in(V) Control
Gch/
40-
Muscarine
20 I 1
-70 -60 -50 -40 -30 mV
V
Fig. 3. Effect of muscarine on membrane chord conductance. Average chord conductance
(G0h) at the holding potential (VH = -30 mV) was calculated from the amplitude of the
instantaneous current-steps Iin (VH) at the onset of a series of hyperpolarizing commands
to different command potentials (V) as shown in the diagram. (See Fig. 1 for an example
of a current record). Thus Gch = Iin(VH)/(V- VH). Qch at each command potential was
calculated from the amplitudes of the instantaneous repolarizing current steps Iin (V):
0ch = In (V)/( VH - V). Measurements were made before (A), during (V) and after (A)
application of 10 /SM-muscarine.
IM kinetics. Muscarine might inhibit IM by shifting its activation curve to a nuch

more depolarized level. This is difficult to assess from steady-state current-voltage
curves of the type shown in Figs. 1 and 2, since, at membrane potentials appreciably
more positive than -30 mV, IM becomes obscured by the much larger delayed
rectifier and Ca2+-activated K+-currents. The time course of the M-current relaxation
provides a more sensitive index of a shift in the activation range, since the relaxation
time-constant shortens appreciably as the cell is hyperpolarized beyond the normal
half-activation voltage of -35 mV (see Adams et al. 1982 a). Thus a positive shift
in the activation curve will be manifest as a faster off-relaxation and a larger
difference between the off- and on-relaxations on applying a hyperpolarizing command
from a membrane potential near to the half-activation voltage. This was tested using
modest concentrations of muscarine so that IM was only partly inhibited and the
residual IM relaxations were sufficiently large to measure. Fig. 4. shows one such test.
It is clear, that, although the amplitudes of the IM relaxations were reduced by some
A B
Control
nA
2
0*5
Muscarine aim 02
1-25
0.1
0 00 2 0O4 sec
Wash __ nA
2 -
I1 nA 1 X
A
02
0-5
I'
-30
mV
0'S sec
0.i-1- As ,\
0 0O2 sec
Fig. 4. Effect of muscarine on the time course of the voltage-dependent M-current
relaxations. The records in A show inward and outward IM relaxations on jumping to
-50 mV (Im(50)) and repolarizing to -30 mV (IM(30)) respectively before, during and
after a brief application of 1-25,uM-muscarine. The relaxation amplitudes are plotted
semi-logarithmically against time in B. Control before muscarine: -V-, -A-; during
muscarine perfusion: -V-, -A-; washout: -- V --,-- A --. The initial inward
current 'spike' immediately after the repolarizing voltage jump is an anode-break axonal
spike.
50-60 %, the time courses of both the off- and on-relaxations were unchanged. This
was confirmed in several other tests of this type, and indicates that muscarine has
not affected the kinetics of IMP
Effect of muscarine in raised K+. In normal Ringer solution containing 2'5 mM-K+
the ionic current through the M-channels is outward at all membrane potentials at
which the M-channels are open, since the reversal potential is about -90 mV.
However, when external [K+] is raised to 25 mm, open channels can carry an inward
current flow between about -45 and -60 mV. Hence a voltage jump from -30 mV
or thereabouts to a potential more negative than -45 mV induces an outward current
relaxation during the ensuing M-channel closure. Muscarine is equally effective in
blocking this outward relaxation as in blocking the inward relaxation seen previously
(Fig. 5: cf. Fig. 1). This implies that the action of muscarine is not dependent on the
direction of current-flow through the M-channels.
Concentration dependence of muscarine action. In a number ofexperiments the effects
of several different concentrations of muscarine were explored using the clamp
protocol illustrated in Fig. 1. Fig. 6A shows a representative selection of such
responses. The depression of IM was calculated from the change in amplitude of the
outward relaxation following cessation of the voltage step. (This off-relaxation is
25 mM-K' VH = -25 mV 10pM-muscarine
1 X 1 1 1 1 1 1 1 1 t JG |~~~~~50mV
_
n = -Tan i ~~~~~-
- -- -- -- -- -- -- -- -
j5nA
100 msec 10 sec 100 msec
Fig. 5. Effect of muscarine (10 #UM) on clamp currents recorded in 25 mM-K+ solution (i.e.
in normal Ringer solution with 22-5 mM-extra KCl). Experimental protocol as in the upper
part of Fig. 1 except that the holding potential was -25 mV and the command potential
-75 mV.
larger than the closure relaxation during the command itself, because the repolarizing
potential is further away from the current reversal potential, and hence is easier to
measure). Fig. 6B shows the relationship between this measure of IM depression and
the concentration of applied muscarine. Half-maximal depression occurred at about
3/uM. In these experiments, a ceiling of about 800 depression appeared to have been
attained at > 10 /LM-muscarine. However, in many other experiments (as in Fig. 1)
virtually full suppression of IM was frequently attained at 10 /aM-muscarine, the
residual currents being purely 'ohmic'. Hence it seems unlikely tha t any substantial
fraction of IM can be regarded as insensitive to muscarinic agonists. As indicated by
the crosses in Fig. 6B, atropine effectively antagonized the action of muscarine:
atropine itself did not affect IM.
With prolonged ( ) 10 min) application of muscarine, some recovery of IM was
frequently noted, suggesting some slow 'desensitization'. Slow desensitization has
been reported in connexion with the muscarinic effects of acetycholine on action
potentials in bullfrog sympathetic neurones (Akasu & Koketsu, 1981).
Other muscarinic agonists. Effects of muscarine were replicated by methacholine
(0-1-1 mM). However, though in agreement with Ginsborg (1965), pilocarpine was
virtually inactive up to 200 gM. Indeed, pilocarpine appeared to exert some antagonism
towards muscarine, since addition of muscarine (10 uM) with pilocarpine (200 SM)
produced less inhibition than 10 IM-muscarine alone. In rat ganglia pilocarpine
appears to behave as a 'partial agonist' at muscarinic receptors (Brown et at. 1980):
in frog ganglia it may be an even weaker partial agonist. The action of muscarine
was also antagonized by high concentrations of tetraethylammonium (see below).
10 fm-muscarine
1 -
a J LJ_ E fJ /'M(30)
3
Am
0q3 M "-02 £
-7- y Ao03 1 3 10 30
100c I sac
L4
wi nA
~~~~~(Muscarin'e] (pm)
Fig. 6. Concentration dependence of IM inhibition by muscarine. The records in A show
examples of responses to: (a), 10/sM; (b), 3 j1M and (c), 03 ,uM-muscarine, using the
experimental protocol illustrated in Fig. 1 (holding potential -30 mV, command potential
-60 mV). Records b and c were obtained in the same cell, record a from another cell in
the same ganglion. In B, IM amplitude is plotted against muscarine concentration.
Ordinates show the amplitude of the repolarizing IM relaxation (IM(30), see Fig. 4)
recorded in the presence of muscarine as a fraction of that immediately before adding
muscarine. Each point is a single measurement, but all were obtained in the same ganglion.
Crosses show responses after switching to 1 /SM-atropine solution.
Relationship between Im-inhibition and net inward current. The records shown in Fig.
1, and the I/ V curve in Fig. 2, indicate that, in this experiment, muscarine did not
produce any inward shift in the steady current level at -60 mV, when the M-channels
were shut. This suggests that the inward current (and membrane depolarization)
produced by muscarine might stem entirely from the suppression of the resting
outward M-current. We explored this hypothesis in more detail, using several
different approaches.
The first approach was simply to relate the amount of inward current developed
at the holding potential with the amount by which the outward relaxation was
depressed on restoring the holding potential from a command potential to -60 mV.
This assumes that the M-channels are entirely shut at -60 mV, so that the
repolarizing relaxation amplitude measures the total M-current generated at the
holding potential. (This may not be strictly true, since some 5 % of M-channels might
remain open at -60 mV - see Fig. 9 in Adams et al. (1982 a) - but the error involved
is not serious, and larger jumps induced greater errors due to the advent of the
transient outward current IA on repolarizing the cell.) Fig. 7 shows the relationship
A B
nA 9 nA
-3 -0 /
-2 -, -30
A -25
-2 -4
0 gd-40
p ,0~~~~~~~~~~~35
50 -
/~~~~~~~~~~~~~~~~~O
I I +02 0~~~~~~-55
0 -1 -2 -3 nA 0 -1 -2 nA
A/M(30) A/M( VH)
Fig. 7. Relationship between IM inhibition and inward current produced by muscarine.
In A the net inward increase in holding current produced by muscarine at a constant
holding potential of -30 mV (AIss (30)) is plotted against the amount by which muscarine
reduced the outward IM relaxation on repolarizing to -30 mV from a command potential
of -60 mV (AIM (30); see Fig. 4). (Since the M-channels are essentially closed at -60 mV,
IM (30) measured from -60 mV approximates to the total amount of outward IM at the
holding potential.) The amount of M-current inhibition was varied by varying the
concentration of muscarine (see Fig. 6). Symbols for different muscarine concentrations
(/uM): 0, 0-3; A, 1; V, 3; E[, 10; O, 30. The graph in B shows results from two neurones
in which the holding potential VH was reset at different values (indicated against the points)
before and during perfusion with 10lM-muscarine. At each holding potential a single
command pulse to -60 or -65 mV was used to close the M-channels and the contribution
of IM to the net current at each holding potential was measured from the amplitude of
the repolarizing outward relaxation, AIM (VH). The change in holding current produced
by muscarine is plotted against AIM for each holding potential. Dashed lines show the
relation expected if the steady inward current produced by muscarine was due entirely
to inhibition of IM-
between the steady inward current (AIsS) and the fall in IM (AIM) derived from two
different protocols: by applying varying concentrations of muscarine at a fixed
(-30 mV) holding potential, so producing a variable reduction in IM (Fig. 7 A) and
by varying the holding potential during the application of a constant concentration
of muscarine (Fig. 7 B). In both cases there is a direct and, more importantly, unitary
proportionality between the inward current and the decline in M-current. The
deviation at the high concentrations of muscarine might reflect some small additive
effect of muscarine, or might be simply a consequence of the small underestimate of
the total contribution of IM to be the steady current at -30 mV resulting from the
incomplete channel closure during the voltage jump referred to above.
This clearly indicates that the inward current developed within the M-current
voltage range can be attributed entirely to M-channel closure. However, it does not
answer the question of whether muscarinic agonists might induce additional inward
currents, of other origin, outside this voltage range. This is a matter of some
significance because there is evidence that, in some cells at least, the muscarinic slow
e.p.s.p. in bullfrog neurones can be recorded at membrane potentials outside the
M-current range (see, for example, Kuba & Koketsu, 1976a), for which additional
sources of inward current to IM suppression are necessary.
To explore this, brief applications of a fixed dose of muscarine were made at
various holding potentials and the peak inward current developed was compared with
that at a holding potential of -30 mV. The application had to be brief in order to
obtain adequate recovery since tests at two potentials at least (more usually three of
four) were necessary in the same cell. Two forms of brief application were used:
ionophoresis with constant ejecting current, and bath perfusion for a short pre-
determined period (1-5 sec) using a stimulator to trigger the electronic flow valves
(see Methods).
Fig. 8 illustrates the results of an ionophoretic test. Voltage pulses were applied
to close (or open) M-channels from each of a series of holding potentials, so allowing
an internal check on the fractional IM suppression produced by the ionophoretic pulse
of muscarine. This was fairly constant at about 30 %, indicating that the dose of
muscarine applied did not vary greatly. Nevertheless, the net current generated at
each holding potential changed substantially, from about 3 nA at -30 mV to
< 0-2 nA at -60 and -80 mV.
Although successful in this experiment, ionophoretic application was rather
ineffective compared with bath perfusion, and the response was not appreciably
faster. Hence for most other tests brief (1-5 sec) rapid bath-perfusion was preferred.
Fig. 9A and B exemplifies one such experiment and Fig. 9C summarizes the results
of bath-perfusion experiments on nine neurones. Although the results are somewhat
scattered, the inward current diminished with membrane hyperpolarization approxi-
mately as predicted from the voltage dependence of IM (continuous line).
There were two striking exceptions to this trend, in which 10 /SM-muscarine produced a clear and
reversible inward current of appreciable magnitude at membrane potentials of -80 and -100 mV.
Both were in neurones of the same ganglion which had not been predigested with trypsin. Fig. 10
illustrates one of these neurones. This cell clearly showed conventional IM relaxations following
stepped hyperpolarizing commands from -30 mV (upper record), and these were greatly reduced
by muscarine just as in other cells. However, on setting the holding potential to -80 mV
application of muscarine still produced a substantial (though smaller) inward current. This could
not be attributed to 1M suppression since the current responses to small (20 mV) depolarizing
commands were essentially 'ohmic', i.e. the M-channels were already shut. Instead, there is a clear
increase in conductance, since these ohmicc' responses are larger in muscarine solution. At -100 mV
a comparable increase in ohmic conductance is seen, but the inward current is even larger. Further
inspection of the upper trace suggests that, unlike the situation depicted in Fig. 1, muscarine also
produced a small net inward current at -60 mV, as judged by the change in the current level at
the end of the 30 mV hyperpolarizing commands from -30 mV: this amounted to some 0 65 nA,
i.e. about one-fifth of that developed at the holding potential of -30 mV and about half of that
at -80 mV.
This extra inward current does not appear to be related solely to the use (or avoidance) of trypsin,
since two other cells in the same ganglion (and a third in another non-trypsinized ganglion) showed
no evidence of any inward current beyond -60 mV. It may be relevant that such currents were
Muscarine
VH V a
-30
-OV -
-80
r-
-
r
i-FLIZI
7
L-
-
*LL iL
IJ'Jw! ~j, r jjjH A
-60 -=° LU rFL §L LJLJLF
_50 -~ =°0 _ , -
6'
.,
-30-~~~~-
604
-P
-3 0
-_
5 nA [ ' 7r r %r <
a"
10 sec 1 sec
Fig. 8. Responses of a ganglion cell to ionophoretically applied muscarine at different

holding potentials. At each holding potential (VH) voltage jumps were applied of sufficient
amplitude (to command potential V) to open or close M-channels: the effectiveness of
