A muscarine-sensitive, slow, transient outward current in frog autonomic neurones

Brain Research, 524 (1990) 236-243

236

Elsevier BRES 15749

A muscarine-sensitive, slow, transient outward current in frog autonomic neurones Alexander A. Selyanko 1, Jeffrey A. Zidichouski 2 and Peter A. Smith 2 l Bogomoletz Institute of Physiology, Kiev ( U. S. S. R. ) and 2Department of Pharmacology, University of Alberta, Edmonton (Canada) (Accepted 6 February 1990)

Key words: Sympathetic neuron; Parasympathetic neuron; A-current; Aminopyridine; Muscarinic response; Autonomic ganglion; K÷ current; Delayed rectifier

A slow, 4-aminopyridine-sensitive K+ current was observed in Rana pipiens autonomic neurones when they were studied under whole-cell patch-clamp recording conditions. This 'slow-A' current (IsA), which was independent of extracellular Ca2÷, exhibited a similar voltage dependence to a classical A current (IA) yet inactivated with an 80-fold slower time course. Although ISA is difficult to distinguish from the delayed rectifier K÷ current (1K), muscarine enhanced the current in sympathetic neurones and either enhanced or suppressed the current in parasympathetic neurones. Effects on slow transient outward currents must therefore be considered when attempting to understand cholinergic modulation of repetitive discharge in autonomic neurones. INTRODUCTION Ionic currents flowing through n e u r o n a l K + channels are involved in action potential repolarisation and/or in the coding of action potential discharge rates in response to depolarising stimuli 1'3'8. Suppression of one such current, the fast transient outward current (IA) 7 by various n e u r o m o d u l a t o r s 4'6 or 4-aminopyridine (4-AP) 2°" 23,26 results in increased n e u r o n a l discharge rates. We used the whole-ceU patch-clamp technique to study n e u r o n e s from Rana pipiens autonomic ganglia and found that only the large sympathetic ganglion B cells exhibited I g. This current was not found in small sympathetic C cells and was often absent from parasympathetic ganglion neurones. In all 3 n e u r o n e types however, our methodology revealed a n o t h e r transient, 4-AP-sensitive potassium current which exhibited similar voltage dependance to I A yet inactivated with an 80-fold slower time course. This paper describes some of the characteristics of this 'slow A ' current (IsA) which corresponds to the current previously termed /KP by Pfaffinger 19.

solution contained (mM), NaCI 117, KCI 2, MgCI2 2, CaCI2 2, HEPES/NaOH (pH 7.2) 5 and D-glucose 10. In experiments where KCl was increased to 6, 20 or 60 mM, the concentration of NaCl was appropriately reduced to balance osmolarity. Although an internal solution containing (mM) KCl 110, NaC! 10, MgCl2 2, CaCl2 0.4, EGTA 4.4, HEPES/KOH (pH 7.2) 5 and D-glucose 10 was adequate for recording ISA, patch pipettes in some experiments contained 0.5 mM inositol 1,4,5-trisphosphate, 100 pM cyclic AMP, ATP and/or GTP (Na + salts). In other experiments, the protein kinase C inhibitors, H-7 (1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine, 50 pM) or gold sodium thiomalate (50 pM) were included. Extracellular drug application was by the 'U-tube' method 15. Whole-cell currents were studied in the voltage range -140 to -30 mV in an attempt to avoid currents through voltage-dependent Ca2+-sensitive K+ channels (lc) and delayed rectifier K÷ channels (IK) which have been reported to activate only at potentials more positive than -25 mV 1'12'13"16. IM1-3'14'21 and I A are the predominant K+ currents known to operate in the experimental voltage range. Whole-cell recordings were obtained as previously described21 using 5-10 M,Q electrodes, an Axopatch 1B amplifier, 'pCLAMP' software, a Labmaster interface and an IBM XT computer. Filters were set to -3 dB at 500 Hz. Neuronal input capacitance was estimated by noting the amplifier setting for whole-cell capacitance compensation. Data were displayed on a Gould-Brush 2400 rectilinear pen recorder (pen rise time < 8 ms) or stored on removable hard discs (10 megabyte 'Bernoulli box') and hard copies obtained from an X - Y plotter.