muscarine in suppressing IM could then be judged from the depression of the slow IM
relaxations (as shown in the faster speed records on the right); this was approximately
the same (- 300% inhibition) with each ejection, irrespective of holding potential.
Nevertheless, the change in holding current increased as the holding potential became less
negative. (Muscarine was ionophoresed from a micropipette filled with 100 mM-muscarine
solution using a constant positive ejecting current applied for 2 see).
A B
10pM-muscarine, 1 sec
VH
-30
-40
-60
-30
-
-20 >mm
-83 m
-30 m y _ ] 1 nA .-.! I]1 nA
30 sec .1 sec
2 C
:r 0
.. CV)I
0
-, 1
I(n cn
'I .1 0 0
* 0
. 0
0
-20 -40 -60 -80 -100

VH (mV)
Fig. 9. Voltage dependence of inward currents evoked by brief, rapid bath-perfusion of
muscarine. The records in A show sample currents induced by repeated application of
10 /SM-muscarine for 1 sec at different holding potentials (VH) in a single cell. The records
in B show currents elicited by 0-8 sec voltage jumps from a holding potential of -30 mV:
IM closure relaxations reversed in direction at between -60 and -83 mV command
potential. The graph in C shows pooled results from nine cells, four in ganglia which had
not been pre-digested with trypsin (0). Peak inward currents at each holding potential
(AISS (VH) were normalized with respect to that at a holding potential of -30 mV
(AISS (30)) in the same cell. The curve is drawn on the assumption that the inward current
results entirely from suppression of steady outward IM' i.e., that AISS = AG (V- VM)
where VM is the reversal potential for IM (-90 mV average) and AG varies with V as
described in Fig. 2.
only observed using fairly high (10juM) concentrations of muscarine. They may relate to the
subpopulation of neurones referred to as type 1 by Kuba & Koketsu (1976a) iii which the steady-state
current-voltage curves in the absence and presence of high concentrations (0-27 mM) of acetycholine
diverged at hyperpolarized levels. As indicated above, this represents a small minority of cells
studies in our experiments. Further, even in these cells, the net inward current developed at normal
resting potentials (< -60 mV) can be ascribed overwhelmingly to IM inhibition. Thus, in Fig. 10,
at least 85 % of the inward current at -30 mV appears to result from IM suppression.
-30 10 JuM-muscarine, 2 sec
Ad ~ ~~~~~i i HtMT- I II IM
f .LIII
-,;
-80c- -L - I- I I I
-100
A r--~ nA
----il J iLLPIIII Li JJJ f l20 mV
1 min 0 5 sec
Fig. 10. Inward current induced by 10 ,uM-muscarine at hyperpolarized potentials,
recorded with a single micro-electrode voltage clamp from a cell in a non-trypsinized
ganglion. See text for further details. Note: at a holding potential of -80 mV a small
(± 20 mV) depolarizing command yielded only 'ohmic' responses; a larger (+ 35 mV)
command evoked an initial transient outward current (IA: see Adams et al. 1982 a)
followed by an outward M-current.
Finally, it should be noted that in no cell was any reversal of the muscarine induced current (Imus)
to an outward current observed in normal Ringer solution (cf. Kuba & Koketsu, 1976a: 'type 3'
cells). This is also to be expected if ImU8 is due to IM suppression, since the M-channels are shut
in the steady state some 20 mV positive to the reversal potential for IM in normal Ringer solution.
Reversal of Imup could only be discerned when external [K+] was raised to 25 mm, so shifting EK
to a point (about -40 mV) within the range of IM activation.
Effect of muacarine on other K+-currents

Three other voltage-sensitive K+-currents can be recorded in voltage-clamped frog
sympathetic neurones: a delayed rectifier current, IK; a Ca-activated current, IC; and
a transient current, IA (Brown et al. 1981; Adams et al. 1982 a). These were generally
insensitive to muscarine.
IA in frog neurones is analogous to the corresponding current originally described
in molluscan neurones by Neher (1971) and Connor & Stevens (1971). It is totally
inactivated at resting potentials positive to -60 mV, where IM is activated, and
hence cannot contribute to the effects of muscarine on steady membrane currents
within the range of IM activation. Inactivation of IA is removed by holding the
membrane potential at a value more negative than -60 mV, or by applying a
hyperpolarizing pre-pulse: IA then appears as a fast, transient current on depolarizing
to a potential more positive than -60 mV. The middle trace in Fig. 10 shows an
10 MM-muscarine
-30 mV
-60
-120
/M~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-
W1W> _] 1 nA
'A j5 nA
0-5sec
Fig. 11. Effects of muscarine on IM (upper current record) and IA (lower current record),
recorded concurrently in the same neurone. The neurone was subjected to regular 0-65 sec
hyperpolarizing commands to -60 mV from a holding potential of -30 mV, to deactivate
and reactivate IM in the manner shown in Fig. 1, with intermittent commands to
- 120 mV. The latter removed the steady inactivation of IA present at the holding
potential, so that IA was activated on subsequently repolarizing to -30 mV and appears
as a large transient outward current in the lower record (arrowed). The two currents were
recorded simultaneously at different gains (note) on two separate recorder channels.
example of IA activated by depolarizing a neurone from -80 to -45 mV. Fig. 11

illustrates another example in which IA is activated on repolarizing to -30 mV after
a large hyperpolarizing pre-pulse to - 120 mV, whereas, on repolarizing from
-60 mV only a slow outward IM is seen. In both examples IA was clearly insensitive
to muscarine whereas IM in the same neurone was depressed. This insensitivity of
IA to muscarine was confirmed in all but one of a large number of cells; indeed, to
study the activation of IA in isolation from IM, we usually inhibited IM with
muscarine (see Adams et al. 1982a).
IK and Ic are activated in parallel and are responsible for the large outward
currents observed on depolarizing to membrane potentials more positive than
-25 mV from holding potentials of -30 mV or more. As shown in Fig. 12, the large
outward currents activated near to (or above) 0 mV were not materially affected by
muscarine, the effect of muscarine on the current-voltage cui Je being confined to that
region (between -60 and -30 mV) where the rectification derived solely from IM*
a b
JL I ]0mV
Control --
A
]2onA 3 2 nA
+Muscarine
0 5 sec
nA
40
VH -30
30
B
20
10 -k
-60 -40 -20 0 mV

Fig. 12. A, effects of muscarine (10 /LM) on (a) large outward currents produced by 100 msec
depolarizing commands to O mV and (b) IM, produced by 1 see command to -30 mV, both
from a holding potential of -50 mV, and recorded alternatively. B, 'steady-state'
current-voltage curves obtained (sequentially) in normal Ringer solution (@), 10 /M-
muscarine (A), after washing out muscarine (A) and then in 5 mM-TEA (V). Curves were
constructed as shown in Fig. 2, using 0 5 see pulses from a holding potential of -30 mV,
but after subtraction of the 'leak' current extrapolated from the linear part of the curve
between -70 and -100 mV. There was a gradual decline in the outward currents elicited
with depolarizing commands during this fairly prolonged experimental sequence. This was
commonly observed even in the absence of added drug (another instance is shown in Fig.
13) particularly where there was large Ca2+-dependent component to the currents: cf.
Constanti et al. (1981 b). i
(Indeed, we frequently noted a small increase in the outward current at -20 to

-10 mV in the presence of muscarine: the origin of this is unclear). In contrast,
tetraethylammonium (TEA, 5-10 mm) strongly reduced the large outward currents
and profoundly depressed the current-voltage curve positive to -30 mV.
Muscarine also discriminated between IM and the other outward currents in that
range (-25 to -20 mV) where the currents overlapped and were of comparable
magnitude. Thus, as shown in Fig. 13A, a jump from -50 to -30 mV evoked only
an M-current, and the repolarizing tail-current (Fig. 13B) assumed a single
exponential; this current was reduced some 40-60 % by 10 /LM-muscarine in this cell.
± 10 pM-muscarine ±10 pM-TEA

A
-20
I;
r 1~ ~n5A
..........<
I
-25
-30
-20
mV 1I
-50-
0-5 sec
B ±Muscarine ±TEA
nA
5-
4
3
10
05
0 100 200 0 100 200 msec
Fig. 13. A, effects of muscarine (10 uM) and tetraethylammonium (TEA, 1O mM) on
outward currents elicited by depolarizing commands to -30, -25 and -20 mV from a
holding potential of -50 mV. Pen-tracings of superimposed control ( ) and test (- )
currents. At -30 mV the slow outward current comprises IM: this is reduced 60 by
-
muscarine and somewhat less by TEA. At -20 mV faster and larger currents are
superimposed: these are not reduced by muscarine but severely reduced by TEA. B,
semi-logarithmic plots of repolarizing tail-current time course at -50 mV following
depolarization commands of the type shown in A, in the absence (@) and presence (0)
of 10 ,M-muscarine or 10 mM-TEA. Note the fast initial component to the tail after
commands to -20 mV and its selective suppression by TEA.
Larger depolarizations, to -25 and -20 mV, induced a superimposed faster current,
and the repolarizing tail-current now showed a rapid initial component (see also
Adams et al. 1982 a: Fig. 8). This faster component was resistant to muscarine: hence,
because the slower M-current was reduced, the fast component to the tail-current is
exaggerated. In contrast, a high concentration of TEA (10 mM) preferentially reduced
the larger outward currents at -25 and -20 mV, and eliminated the fast component
of the repolarizing tail-current.
It should be noted that TEA is a relatively unselective K+ current blocker in these neurones since,
at 5-10 mm, it not only reduces both IK and Ic (see Constanti, Adams & Brown, 1981 b; Adams
et al. 1982b) but also weakly inhibits IM as shown in Fig. 13. We also noted that 5 mM-TEA
antagonized the effect of muscarine on IM, presumably by blocking the muscarinic receptors. A
combination of weak IM inhibition and muscarine antagonism accord with previous observations
on rat sympathetic neurones (Brown & Constanti, 1980). Partial IM inhibition might also contribute
to the potentiation of the slow e.p.s.p. by TEA reported by Kuba & Koketsu (1977).
As pointed out above, the large outward currents positive to -25 mV contain two
components, IK and Ic, whose relative magnitude varies from cell to cell (Constanti
et al. 1981 b). IK can be recorded in the absence of Ic by suppressing the inward
Ca2+-current necessary to generate the latter with a Ca2+-channel blocker such as
Cd2+. Conversely, Ic can be generated separately from IK by injecting Ca2+ ions into
the neurone at a membrane potential below the threshold for IK, or at more
depolarized levels if time is first allowed for IK to inactivate (Adams et al. 1982b).
Separate tests on these two components indicated that neither was sensitive to
inhibition by muscarine, whereas Ic was extremely sensitive to external TEA, being
half-blocked at 1 mm.
Luteinizing-hormone releasing hormone (LHRH)

This compound, or a structurally-related peptide (Eiden & Eskay, 1980) is
probably the mediator of the non-cholinergic 'late slow e.p.s.p.' in bullfrog lumbar
sympathetic ganglia (Jan et al. 1979, 1980). The nature of the late slow e.p.s.p., a
slow depolarization accompanied by a rise in input resistance, resembles that of the
muscarinic slow e.p.s.p. and the response to muscarinic agonists, so we wondered
whether LHRH could also inhibit IM. We have previously reported that LHRH itself
could indeed inhibit IM (Adams & Brown, 1980), but this natural peptide is rapidly
degraded: hence relatively high concentrations () 5,M) were necessary and the
effects were rather variable in magnitude (as also noted by Jan et al. 1979). We have
now extended our study to include two more stable analogues: D-Trp6-Pro9-N-
ethyl-LHRH and D-Ala6-LHRH.
Fig. 14 shows the effect of a large dose (5 /M) of D-Ala6-LHRH on clamp currents
at a holding potential of -30 mV, using a similar pulse protocol to that in Figs. 1
and 2. The following information may be extracted from this experiment.
(i) The slow time-dependent current relaxations during the voltage-steps associated
with the closure' and opening of the M-channels were almost abolished.
(ii) At the holding potential of -30 mV, D-Ala6-LHRH produced a steady inward
current of about 2-3 nA. In contrast there was no change in the steady current level
attained during a 0-8 sec command to membrane potentials negative to -50 mV.
Thus inward current development was restricted to membrane potentials more
positive than -60 mV.
-30
B
-60
mI r Il i li l ~ 310mV
PHARMACOLOGY OF M-CURRENT
(iii) The chord conductance at -30 mV, measured from the amplitude of the
T f b
5pM-D-Ala6-LHRH
50 sec
243
instantaneous current steps at the beginning of the hyperpolarizing commands, was

reduced from 103 to 60 nS in the presence of D-Ala6-LHRH. The chord conductance
at -60 mV (measured from the amplitude of the repolarizing instantaneous current
step remaining after extrapolating the slow repolarizing IM tail) remained constant
i ~~~~~~~~~~~31
nA
0 5 sec
mV -90 -60 -30

I
~~~~0
-2
-4
-6
nA
Fig. 14. Effect of D-Ala6-LHRH (5 /M) on clamp currents, using the same experimental
protocol as that shown in Figs. 1 and 2. A, sample records at a holding potential of
-30 mV, with voltage jumps for 0 8 sec to -60 mV. B, 'steady-state' current-voltage
curves in the same cell (measured from currents attained at the end of each of a series
of voltage commands to potentials of -40 to -100 mV) before (U) and during (El)
perfusion with D-Ala6-LHRH (cf. Fig. 2). The reversal potential for IM was -90 mV in
the control run.
at 53 nS in the presence and absence of the agonist. Assuming the latter to represent
the leak conductance remaining when the M-channels have been closed, the contri-
bution of GM to the chord conductance at -30 mV has been reduced from
(103-53) = 50 nS to (60-53) = 7 nS by the agonist, i.e. 51xM-D-Ala6-LHRH has
closed about 86 % of the M-channels open at -30 mV.
(iv) In the control responses, the current trace became flat at about -90 mV. This
gives the reversal potential (VM) for IMP The expected net inward current induced
by D-Ala6-LHRH at -30 mV can then be calculated from the product of the fall in
chord conductance (AG) and the driving force (VH-VM): I = AG(VH-VM) =
_~ ~ ~
(-43 x 10-9 x 60 x 10-3) =--26 nA. This agrees well with the observed inward
current of about -24 nA.
In further experiments D-Ala6-LHRH was applied by brief bath-perfusion to
neurones in non-trypsinized ganglia at different holding potentials, in the manner
described above for muscarine. The inward current diminished with hyperpolarization
5%M-D-Asg'-LHRH, 5
VH-60
~J---uumufluurL-Lj O 1J 120 mv
A
I02 nA
VH-32
L J IfiUtLlD1J - UIEU I 120 my
B
lOse I sec
Fig. 15. Effects of brief (5 see) perfusion with 5 ,uM-D-Ala6-LHRH recorded in a neurone
in a non-trypsinized ganglion using a single micro-electrode voltage clamp at holding
potentials of A, -60 and B, -32 mV. Note the increase in current gain in A.
as expected were it to be generated exclusively by IM suppression. An example is

shown in Fig. 15. At -60 mV the M-channels are closed, so that a hyperpolarizing
command produced only an ohmic 'leak' current response. At this potential
D-Ala6-LHRH produced no change in the steady current level (as predicted from the
result shown in Fig. 14). A subsequent application of the same dose at -32 mV
produced a clear inward current of - 12 nA, which could be accounted for entirely
by the amount by which the outward IM relaxation on repolarizing from -60 to
-30 mV was reduced. Essentially the same result was obtained in three other
neurones.
Jan et al. (1980) and Sejnowski (1982) observed comparable effects of LHRH in a minority of
bullfrog lumbar sympathetic B neurones; however, in others they described either an increased
depolarization (or inward current) at hyperpolarized levels, with an increased conductance, or, in
some cases, reversal to an outward current at around -60 mV. We did not test LHRH or its
analogues at membrane potentials beyond -60 mV, so may have missed these phenomena.
However, it is clear from the results shown in Figs. 14 and 15 (and from our previous results: Adams
& Brown, 1980; Brown et al. 1981) that the current developed at membrane potentials positive
to -60 mV is fully accounted by IM inhibition. Since the resting potential of our cells was normally
Vh
1
100 pM-UTP
fimy-
-1]-~ 60
less than -60 mV (in agreement with Jan et al. 1980), any additional voltage-sensitive current
developed beyond -60 mV is probably irrelevant to the normal effect of LHRH; and so it seems
/(nA)
+4
<
sec
mV
]1 nA
245
reasonable to conclude that the depolarization produced at rest potentials results exclusively from
M-channel closure. This would certainly explain the occlusion between the acetylcholine-mediated
slow e.p.s.p. and the LHRH-mediated late-slow e.p.s.p. described by Jan et al. (1980), if both
resulted from closure of the same species of ionic channels.
V(mV) -100 -80 -60 -40 -20
-5
Fig. 16. IM inhibition by 100 /sM-uridine triphosphate (UTP) demonstrated using the same
protocol as that illustrated in Figs. 1, 2 and 14. Circles (@, control; 0, 100 /SM-UTP) show
steady-state currents ISS; crosses (x) show instantaneous currents Ijn (see diagram).
LHRH and its stable analogues did not affect the large outward currents generated
by depolarizing the neurone to < -20 mV, nor the transient A-current generated by
depolarizing from very negative potentials.
Uridine triphosphate (UTP)
Siggins et al. (1977) and Gruol et al. (1981) made the interesting observation that
certain nucleotides (particularly 5'uridine di- and triphosphates) depolarized ex-
planted frog sympathetic ganglion cells in a manner closely resembling muscarinic
agonists: that is, the depolarization was accompanied by repetitive spike discharges
and decrease in input conductance (see also Fig. 2 of Siggins, 1978). This suggested
that UTP might also suppress IM. As shown in Fig. 16 this was indeed the case: UTP
produced the same voltage-dependent inward current as muscarine or LHRH, and
selectively reduced the amplitude of the IM relaxations during and after hyper-
~ ~ ~ ~ ]-10
polarizing commands. IM reduction at 50-100 /LM-UTP was between 30 and 60 %.
Again the currents evoked by depolarizing commands to -20 mV and the tran-
sient A-currents were unaffected (Fig. 17).
Uridine diphosphate appeared somewhat less effective than UTP. The metabolic
precursors uridine 5'-monophosphate, oritidine-5'-monophosphate and orotic acid
were ineffective up to 1 mM.
50 MM-UTP
________ ~~~~~~~~~~~~~~~~~~~~
mV
1111111111 L _ ]~~~~-30
, ,_J 31 nA
____________________ sec
mV
7. Ji<
, ]1 nA
Fig. 17. Effect of a brief application of 50 FM-UTP on (A) holding current at -30 mV
holding potential and fM-relaxation produced by 30 mV hyperpolarizing commands from
-30 mV (see Fig. 1), and (B) the transient outward current IA elicited by repolarizing
to -30 mV after 0-6 sec hyperpolarization to -110 mV (cf. Fig. 11). Note that UTP
reduced the amplitude of the IM relaxation in A (and the inverted outward relaxation
in B), but not IA
Divalent cation8
IM was relatively insensitive to external Ca2+ and Mg2+. Our Ringer solution
usually contained 2 mM-Ca2+ and 10 mMMg2+, but neither cation was necessary for
IM. Thus, as shown in Fig. 18 neither the holding current nor the amplitude or speed
of the IM relaxations were materially altered on separately removing Ca2+ or Mg2+.
In a further series of experiments the effect of several other divalent cations was
assessed using an experimental protocol along the lines of that illustrated in Fig. 19.
The membrane potential was held at -30 mV and subjected to a sequence of
alternate short depolarizing and long hyperpolarizing commands. The latter carried
the membrane potential to -50 or -60 mV, so closing M-channels, and the effect
of the divalent cation on the repolarizing tail-current gave a measure of IM inhibition,
in the manner described above using muscarine, LHRF or UTP. The depolarizing
A
-35
60IOm
ASLJ1li l ~ ~ 130mV
command, to between -10 and + 10 mV, generated a large outward current to which
the Ca2+-activated current Ic contributed a substantial (though variable) proportion
(see Adams et al. 1982b; Constanti et al. 1981 b): this current was greatly depressed
by omitting Ca2+ or adding a potent Ca2+-channel blocker such as Cd2+. Thus, the
depression of this outward current provided an approximate index of the ability of
6 mM-Ca2", 10 mM-Mg2+L
OCa2+, 10 mM-Mg2+1
50 sec
6 mM-Ca2+, 0 Mg2+
162
6 mM-Ca2+, 10 mM-Mg2+
0*5 sec
sec
12 nA
sec
247
-30
30 mV
8 -60
~~1 40
12 |nA
58 55 ___
50 sec 0-5 sec