MATERIALS AND METHODS

RESULTS

The VIIIth to Xth paravertebral sympathetic ganglia9 and/or the intra-atrial parasympathetic ganglia2s were removed from 2-3 pithed Rana pipiens. Neurones were dissociated using trypsin (Sigma type III, 3 mg/ml, 50 min, 37 °C) followed by collagenase (Sigma type la, 1 mg/ml, 25 min, 37 °C). The normal external

Neurones dissociated from Rana pipiens paravertebral sympathetic ganglia were subdivided into putative B and C cells9 on the basis of their input capacitance (Cin; a measure of size). The larger B cells (mean Ci, 40.5 _ 1.5

Correspondence: P.A. Smith, Department of Pharmacology, 9.75 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

237 pF, n = 66) exhibited an inward current in response to 10 g M muscarine 2'3 whereas the smaller C cells (mean Ci, 21.3 + 0.8 p E n = 70) responded with an outward current to the same agonistl°'2L Although the B cells exhibited a c l a s s i c a l / A 1"5'7'17'20"25,this current was absent from all sympathetic C cells and was only detected in 5 out of 24 parasympathetic cardiac ganglion cells. Fig. 1A,a shows the currents evoked by a command pulse from a holding potential of - 3 0 to -100 mV in a sympathetic ganglion C cell, 30 s after establishing whole-ceU recording. This holding potential was chosen as part of another aspect of our work 21 which is concerned with the effect of agonists on another voltagesensitive K + current, the M current (IM)1-3. This current is activated at - 3 0 mV and its properties may be conveniently examined by observing the deactivation produced by using hyperpolarising voltage commands 1-3' 14,21. The initial inward current transient recorded at -100

A

mV represents K + influx through M channels 1'3'14 before they deactivate. U p o n repolarisation to - 3 0 mV, a second transient occurs which may be due to activation of inward INa and/or /Ca" The slow increase in outward current following the termination of the voltage c o m m a n d is due to the slow reactivation of I M. However, as recording is continued, (Fig. 1A,b,c) an additional slow, transient outward current (IsA) appears and its magnitude and decay rate constant increase with time. Fig. 1B shows a similar experiment on a sympathetic ganglion B cell. W h e n the whole-cell patch-clamp recording conditions are first established (Fig. 1B,a), a command pulse to -110 mV elicits a current response similar to that shown in the C cell (Fig. 1A,a) except that a fast, transient outward current (IA, Fig. 1B,a) interrupts the activation of I M which occurs when the membrane potential is returned to - 3 0 mV. This transient results from the removal of A channel inactivation at -110 mV

Sympathetic C - neurone a) 30

s

48

~

~40 pA ls

I00 pA

Is

400ms

mv-1oo -30 B

rain

L~

U

U

U

Sympathetic B - neurone

a)

~__1A 1 min Ji

200pA

IM

mV.11o -30 C

L,[

U

U

II

Parasympathetic Neurone

~ , S A

3~s 100pA

-30-]_.[ mV-120 Fig. 1. Currents recorded in autonomic neurones in response to hyperpolarising voltage commands. A: sympathetic ganglion C cell. Currents evoked by a 400 ms hyperpolarising command pulse to -100 mV from the holding potential of -30 mV. a: 30 s, b: 5 min, c: 24 min and d: 48 min after establishing whole-cell recording conditions. Note appearance and progressive enlargement of slow transient outward current (IsA) upon repolarisation to -30 mV. 40 pA calibration refers to (a) and (b), 100 pA refers to (c) and (d). B: sympathetic ganglion B cell. Currents evoked by a hyperpolarising voltage command to -110 mV from a holding potential of -30 inV. a: 1 min after establishing whole cell recording. Note interruption of I M activation by a rapid, transient, outward I A. b-d: 12, 23 and 47 min after establishing whole-cell recording. 200 pA calibration refers to all records. C: parasympathetic neurone. Currents evoked by hyperpolarising voltage command to -120 mV from a holding potential of -30 mV. Small, transient inward currents result from brief, hyperpolarising 10 mV commands elicited during the main clamp paradigm. The voltage commands produce larger currents during the activation of ISA, indicating an increase in conductance.