Fig. 18. Effects of (A) omitting Mg2+ (in 6 mM-Ca2+ solution) and (B) raising [Ca2+] from
0 to 6 mm (in 10 mMMg2+ solution) on IM. Records show clamp currents recorded as in
Fig. 1. Numbers on current records show time-constants of the current relaxations in
milliseconds, measured from semi-logarithmic plots like those in Fig. 4. The downward
'spike' in the current record after the voltage jump in B is an axonal anode-break spike.
the cation to depress a known Ca2+-activated current, against which effects on IM

could be judged. The solution used for these experiments contained 2 mM-Ca2+ (to
activate Ic) and 10 MM-Mg2+ (to minimize changes in total divalent cation
concentration).
As shown in Fig. 19, Co2+ (4 mM) and Ni2+ (4 mM) greatly reduced Ic but produced
only a small diminution of IM- Fig. 20 summarizes the results of a number of such
tests. There is clearly no correlation between the relative effectiveness of the different
cations in depressing the two currents. The extreme situations are represented by Cd2+
and Ba2+ (see Fig. 2 1). The former was by far the most potent inhibitor of the outward
currents, but produced no significant depression of IM. In contrast, brief applications
of Ba2+ strongly inhibited IM but had negligible effect on IC.
_5 CoorNi
-30- 5
US
A ------- _ W
II
I
1 or 100 sec
I!J! 111? 125 or
Fig. 19. Effects of A, Co2+ (4 mM) and B, Ni2+ (4 mM) on clamp currents recorded following
alternate voltage commands of 60 msec to -5 mV and 600 msec to -50 mV from a
holding potential of -30 mV. Records show currents only, recorded at low and high gain
(upper and lower traces respectively), initially at a fast recorder speed and then at 100
times slower speed just prior to and during cation addition (at the arrow). Each cation
was added for about 1 min. Records A and B are from the same cell but separated by
the several minutes because of slow recovery from Co2+ (partly illustrated at the end of
trace A). Note that both cations depressed the larger outward currents (upper records)
considerably more than the M-currents (lower records: cf. Fig. 1).
mM /M /C+ K
Ba 1 12
Ba4 24 12
Ni4 11 4
Co4 10i 2
Mn4 5 1
Cd 01 3H14 10+
0 20 40 80 60 0 20 40 60 80
Percent reduction
Fig. 20. Comparative effects of some divalent cations on IM and on the large outward
currents (Ic + IK) evoked at command potentials between -10 and + 10 mV (see Fig. 19).
Depression of IM was measured from the amplitude of the repolarizing outward tails at
the holding potential (-30 mV) following commands to -50 or -60 mV (cf. Fig. 5). Each
block shows the mean+ S.E. of the mean of the number of tests indicated. (IM was
measured in more cells than IC+IK-)
The action of Ba2+ is particularly interesting. Its excitatory effect on sympathetic
neurones has been noted by several authors (e.g. Ambache, 1949; Takeshige & Volle,
1964; Tashiro & Nishi, 1972; McLachlan, 1977; McAfee & Yarowsky, 1979). The
analogy with the muscarinic actions of acetylcholine has been documented in most
detail by Krnjevic, Pumain & Renaud (1971), working on cortical neurones. This
/M /C
Ba
nA.
Cd
-Bs 50~50c
50 sec 0-5 SWc
Fig. 21. Distinction between effects of Ba2+ (4 mM) and Cd2+ (0-1 mM) on the M-current
(IM) and the Ca2+-dependent K+-current (IC). Holding potential -30 mV; command
potentials alternately to -60 mV for 05 sec (left-hand panels) and 0 mV for 60 msec
(right-panels). The two currents were recorded simultaneously on two channels at different
amplification; note that the low-gain Ic records on the right are inverted. Brief
applications of 4 mM-Ba2+ reduced the IM relaxations and generated an inward current
(upper and lower panels) but did not affect Ic; Cd2+ (0-1 mm, applied between the two
Ba2+ doses) did not reduce IM but reduced IC.
analogy also holds for sympathetic neurones, as illustrated in Fig. 25 below: in

unclamped neurones the effect of a modest concentration (1-4 mM) of Ba2+ is
essentially identical with that of muscarine, comprising a membrane depolarization,
accompanied by an increased 'input resistance' and increased neuronal excitability.
These effects are fully compatible with the muscarine-like inhibition of IM shown in
Fig. 21. Examination of current-voltage curves over a fairly extensive voltage range
(Fig. 22) confirmed that Ba2+ selectively suppressed the outward rectification induced
by IM over the range -70 to -20 mV, with negligible effect on the 'leak' currents
at more hyperpolarized levels or on the large outward currents up to + 30 mV. The
only caveat to the latter conclusion is that, with prolonged applications of Ba2+ (as
necessary for a full current-voltage curve), large outward currents became depressed
in an essentially irreversible manner, probably because of Ba2+ entry and progressive
intracellular accumulation (Hermann & Gorman, 1979). This would accord with our
earlier conclusion (Constanti et al. 1981 a) that Ba2+ selectively depresses IM through
an action from the outside, rather than via entry into the neurone. In these earlier
experiments we also confirmed Takeshige & Volle's (1964) and Krnjevic et al.'8 (1971)
observations that the post-synaptic action of Ba2+ was not blocked by atropine, and
hence was not due to the release of acetycholine.
nA
+300
+200--
-80 -60 -40 -20 mV
nA or
+10 -
+5 /
0 / +100 *
-5 - --a--
a, ~ ~ ~
/
-100 -50/
+50 +100
t ~~~~~~mV
VH 5
Fig. 22. Current-voltage curves for a single neurone obtained in the sequence: control
Ringer solution (0); Ringer plus 4 mM-BaCl2 (M); Ringer solution (0); and Ringer
solution + 10 mM-TEA (C>). Holding potential -30 mY. Currents were measured at the
end of 700 msec hyperpolarization or 70 msec depolarization. The inset graph shows the
effect of Ba2+ between -80 and -20 mV on an expanded current scale. Note that BaO+
reduced rectification between -70 and -20 mY, like muscarine (compare Fig. 2).
Intracellular control of IM
The ability of such chemically diverse compounds as muscarine, LHRH and UTP
to suppress the same ionic current suggests that the connexion between the external
receptor and the M-channel might be rather remote, and that channel closure might
require some secondary intramembrane or intracellular chemical transduction. The
most intensively studied transduction mechanism in sympathetic ganglia is the
muscarinic activation of guanylate cyclase, with consequent elevation of intracellular
cyclic guanosine monosphosphate (Weight, Petzgold & Greengard, 1974; Kebabian,
Steiner & Greengard, 1975; Volle, Quenzer, Patterson, Alkadhi & Henderson, 1981;
but see Brown et al. 1980). Alternative transducers in other tissues include inhibition
of adenylate cyclase (Hulme, Berrie, Birdsall & Burgen, 1981), raised intracellular
Ca2+ (Putney, 1978, Petersen, Nishiyama, Laugier & Philpott, 1981), accelerated
hydrolysis of phosphatidylinositol (Michell, 1975), possibly leading to Ca2+-influx via
phosphatidic acid (Salmon & Honeyman, 1980; Putney, Weiss, van der Walle &
Haddas, 1980), and transmethylation of phosphatidylethanolamine to phosphatidyl-
choline by S-adenosyl-methionine (SAM) (Axelrod & Hirata, 1981).
In an attempt to test some of these possibilities, we have made a number of
intracellular ionophoretic injections of plausible second messengers while clamping
the cell membrane through a second single micro-electrode (see Constanti et al. 1981 b).
To date these tests have been uniformly negative: using ionophoretic currents of up
to + 10 nA from pipettes filled with 10-100 mm solution, no clear reduction of IM has
been detected following intracellular injection of Ca2+, 8-bromo cyclic GMP, cyclic
AMP, GTP, GTP-y-S or S-adenosylmethionine to activate phosphatidylethanolamine
transmethylation. Further, the action of muscarine was not affected by removal of
external Ca2+, addition of a Ca2+-channel blocker (Cd2+, 0.1 mm) or external addition
of a phosphodiesterase inhibitor (isomethyl butyl xanthine, 1 mM), and was not
replicated by 1 mM-external 8-bromo cyclic GMP or dibutryl cyclic AMP. (Intracellular
injections of muscarine also failed to reduce IM.)
In most cases these negative results are of course, somewhat equivocal, since we
have no way of knowing whether the amounts injected (or added) are appropriate.
However, this stricture does not apply to the effect of Ca2+, because the Ca2+
injections were sufficient to evoke large outward C-currents, Ic (Constanti et al. 1981 b;
Adams et al. 1982b). Notwithstanding, IM relaxations were unaffected and remained
superimposed on the steady outward Ic (Fig. 23). This experiment, coupled with the
persistence of muscarine action in a Cd2+ or Ca-free solution, forms strong evidence
against a role of intracellular Ca2+ or of Ca2+ influx in mediating the action of
muscarine in this tissue.
Fig. 24 illustrates two of our experimental tests with 8'Br cyclic GMP. Addition
of 1 mm-external 8'Br cyclic GMP (Fig. 24A) produced, at most, a gradual inward
current and depolarization but did not clearly inhibit IM, whereas subsequent
application of 10 /LM-muscarine was fully effective in inhibiting IM. Intracellular
injection of 10 nA 8'Br cyclic GMP (Fig. 24B) had no clear effect at all, apart from
a current imbalance during the injection itself. These negative effects accord with the
previous results of Weight, Smith & Schulman (1978) on frog neurones.
IM inhibition and excitability
In addition to the membrane depolarization noted in connexion with Figs. 1 and
2, IM inhibitors usually produced a striking increase in the excitability of frog
neurones. Fig. 25 illustrates two examples, using muscarine and Ba2+ respectively.
In Fig. 25A this took the form of long trains of anode-break spikes following the
injection of short hyperpolarizing current pulses, persisting for 1-2 sec and subsiding
into pronounced and irregular oscillations in membrane potential. (Occasionally these
oscillations gave way to renewed bursts of spontaneous spike discharges.) Fig. 25B
shows briefer repetitive anode-break spiking after Ba2+ application, together with an
increased frequency and duration of repetitive discharges during depolarizing current
injections. (Fig. 27 shows another example ofthis phenomeon, produced by muscarine.)
These effects were not due to the membrane depolarization per se, since they were
not replicated by sustained depolarizing current injection (lower record of Fig. 25B).
As described previously (Brown & Adams, 1980; Constanti et al. 1981 a), the spike
VH -25 Control Ca
AV
-10
-30 T
15nA
-40
]50mV
0-5 sec
Fig. 23. Intracellular ionophoresis of Ca2+. In this experiment clamp currents were
recorded with a single micro-electrode (see Methods) before and during ionophoresis of Ca2+
with positive current through a second intracellular electrode filled with 04 M-CaCl2
solution (cf. Adams et al. 1982 b). From a holding potential of -25 mV a single voltage
command was applied before (control) and during (Ca) 5-10 sec ionophoresis of Ca2+. This
was repeated using the four command steps shown. The Ca2+ injections evoked fairly
consistent outward currents as indicated by the displacement of the current base line, and
increased the membrane conductance at the holding potential (shown by the larger
'instantaneous' current steps). However, the slow IM relaxations were unchanged during
the Ca2+ injections.
configuration itself was not modified by these compounds in a manner other than that
(reduced amplitude, slowed rate of rise and repolarization, and reduced after
hyperpolarization) consequent upon membrane depolarization. In particular repolari-
zation was not delayed in the manner induced by TEA or Cds+.
DISCUSSION
The principal conclusions from these experiments are that muscarinic agonists,
LHRH, UTP and Ba2+ ions all inhibit the outward K+-current we have previously
A
1mM-8'Br cyclic GMP 10 LM-muscarine
Voltageclamp-30 I-
IKj2 nA
0-5 sec
Clamp off -40
o Intracellular 8'Br cyclic GMP
1LJ~ ~ ~~
_ _ = ...s-------Pm~~r--Lw----V 20 mV
-I W~lCla 4lamp, 11 nA
/ion ]lOnA
0 5 sec
Fig. 24. Lack of effect of 8'Br cyclic GMP on IM. In A 1 mM-8'Br cyclic GMP was applied
by bath perfusion for the time indicated to a neurone clamped at -30 mV and commanded
to -60 mV, as in Fig. 1. At intervals the clamp was released and the voltage response
to hyperpolarizing current injections (- 1 nA) was briefly monitored: the initial resting
potential was -40 mV. During 8'Br cyclic GMP perfusion there is a small inward current
drift, accompanied by about 5 mV depolarization, which persisted for several minutes
after ceasing perfusion. After this time, 10 ,sM-muscarine was perfused and produced the
effects previously illustrated in Fig. 1. In B the 8'Br cyclic GMP was applied intracellularly
by ionophoresis (see Fig. 23): neither negative nor positive ionophoresis (+ 10 nA for I min
from 100 mm-solution) produced any discernible effect. Iclamp = voltage clamp current;
=ion= ionophoretic current. (Repolarizing currents in the upper record in B are interrupted
by anode-break axonal spikes.)
designated the M-current (IM: Adams et al. 1982a); and that the concentrations of
agonist effective against IM did not materially or consistently affect the other
K+-currents (IK, IC or IA) so far detected in these cells. Thus IM can be distinguished
pharmacologically, as well as kinetically, from these other currents.
Evidence for IM inhibition is essentially three-fold. Firstly, the slow current
relaxations associated with the opening or closing of the M-channels during a voltage
10 jAM-muscarine
A L mV
WnOm OWN INu -------
to .,- 11 nA
1 min 1 sec
4 mM-Ba2n
m lfnmT- ..rlmlmmlfmfr -
B 1 min sec
Depolarizing current
An_gS 1-Ad4~ 1 20 mVJ