238 A) 20 mM K*

TABLE I

Comparison of lsA with 1m in amphibian sympathetic neurones ,4 a

-30~ mV-90 ~ 1~

Effect of 2 mM 4-AP Effect of muscarine

50 .~wt~_..t,_:_

-''~" "~-:-~--'-,~,,: - . - ~

_;_::~,,.

unaffected antagonized unaffected enhanced in sympathetics, reduced in parasympathetics En EK -60 mV

-60 mV

50 ms

3.58-+0.35 s (n= 19) 4.04-+0.73 s (n=6) c (for B cells) 4.39+0.62 s (n=8) d 100 mV

Voltage for half inactivation -110 mV Time constant for removal of inactivatione 150 ms

B) 60 mM K*

mV-80

Reversal potential Theshold voltage for activation Decay time constant of tail current at -30 mV

= ~ 1 = -70

0

~

l,r-W=I~

250 ] 2s

Fig. 2. Reversal potential for lSA. Inactivation was removed by stepping to -90 mV (A) or -80 mV (B) and ISA activated by returning to the holding potential of -30 mV. The potential then was stepped to a series of values during the resulting IsA transients. A: experiment carried out in 20 mM K÷. B: experiment carried out in 60 mM K ÷. The extrapolated reversal potentials in both cases, -43 mV (in 20 mM K÷) and -14 mV (in 60 mM K÷) correspond to the estimated values of E K for these two different K + concentrations. Experiments were carried out on two different sympathetic ganglion C cells. Records presented are X - Y plots of digitally stored data. followed by activation and rapid inactivation at - 3 0 mV 1. Fig. 1B,b,c shows the progressive development of ISA as recording is continued. The faster 1A transients persist during the development of ISA, showing that the two currents are i n d e p e n d e n t of each other. Fig. 1C shows recordings of ISA from a parasympathetic n e u r o n e . In this experiment, small, brief hyperpolarising voltage commands were superimposed on the usual voltage c o m m a n d paradigm to measure conductance changes. Activation of ISA is clearly associated with an increase in conductance. In a n o t h e r series of experiments, the m e m b r a n e was clamped to a series of different post-pulse potentials following activation of ISA at - 3 0 mV. Since the current reversed polarity at E K in all 4 cells tested, ISA results from an increase in K ÷ conductance (Fig. 2).

]SA b

-

214 + 36 ms (n = 2)

a Data from Adams et al. ~ obtained with two-electrode voltageclamp of bullfrog sympathetic B neurones. b Unless otherwise stated, data from patch-clamped Rana pipiens sympathetic C neurones. c In some C cells, the inactivation was best fit by two time constants. A two-exponential fit was deemed appropriate when the inclusion of a second time constant in the fitting programme reduced the standard error of the fit by 50% or more. The slower time constant corresponded well with the single time constant seen in other cells. The ratio of fast to slow components for the extrapolated zero-time current was 0.51 + 0.2 (n = 6) and the corresponding ratio of the areas under the two exponential curves was 0.13 + 0.05, i.e. the current tail was mainly composed of the slow component. In 2 out of 3 parasympathetic neurones studied, lSA was best fit by two exponentials. The time constant of the first component was 0.27 + 0.014 s and that of the second component was 1.75 + 0.59 s (n = 2). a The decay time constant was also measured for some B cells and was always best described by a single exponential. e See Materials and Methods section.