- ~ §12 nA
1 sec
Fig. 25. Sample effects of (A) muscarine and (B) Ba2+ on spike discharges, recorded under
'current clamp'. Records show responses to hyperpolarizing and depolarizing current
injections before, during and after drug administration in which the recording speed was
periodically accelerated 100-fold. In A, 10 ItM-muscarine depolarized the neurone from
-42 to -28 mV and increased the amplitude of the voltage deflexion produced by
constant hyperpolarizing current pulses, in the manner shown in Fig. 1. In addition
repetitive anode-break spikes were generated, first subsiding to single spikes, and then
disappearing, on washing. In B, 4 mM-Ba2+ produced a similar depolarization during which
short depolarizing pulses generated longer trains of spikes. The lower record in B shows
the effect of an equivalent depolarization produced by injecting sustained positive current.
step are reduced or suppressed (Fig. 1). Secondly, the component of membrane chord
conductance referable to the conductance of the M-system is reduced, without
material change in leak conductance (Fig. 3). Thirdly, an inward current is generated,
the amplitude of which matches the amplitu> of IM inhibition at the same membrane
voltage (Fig. 7 A) and which varies with membrane voltage pari pas8u the change
in IM (Figs. 7A and 9). This evidence is most comprehensive for muscarine, but
appears equally firm for the other agonists listed above (see also Adams & Brown,
1980; Constanti et al. 1981a; Brown et al. 1981).
The mechanism of IM inhibition is not fully clear. It does not reflect a shift in
voltage sensitivity, since the partially blocked currents show the same time course
and voltage sensitivity as the normal current. This contrasts with (for example) the
effect of adrenaline on the cardiac Purkinje fibre IK2 current (Hauswirth, Noble &
Tsien, 1968). It seems as though a variable fraction of the channels (depending on
the concentration of agonist) are rendered into a non-conducting state independently
of voltage. Ba2+ ions might simple 'plug' the channels, but this seems less likely for
the other agonists since they act via quite different receptors yet each can still shut
down all of the channels. Possibly the agonists might trigger some common
intracellular or intramembrane transduction system resulting in a chemical change
in the M-channels. We have made some preliminary tests using various 'second
messengers' previously suggested to mediate muscarinic effects, without success. At
this stage we feel justified in ruling out a rise in intracellular Ca2+ as the final agent,
since IM was unaffected by elevating or reducing extracellular Ca2+ or by injecting
sufficient Ca2+ into the neurones to generate large C-currents. The negative evidence
for cyclic GMP also seems fairly compelling, but further resolution of the mechanism
clearly requires different technical approaches to those we have used so far.
Interpretation of effect in unclamped neurones
In unclamped neurones these agonists all produce a characteristic 'slow depolari-
zation' (muscarinic agonists: Nishi et al. 1969; Kobayshi & Libet, 1970; Kuba &
Koketsu, 1976a, b; Brown & Constanti, 1980; Freschi & Shain, 1980; Gruol et al. 1981;
LHRF: Jan et al. 1979, 1980; Sejnowski, 1982; uridine pho8phates: Siggins et al. 1977;
Gruol et al. 1981; Ba2+: Takeshige & Volle, 1964; Tashiro & Nishi, 1972; McLachlan,
1977; McAfee & Yarowsky, 1979; and, in cortical neurones Krnjevic et al. 1971).
Effects of muscarinic agonists have been studied the most extensively and show the
following seminal features: (i) voltage sensitivity - the depolarization is largest at or
near rest potential, and is reduced by modest membrane hyperpolarization; (ii) an
increase in apparent input resistance, which also varies with voltage; (iii) reduced
outward rectification in the current-voltage curve; and (iv) increased excitability,
such that action potentials are more readily generated by a depolarizing current
injection or by synaptic excitation, and persist for longer, or spontaneous firing may
be engendered.
We suggest that all of these effects can be explained by the selective M-current
inhibition observed under voltage clamp.
(i) Depolarization. According to the scheme proposed previously (Adams et al.
1982a), the resting membrane potential is set by the balance between a voltage-
insensitive inward leak current and a voltage-sensitive outward K+-current through
the M-channels. (The voltage sensitivity of the M-channel conductance is the crucial
difference between this model and that of Weight & Votava, 1970.) When IM is
inhibited, depolarization ensues because of an unbalanced inward leak current. The
strongest evidence for this is that within the range -70 to -30 mV the net inward
current induced by muscarine matches the net suppression of IM (Fig. 7) and varies
with voltage in proportion to the change in IM (Fig. 9). We do not need to postulate
any increase in the leak conductance itself; indeed, no change in leak conductance
could be detected (from measurements of chord conductance, or of the slope of the
current-voltage curve at potentials more negative than -60 mV where the M-
channels are shut) in nearly all of the cells tested using concentrations of muscarine,
LHRH, UTP or Ba2+ which suppressed IMP
A V/A Vm ax mV
1 ~~~~~~~~~~~~~-10
-20
0*5 -30
-40
-50
0-
0 -0 25 -0-5 -0-75 -1
-AGM/GM
Fig. 26. Predicted relationship between membrane depolarization (A V, expressed as a
fraction of maximal depolarization A Vmax), or membrane potential, and the fractional
reduction of the maximum M-conductance, GM. Calculations were based on the equivalent
circuit in Adams et al. (1982a) (see legend to Fig. 1). Values for conductances and reversal
potentials are average values used by Adams et al. (1982a), namely GL = 1O nS,
GM = 84 nS, VL = -10 mV, VM(= VK) = -90 mV. The voltage sensitivity of GM is that
given in the legend to Fig. 2.
Kuba & Koketsu (1976a) reported an increased depolarization by (high) acetylcholine concen-
trations at very hyperpolarized membrane potentials in a proportion of bullfrog sympathetic
neurones, with a reduction in the slope of the voltage-current relations equivalent to an increased
membrane conductance. We detected an equivalent inward current and increased 'leak conduc-
tance' in two neurones at membrane potentials > -70 mV following 10 ItM-muscarine. However,
even when present, this current is unlikely to contribute significantly to the depolarization produced
at normal rest potentials (between -50 and -60 mV) since itbecame negligible above -60 mV
compared with that generated by IM inhibition. The same consideration applied to the inward
current and conductance increase sometimes produced by LHRF at hyperpolarized potentials (Jan
et al. 1980; Sejnowski, 1982). This we have not so far confirmed, but we reiterate that the inward
current generated at normal membrane potentials (< -60 mV) was quantitatively accountable
by the amount of IM suppression (see also Adams & Brown, 1980; Brown et al. 1981).
This mechanism of depolarization has some interesting pharmacological consequences concerning
the expected relationship between receptor occupancy and measured response. IM normally exerts
a very strong potential-clamping effect because, as the cell depolarizes, more M-channels open and
the outward current increases (see Adams et al. 1982a). Thus, the anticipated relationship between
depolarization and fractional reduction of M-conductance is highly asymmetric, such that some 90 %
of the M-channels must be shut to produce half the maximal depolarization (Fig. 26). This is quite
opposite to the effect predicted for a channel-opening mechanism (cf. Bolton, 1972; Brown et al.
1980). Unfortunately, this relationship is difficult to verify experimentally because large depola-
rizations open other slowly inactivating K+-channels, such as the Ca2+-dependent and delayed
rectifier channels.
(ii) Resi8tance measurements. Inhibition of IM provides a clear explanation for the
effects of muscarinic agonists on cell 'input resistance' when measured from the
voltage deflexions produced by current injection in unclamped neurones. Under
voltage clamp, when the membrane potential is abruptly increased there are two clear
stages in the resultant current flow (see Fig. 1): an 'instantaneous' step followed by
a slow relaxation to a new steady state. The former represents the current flow
through the channels which were open before applying the voltage step, because they
do not have time to close, and so provides a measure of the pre-existing chord
conductance. The slow step represents the secondary current flow accompanying the
closure of the M-channels. Under 'current clamp', however, the two events merge:
the initial voltage deflexion produced by a stepped current injection is slowed by the
membrane capacitance to such an extent that the M-channels begin to close before
the peak voltage deflexion is attained. This is shown in the reconstruction illustrated
in Fig. 27A. The current-flow induced by the M-channel closure then leads to a
secondary voltage deflexion whose amplitude and time course is dependent primarily
on the voltage attained and not on the applied current. With moderate initial voltage
shifts (such that the voltage attained is less than EK), the secondary voltage deflexion
tends to restore the membrane potential towards its initial value, i.e. the 'resistance'
appears to fall (although it has actually risen!). 'Current clamp' records of this type
are illustrated in Figs. 1 and 2, and further exemplified in Fig. 27B. If the M-current
is inhibited, these secondary voltage shifts are suppressed, and the voltage trajectory
should then take the simpler form shown in the right-hand panel of Fig. 27A. This
is precisely the effect of adding a large dose of muscarine, as shown in Fig. 27 B. Thus,
the changes in the shape of the voltage responses accord with the M-current
hypothesis.
It is clear from Figs. 1, 25A and 27B that, even in the unclamped state, the apparent input
resistance of the cell has been increased by muscarine, but how should this be measured? The most
useful measurement is the chord conductance but, as pointed out above, this cannot be measured
in the unclamped state. The best alternative is to measure the voltage deflexion at the end of a
pulse sufficiently long for the secondary voltage deflexions to have settled, i.e. for GM to have
attained a new steady-state. When the membrane potential attained at this time is plotted against
the current injected, the resultant current-voltage curve agrees well with the steady-state I/ V curve
obtained using voltage clamp (see Fig. 2). Tangents drawn to the curve will then indicate the 8lope
resistance. Previous use of this method showed clearly the voltage sensitivity of both the resting
input resistance and also of the resistance change produced by muscarinic agonists (Kuba &
Koketsu, 1976a; Brown & Constanti, 1980), qualitatively, comparable with the present data. In
fact, these slope resistance measurements over-estimate the true voltage sensitivity of the resting
M-conductance (q.v., Horn & Brodwick, 1980) and, in consequence, that of muscarinic action. Thus,
our previous conclusion (Brown & Constanti, 1980) that the peak slope of the input conductance
corresponds to an e-fold change per 4-6-5 mV depolarization is not incompatible with the
voltage-clamp data (cf. Adams et al. 1982a).
There are of course, other difficulties in interpreting the effect of input resistance measurements
using constant current pulses. One problem is that the depolarization induced by IM inhibition itself
opens more M-channels, so offsetting the initial fall in conductance. This is usually accommodated
by resetting the membrane potential with sustained current injection. However, this, of itself, is
insufficient to allow a correct comparison, because the final state of the M-system is governed by
the final potential attained during the current pulse. Thus, a proper correction for the effect of
voltage should also incorporate an adjustment of the current pulse to obtain the same final
potential.
PHY 332
+GM -GM
.-~~---- ] 1- nA
Control Muscarine
vE] 20 mV
500 msec
Fig. 27. A, computer-predicted time-dependent responses of the membrane (V) to
+0-25 nA current pulses (I) based on the equivalent circuit model in Adams et al. (1982 a)
under normal conditions (+GM) and under conditions where all of the M-channels are
occluded (-OM) (see legend to Fig. 2). The time and voltage dependence of the opening
and closing rate constants for the OM (aM and flM respectively) are given by
{[e 1]2T
[M] -33exp (V+35)} sec1,
where V = membrane potential. A capacitance of 0 4 nF is assumed. Computational
procedures are described in Adams et al. (1982 a). GL and GM were set at 5 and 20 nS
respectively, for a high-resistance cell. Predicted rest potential is -44 mV. B, examples
of observed voltage deflexions in a high resistance cell in the absence and presence of 10
/SM-muscarine. Note that the responses to hyperpolarizing current injections accord in
general form with those predicted in A. (The response to depolarizing pulses may be
modified by other currents not incorporated into the simulation.)
(iii) Rectification. Progressive opening of the M-channels with depolarization

explains the outward rectification in the steady-state I/ V curve between -70 and
-25 mV, as shown in Fig. 2. The effect of muscarinic agonists, LHRH, UTP or Ba2+
on the I/ V curve are fully consistent with reduction or suppression of IM. Rectification
outside this range results from the advent of other currents - a variable slow inward
current at very hyperpolarized potentials (see Fig. 2), and IK + IC positive to
-25 mV. These currents, and the rectification they confer, were unaffected by the
above agonists.
Kuba & Koketsu (1976a) ascribed this change in the I/V curve to a shift in the threshold for
delayed rectification. In a subsequent paper (Kuba & Koketsu, 1976b) they described changes in
spike configuration (slowed repolarization and reduced after-hyperpolarization) following appli-
cation of high concentrations (0-27 mM) of acetycholine which appeared compatible with this
suggestion. Akasu (1981) has recently reported a reduced amplitude of both delayed rectifier and
Ca2+-dependent K+-current in voltage-clamped bullfrog ganglion cells by acetycholine, but also
stated that, at the necessary concentrations, acetycholine preferentially depressed IM. Our results
have been quite unequivocal on this point: we have not detected any clear depression of Ic or IK
by muscarine in concentrations inhibiting IM by 80-90%; indeed, in many cells (e.g. Fig. 2) the
IC-IK currents evoked by just suprathreshold voltage commands (to -25 or -20 mV) were
increased (though it would be difficult to exclude a very small (1-2 mV) drift in holding potential
as a contributory factor). Further, we have not observed any change in spike configuration following
application of muscarine or Ba2+ comparable to that produced by TEA or Cd2+ ions, which inhibit
the larger outward currents (see Brown & Adams, 1980; Constanti et al. 1981 b; Adams et al. 1982 b).
A pertinent question in this respect is whether, in fact, IM could contribute to the repolarizing
phase of the spike. We do not have direct knowledge of IM-kinetics at positive membrane potentials,
but, if the voltage sensitivity of the M-channel opening rate mirrored that of the closing rate, the
time-constant for GM activation should shorten from 150 msec at -35 mV to 50 msec at 0 mV
and 10 msec at + 40 mV (legend to Fig. 27). Thus GM should increase rapidly during the spike
but decrease slowly on repolarizing. Computer simulation suggests that, in the absence of any other
currents, GM would then accelerate the restoration of potential after a brief (2 msec) depolarization
to - 0 mV and induce a subsequent after-hyperpolarization. However, the important condition
for this is that no other outward currents are present: since the maximal value of OM is only about
3 % or so of that ofthe delayed rectifier or Ca2+-dependent K+-currents the latter would predominate
unless they were inactivated or inoperative for some other reason. Under normal conditions,
therefore, we would not expect IM inhibition to affect spike configuration directly if adequately
compensated for the membrane depolarization.
(iv) Excitability. Muscarinic agonists, UTP and Ba2+ all increase the excitability
of sympathetic neurones. This usually took the form of an increase in the duration
of the spike trains during depolarizing current injection, with a reduced inter-spike
interval, or the generation of repetitive spikes at the anode-break of a hyperpolarizing
current pulse, (see Figs. 25 and 27). Occasionally sustained trains of spikes and
membrane potential oscillations occurred during muscarinic perfusion. Comparable
effects have been reported in cultured frog neurones (Gruol et al. 1981), and in
mammalian sympathetic neurones in situ (Kobayashi & Libet, 1970; Brown &
Constanti, 1980) and in culture (Freschi & Shain, 1980). Another form of excitability
increase is a reduced threshold for synaptic excitation and increased e.p.s.p.
amplitude (Schulman & Weight, 1976).
Data regarding the kinetics of IM, and of some of the other membrane currents,
at depolarized potentials are not yet precise enough to allow a detailed interpretation
of changes in spike pattern, but it is fairly easy to envisage how IM inhibition could
explain these effects in a qualitative manner. Although (as pointed out above) IM
is unlikely to affect spike configuration, it would be expected to have a profound effect
on spike threshold because it is the only voltage-sensitive outward current operating
between -60 and -25 mV and, at the more depolarized end of this range, contributes
> 90 % of the total membrane current. As indicated in Fig. 27A, GM is activated
slowly during a depolarizing current injection to provide a repolarizing drive. In
addition, however, if the kinetics of GM further shortened with depolarization in the
manner discussed above, activation of GM would be accelerated and intensified by
spikes, such as to become complete after the first one or two spikes, and would be
maintained during the subsequent depolarization because of its slow offset kinetics.
The consequent sustained rise in spike threshold, if sufficiently great, would then
9-2
arrest the spike train after a relatively few spikes. Inhibition of IM would prevent
this rise in threshold, so that spikes would be more easily generated by the initial
depolarization and the length of the spike train increased to an extent determined
by the normal accommodation processes. Computer simulation also suggests sub-
stantial activation of IM during a normal e.p.s.p. so inhibition of IM could explain
the increased e.p.s.p. amplitude by methacholine reported by Schulman & Weight
(1976).
Viewed in this context, IM can be regarded as a natural 'braking' current,
controlling neuronal excitability and limiting repetitive spike trains. Thus, removal
of this' brake' and the consequent increase in excitability - rather than the membrane
depolarization or passive resistance increase - probably represents the physiologically
significant aspect of IM suppression.
Supported by NIH grants N.S. 14986, and the Sloane Foundation and the Klingenstein Fund
(P.R.A.) and by the U.K. Medical Research Council and the Wellcome Trust (D.A.B. and A.C.)
We thank Professor K. Krnjevic for suggesting the experiments with Ba2+.
REFERENCES
ADAMS, P. R. (1981). The calcium current of a vertebrate neuron. In Advances in Physiological
Science, vol. 4, ed. SALANKI, J., pp. 135-138. Budapest: Akademai Kiado.
ADAMS, P. R. & BROWN, D. A. (1980). LHRF and muscarinic agonists act on the same voltage-
sensitive K+-current in bullfrog sympathetic neurones. Br. J. Pharmac. 68, 353-355.
ADAMS, P. R., BROWN, D. A. & CONSTANTI, A. (1982a). M-currents and other potassium currents
in bullfrog sympathetic neurones. J. Physiol. 330, 537-572.
ADAMS, P. R., CONSTANTI, A., BROWN, D. A. & CLARK, R. B. (1982b). Intracellular Ca activates
a fast voltage-sensitive K-current in vertebrate sympathetic neurones. Nature, Lond. 296, 746-749.
ADAMS, P. R. & HALLIWELL, J. V. (1982). A hyperpolarization-induced inward current in
hippocampal pyramidal cells. J. Physiol. 324, 62P.
AKASU, T. (1981). Voltage-clamp analysis of muscarine effects on action potentials in sympathetic
ganglion cells of bullfrogs. Neurosci. Lett. S59, Suppl. 6.
AKASU, T. & KOKETSU, K. (1981). Desensitization of the muscarine receptor controlling action
potentials of sympathetic ganglion cells in bullfrogs. Life Sci., Oxford 27, 2261-2267.
AMBACHE, N. (1949). The nicotinic action of substances supposed to be purely smooth-muscle
stimulating. (B) Effect of BaCl2 and pilocarpine on the superior cervical ganglion. J. Physiol. 110,
164-172.
AMBACHE, N., PERRY, W. L. M. & ROBERTSON, P. A. (1956). The effect of muscarine on perfused
superior cervical ganglion of cats. Br. J. Pharmac. 11, 442-448.
AXELROD, J. & HIRATA, F. (1981). Phospholipid methylation and receptor-mediated transmission
of biological signals through membranes. In Drugs Receptors and their Effectors, ed. BIRDSALL,
N. J. M., pp. 51-57. London: Macmillan.
BOLTON, T. B. (1972). The depolarizing action of acetylcholine or carbachol in intestinal smooth
muscle. J. Physiol. 220, 647-671.
BROWN, D. A. & ADAMS, P. R. (1979). Muscarinic modification of voltage-sensitive currents in
sympathetic neurones. Neurosci. Abstr. 5, 585.
BROWN, D. A. & ADAMS, P. R. (1980). Muscarinic suppression of a novel voltage-sensitive K+-current
in a vertebrate neurone. Nature, Lond. 283, 673-676.
BROWN, D. A. & CONSTANTI, A. (1980). Intracellular observations on the effects of muscarinic
agonists on rat sympathetic neurones. Br. J. Pharmac. 70, 593-608.
BROWN, D. A., CONSTANTI, A. & ADAMS, P. R. (1981k. Slow cholinergic and peptidergic transmission
in sympathetic ganglia. Fedn Proc. 40, 2625-2630.
BROWN, D. A., FATHERAZI, S., GARTHWAITE, J. & WHITE, R. D. (1980). Muscarinic receptors in
rat sympathetic ganglia. Br. J. Pharmac. 70, 577-592.
CONNOR, J. A. & STEVENS, C. F. (1971). Voltage-clamp studies of a transient outward membrane
current in gastropod neural somata. J. Physiol. 213, 21-30.
CONSTANTI, A., ADAMS, P. R. & BROWN, D. A. (1981 a). Why do barium ions imitate acetycholine?
Brain Res. 206, 244-250.
CONSTANTI, A., ADAMS, P. R. & BROWN, D. A. (1981 b). Calcium activated outward currents (IC)
in voltage-clamped bullfrog sympathetic neurones. Neurosci. Abstr. 7, 14.
CONSTANTI, A. & BROWN, D. A. (1981). M-current in voltage-clamped mammalian sympathetic
neurones. Neurosci. Lett. 24, 289-294.
EIDEN, L. E. & ESKAY, R. L. (1980). Characterization of LRF-like immunoreactivity in the frog
sympathetic ganglia: non-identity with LRF decapeptide. Neuropeptides 1, 29-37.
FRESCHI, J. E. & SHAIN, W. G. (1980). Slow muscarinic depolarization in neurons of dissociated
rat superior cervical ganglia can be evoked by iontophoresis of acetycholine. Brain Res. 185,
429-434.
GINSBORG, B. L. (1965). The action of McN-A-343, pilocarpine and acetyl-fl-methycholine on
sympathetic ganglion cells of the frog. J. Pharmac. exp. Ther. 150, 216-219.
GRUOL, D. L., SIGGINS, G. R., PADJEN, A. & FORMAN, D. S. (1981). Explant cultures of adult
amphibian sympathetic ganglia: electrophysiological and pharmacological investigation of
neurotransmitter and nucleotide action. Brain Res. 223, 81-106.
HALLIWELL, J. V. & ADAMS, P. R. (1982). Voltage-clamp analysis of muscarinic excitation in
hippocampal neurones. Brain Re8. (in the Press).
HAUSWIRTH, O., NOBLE, D. & TSIEN, R. W. (1968). Adrenaline: mechanism of action on the
pacemaker potential in cardiac Purkinje fibers. Science, N.Y. 162, 916-917.
HERMANN, A. & GORMAN, A. L. F. (1979). Blockade of voltage-dependent and Ca2+-dependent
K+-current components by internal Ba2+ in molluscan pacemaker neurones. Experientia (Basel)
35, 229-231.
HORN, R. & BRODWICK, M. S. (1980). Acetycholine-induced current in perfused rat myoballs. J.
gen. Physiol. 75, 297-322.
HULME, E. F., BERRIE, C. P., BIRDSALL, N. J. M. & BURGEN, A. S. V. (1981). Interactions of
muscarinic receptors with guanine nucleotides and adenylate cyclase. In Drug Receptors and their
Effectors, ed. BIRDSALL, N. J. M. pp. 23-24. London: Macmillan.
JAN, Y. N. JAN, L. Y. & KUFFLER, S. W. (1979). A peptide as a possible transmitter in sympathetic
ganglia of the frog. Proc. natn. Acad. Sci. U.S.A. 76, 1501-1505.
JAN, Y. N., JAN, L. Y. & KUFFLER, S. W. (1980). Further evidence for peptidergic transmission
in sympathetic ganglia. Proc. natn. Acad. Sci. U.S.A. 77, 5008-5012.
KEBABIAN, J. W., STEINER, A. L. & GREENGARD, P. (1975). Muscarinic cholinergic regulation of
cyclic guanosine-3'5'-monosphosphate in autonomic ganglia: possible role in synaptic trans-
mission. J. Pharmac. exp. Ther. 193, 474-488.
KOBAYASHI, H. & LIBET, B. (1970). Action of noradrenaline and acetylcholine on sympathetic
ganglion cells. J. Physiol. 208, 353-372.
KONZETT, H. & WASER, P. G. (1956). Zur ganglioniiren Wirkung von Muscarin. Helv. physiol. Acta
14, 202-206.
KRNJEVI6, K., PUMAIN, R. & RENAUD, L. (1971). Effects of Ba2+ and tetraethylammonium on
cortical neurones. J. Physiol. 215, 223-245.
KUBA, K. & KOKETSU, K. (1976a). Analysis of the slow excitatory potential in bullfrog sympathetic
ganglion cells. Jap. J. Physiol. 26, 651-669.
KUBA, K. & KOKETSU, K. (1976b). The muscarinic effects of acetycholine on the action potential
of bullfrog sympathetic ganglion cells. Jap. J. Physiol. 26, 703-716.
KUBA, K. & KOKETSU, K. (1977). Postsynaptic potentiation of the slow muscarinic excitatory
response by tetraethylammonium chloride in the bullfrog sympathetic ganglion cells. Brain Res.
137, 381-386.
MCAFEE, D. A. & YAROWSKY, P. J. (1979). Calcium-dependent potentials in the mammalian
sympathetic neurone J. Physiol. 290, 507-523.
McLACHAN, E. M. (1977). The effects of strontium and barium ions at synapses in sympathetic
ganglia. J. Physiol. 267, 497-518.
MICHELL, R. H. (1975). Inositol phospholipids and cell surface receptor function. Biochim. biophys.
Acta. 415, 81-147.
NEHER, E. (1971). Two fast transient current components during voltage clamp on snail neurons.
J. gen. Physiol. 58, 36-53.
NISHI, S., SOEDA, H. & KOKETSU, K. (1969). Unusual nature of ganglionic slow EPSP studied by
a voltage-clamp method. Life Sci., Oxford 8, 33-42.
PETERSEN, O., NISHIYAMA, A., LAUGIER, R. & PHILPOTT, H. G. (1981). Hormonal control of ion
permeability of the pancreatic acinar cell membrane mediated by intracellular calcium. In Drug
Receptors and their Effectors, ed. BIRDSALL, N. J. M., pp. 63-73. London: Macmillan.
PUTNEY, J. W. (1978). Stimulus-permeability coupling: role of calcium in the receptor regulation
of membrane permeability. Pharmac. Rev. 30, 209-245.
PUTNEY, J. W. JR.. WEISS, S. J. VAN DER WALLE, C. M. & HADDAS, R. A. (1980). Is phosphatidic
acid a calcium ionophore under neurohormonal control? Nature, Land. 284, 345-346.
SALMON, D. M. & HONEYMAN, T. W. (1980). Proposed mechanism of cholinergic action in smooth
muscle. Nature, Lond. 284, 344-345.
SCHULMAN, J. & WEIGHT, F. (1976). Synaptic transmission: long-lasting potentiation by a
postsynaptic mechanism. Science, N. Y. 194, 1437-1439.
SEJNOWSKI, T. J. (1982). Peptidergic synaptic transmission in sympathetic ganglia. Fedn Proc. (In
the Press).
SImGINS, G. R. (1978). Electrophysiological assessment of monucleotides and nucleosides as first
and second messengers in the nervous system. In Neuronal Information Transfer, ed. KARLIN, A.,
pp. 339-355. New York: Academic Press.
SIGGINS, G. R., GRUOL, D., PADJEN, A. & FORMAN, D. (1977). Purine and pyrimidine mono-
nucleotides depolarize neurones of explanted amphibian sympathetic ganglia. Nature, Lond.
270, 263-265.
TAKESHIGE, C. & VOLLE, R. L. (1964). The effects of barium and other inorganic cations on
sympathetic ganglia. J. Pharmac. exp. Ther. 146, 327-334.
TASHIRO, N. & NISHI, S. (1972). Effects of alkali-earth cations on sympathetic ganglion cell of the
rabbit. Life Sci., Oxford 11, 941-948.
VOLLE, R. L., QUENZER, L. F., PATTERSON, B. A., ALKADHI, K. A. & HENDERSON, E. G. (1981).
Cyclic guanosine 3'5'-monophosphate accumulation and 45Ca-uptake by rat superior cervical
ganglia during preganglionic stimulation. J. Pharmac. exp. Ther. 219, 338-343.
WEIGHT, F., PETZGOLD, G. & GREENGARD, P. (1974). Guanosine-3'5'-monophosphate in sympa-
thetic ganglia: increase associated with synaptic transmission. Science, N. Y. 186, 942-944.
WEIGHT, F., SMITH, P. A. & SCHULMAN, J. A. (1978). Postsynaptic potential generation appears
independent of synaptic elevation of cyclic nucleotides in sympathetic neurones. Brain Re8. 158,
197-202.
WEIGHT, F. & VOTAVA, Z. (1970). Slow synaptic excitation in sympathetic ganglion cells: evidence
for synaptic inactivation of potassium conductance. Science, N. Y. 170, 755-758.