ISA kinetics The activation and inactivation of/SA were studied in sympathetic ganglion C cells so as to avoid contamination of the current by I A. Also, I M progressively declined during the time course of experiments and although the currents accompanying I M activation at - 3 0 m V were very small compared to ISA, accurate m e a s u r e m e n t s were best obtained after I M had abated. As with the classical /A 1'5'7'17'20'25, longer hyperpolarising c o m m a n d pulses produced larger ISA tails as inactivation was progressively removed at hyperpolarised potentials. The time constant for the removal of inactivation was determined by using a series of c o m m a n d pulses to - 1 1 0 mV from the holding potential of - 3 0 mV. The duration of the c o m m a n d pulse varied from 30 ms to 20 s and the magnitude of the ISA transient seen upon repolarisation to - 3 0 m V normalised to the magnitude of the maximal tail current produced by a long (12 s) c o m m a n d pulse (i.e. [I12max--I(o]/[I12max] × 100%). The time constant which as calculated by plotting log normalised current vs c o m m a n d pulse duration 1'7 was of the same order as that previously published for the I A of bullfrog sympathetic ganglia I (Table I).

239 C 3S~100 pA

a) ~ . ~ _ o l 2.0

- 0.5

A

I

mV I -110

3omv .ssmv Ico~

1.5

-30--

0.4 0.3

Iv/l(.~oo)

I(nA)

1.0 0.2 0.5.

0.0 . . . . . -150 -130

c) \

0.1 • -110

. . . . . -90 -70

• -50

~

, -30

Recovery

lO ]aM M'~'~usc

60--'J:'4n

0.0

v (mv)

B -30- -

B

Al___ ,oo 800 ms

Fig, 3. A: activation and inactivation curves for lsA. The activation curve obtained by measuring absolute currents evoked by terminating a 400 ms hyperpolarising command to -100 mV and stepping to a range of postpulse potentials as shown in the inset. Curve for (the removal of) inactivation obtained by stepping from a series of hyperpolarised potentials and measuring the currents flowing upon repolarisation to -30 mV (see inset). Currents were normalised to the current produced from the -100 mV command pulse. Note the small overlap between this curve and the activation curve. Data obtained from a sympathetic ganglion C cell following the abatement of IM. B: kinetics of IsA. Data from sympathetic ganglion C cell. In this cell, a computer-generated line derived from a single exponential accurately described the digitalised data record obtained using pCLAMP software (~ = 2.7 s). In some cases, the data were best fit by two exponentials (see Table I).

The activation and inactivation curves for/SA (Fig. 3A) also showed a striking similarity to those reported for IA ] in a m p h i b i a n sympathetic n e u r o n e s (Table I). IsA only activates at potentials more positive than - 6 0 mV. The current displays a strong voltage dependence, more than doubling in intensity over the range of - 4 0 to - 3 0 mV. Inactivation was r e m o v e d by a 1 s voltage c o m m a n d to potentials more negative than - 7 0 m V with half-inactivation at about - 1 0 0 mV. This is somewhat more negative than the voltage for half-inactivation of the classical I A in other n e u r o n a l types 5'6'17A8. Since the activation and inactivation curves showed little overlap (Fig. 3A), ISA is inactivated at all potentials in its activation range. Thus ISA, like IA, does not contribute to steady-state memb r a n e current. The main differences between the two currents were that ISA was about 80× slower in decaying than I A (Fig. 3B) and in 6 out of 25 C cells, this slow inactivation involved two time constants (Table I).

Pharmacology of ISA Fig. 4A illustrates the inhibition of ISA by 2 m M 4-AP.

---- mV

-30

-110~J

my [ -110

I

~

'i 2so]au6~,.

0.4 nA

20S

Fig. 4. Effects of drugs on ISA. A: effect of 2 mM 4-AP, a: control current response of a sympathetic ganglion C cell to a voltage command from the holding potential of -30 to -110 inV. b: same response recorded in the presence of 2 mM 4-AP. Note attenuation of peak amplitude of/SA and biphasic decline of current, c: recovery

of lSA following 3 min 15 s washout of 4-AP. B: lack of effect of 250 /~M Cd2+ on ISA evoked in a sympathetic ganglion C cell following a voltage command from -110 to -30 inV. C: induction of lsg by muscarine in a sympathetic ganglion B cell. Initial voltage command from a holding potential of -30 to -110 mV produces an inward current followed by a very small, transient ISA' Application of 10 /~M muscarine produces an increase in steady-state inward current as a result of IM suppression, but also significantly increases the amplitude of ISA' Upon the removal of muscarine, the steady-state current recovers to its control levels and lsA becomes less prominent. Traces from rectilinear pen recorder.

Note attenuation of the peak current amplitude, initial rapid decline followed by a second phase of very slow decay of the current in the presence of the blocker. Prior to treatment with the aminopyridine, the inactivation of

TABLE II Effects of muscarine on Jsm in amphibian autonomic neurones n

Sympathetic B cells Sympathetic C ceils All sympathetic neurones Parasympathetic neurones

39 37 76 25

No effect Increased Decreased 6 8 14 5

lsA

I~A

32 23 55a 10c

1 6 7b 10d

" Muscarine induced ISAin 13 of these 55 cells where the current was previously absent (i.e. infinite potentiation). In the remaining cells, ls,~ was potentiated by 203.6 + 11.8% (n = 42). There was no obvious difference in the ability of muscarine to induce ISA in B cells compared with C cells. b Muscarine reduced lsA to 79.0 + 3.1% of control (n = 7). Muscarine only induced in /SA in one cell and potentiated the current by 136.5 + 6.6% in 9 others. d Muscarine reduced/SA to 41.9 + 7.7% of control (n = 10).

240

A

Musc

B

Control

\_

!

.~ .

.~ .

--0.5s Jj200pA

IM Fig. 5. Suppression of ISA by muscarine in a parasympathetic neurone. A: cell was held at -30 mV and a series of voltage commands to -110, -90, -70, -50 and -10 mV applied, before, during and after the application of 10/~M muscarine (voltage command record omitted for clarity). The commands to the more negative potentials were followed by ISA transients. Muscarine produced an inward current associated with decreased conductance. ISA transients were reversibly suppressed in the presence of muscarine. B: current response to the -50 mV command shown on a faster sweep speed. The instantaneous current response results from current through open M channels and 'leak' channels and the slow inward relaxations result from IM deactivation. In the presence of muscarine, the I M relaxations are unaffected whilst the instantaneous conductance is decreased. This suggests that muscarine produced an inward current in this parasympathetic neurone by decreasing instantaneous leak conductance. Records in (A) from rectilinear pen recorder, records in (B) from X - Y plots of digitally stored data.

TM

B

A

-30

JI

'1

-5o mV -110

Ba2~/Musc . ~ ] 200 pA 0.2s

i i

i,

BaZ+

__1200 i

t

Musc Ba2*

pA

60 s

Fig. 6. Ba 2+ fails to induce ISA" Current responses evoked in a sympathetic ganglion B cell following voltage commands to -110 and -50 mV from a holding potential of -30 mV. Commands were applied before and during the application of drugs (recovery records omitted for clarity). A: Ba 2+ (5 mM) produces inward current as a result of 1M suppression yet fails to induce ISA transients. The insets show the current responses to the -50 and -110 mV commands. Note suppression of the 1M relaxations by Ba 2+. B: application of a mixture of 5 mM Ba 2÷ and 10/zM muscarine to the same cell. An inward current results from lr~ suppression and is accompanied by inhibition of I M relaxations in the records illustrated in the insets. The small, transient ISA which follows the command to -110 mV is almost doubled in amplitude following the application of the Ba2+/muscarine mixture. Records in (A) and (B) from rectilinear pen recorder, insets are X - Y plots of digitally stored data. 200 pA, 60 s calibration refers to records in (A) and (B), 200 pA, 0.2 s calibration refers to insets.