Adams, P.R., Brown, D.a., and Constanti, A. (1982b) - Pharmacological Inhibition of The M-Current

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Adams, P.R., Brown, D.a., and Constanti, A. (1982b) - Pharmacological Inhibition of The M-Current

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J. Phy8iol. (1982), 332, pp.

PHARMACOLOGICAL INHIBITION OF THE M-CURRENT

We have previously reported the presence of a time and voltage-dependent

Control Muscarine Control Muscarine

-60 3.4L --~

where e = + 25, V0 =-35 mV and V is the membrane potential. The maximum M-

IM kinetics. Muscarine might inhibit IM by shifting its activation curve to a nuch

25 mM-K' VH = -25 mV 10pM-muscarine

IJ'Jw! ~j, r jjjH A

-60 -=° LU rFL §L LJLJLF

Fig. 8. Responses of a ganglion cell to ionophoretically applied muscarine at different

-30 m y _ ] 1 nA .-.! I]1 nA

-20 -40 -60 -80 -100

-30 10 JuM-muscarine, 2 sec

----il J iLLPIIII Li JJJ f l20 mV

Effect of muacarine on other K+-currents

example of IA activated by depolarizing a neurone from -80 to -45 mV. Fig. 11

-60 -40 -20 0 mV

(Indeed, we frequently noted a small increase in the outward current at -20 to

± 10 pM-muscarine ±10 pM-TEA

0 100 200 0 100 200 msec

Luteinizing-hormone releasing hormone (LHRH)

instantaneous current steps at the beginning of the hyperpolarizing commands, was

mV -90 -60 -30

as expected were it to be generated exclusively by IM suppression. An example is

V(mV) -100 -80 -60 -40 -20

50 sec 0-5 sec

the cation to depress a known Ca2+-activated current, against which effects on IM

I!J! 111? 125 or

analogy also holds for sympathetic neurones, as illustrated in Fig. 25 below: in

Clamp off -40

o Intracellular 8'Br cyclic GMP

An_gS 1-Ad4~ 1 20 mVJ

(iii) Rectification. Progressive opening of the M-channels with depolarization

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