241 ISA in this C cell was described by a single time constant (7.7 s) but in the presence of 4-AP, the response was bi-exponential exhibiting r values of 0.8 and 13.2 s. ISA was independent of extracellular Ca 2+ since it was not affected by external solution containing 2 mM Mg2+/0 Ca 2÷ or 0.25 mM Cd 2+ (Fig. 4B). There was also less than 10% depression of ISA by the K ÷ channel blockers, Ba 2+ (2-5 raM) 2 or D-tubocurarine (70 tiM) 13. Similarly, tetraethylammonium 16'2° (3 mM) usually had little effect on ISA although in 2 cells, a significant depression was observed. One possible explanation for this could be that the ISA in these cells was contaminated by I c. This current could have been activated by Ca 2+ influx following the step depolarisation from -110 to -30 mV. Alternatively, the current we are describing as ISA could simply reflect activation of I K at a slightly more negative potential than has previously been reported 1'13"16 (see Discussion). The development of ISA after the establishment of whole-cell patch recording conditions suggests that some intracellular factor may prevent the expression of the current in 'freshly patched' cells or in cells studied with conventional microelectrodes 1. In an attempt to replenish such factors, we included ATP, GTP, cyclic AMP, inositol 1,4,5-trisphosphate or protein kinase C inhibitors in the patch pipette, but were unable to prevent the development of ISA" The current was also unaffected by the extracellular application of phorboi esters. Despite this, it is unlikely that ISA is an artifact produced by the wash-out of cytoplasmic components because muscarine reversibly induced or enhanced ISA in sympathetic B and C neurones, even immediately after the establishment of whole-cell recording conditions. This effect was seen in 55 out of 76 cells. When ISA had developed prior to the addition of muscarine, the current was usually potentiated by about 200% (Table II). In the experiment illustrated in Fig. 4C, which was carried out on a sympathetic ganglion B cell, a voltage command from the holding potential o f - 3 0 to -110 mV is initially followed by only a small, transient ISA. In the presence of 10/~M muscarine however, a steady-state inward current develops as a result of Ira suppression 2'3 and ISA is clearly enhanced during the action of the drug. ISA abates once muscarine is removed. Although muscarine also potentiated ISA in 10 Out of 25 parasympathetic neurones, the reverse effect (i.e. inhibition of the current) was seen in another 10 neurones (Table II). A typical experiment is illustrated in Fig. 5A. Hyperpolarising voltage commands to -110, -90 and -70 mV are followed by ISA transients upon return to the holding potential of -30 mV. Application of 10 /~M muscarine produces a steady-state inward current (due to a decrease in leak conductance, see ref. 22 and legend to

Fig. 5B) during which the amplitudes of ISA transients are reduced. Although muscarine promotes the appearence of ISA in both the B and C cells of sympathetic ganglia, it might be argued that the effect in the first case is a consequence of changes in space clamp resulting from the increase in membrane resistance following inhibition of IM. This could result in better voltage control so that the potential at the membrane approached that required for activation of I K, i.e. the novel current we are refering to as ISA could simply be I K. We therefore examined whether ISA could be evoked when I M was inhibited using Ba 2÷ (5 mM) e. Ba 2÷ failed to induce ISA in 9 tests on 5 B cells. Furthermore, when 10 gM muscarine was applied in the presence of Ba 2÷, ISA was induced in 6 out of 9 experiments on 4 B cells tested. In the typical experiment illustrated in Fig. 6A, Ba 2÷ produces an inward current at -30 mV as a result of I M suppression. Voltage commands to -110 and -50 mV were applied before and during the application of Ba 2+ and the currents resulting from the steps are illustrated in the insets. Although the Ira relaxations recorded at -50 mV are suppressed by Ba 2÷, there is no trace of ISA in either the steady-state record or in the insets. When a mixture of 10 /~m muscarine and 5 mM Ba 2÷ were applied to the same cell (Fig. 6B), Ira relaxations were suppressed and the small ISA which had started to develop spontaneously, was enhanced by about 200%. DISCUSSION Adams et al. 1 have shown that IK, (the delayed rectifier K ÷ current) in bullfrog sympathetic neurones inactivates with a time course similar to ISA and more recent work has shown that I K is aminopyridine sensitive 11. Although I K is assumed to only activate at potentials positive to -25 mV 1'16, it is possible that our voltage commands to -30 mV have caused the activation of I K. This is especially likely if internal perfusion of the cells with the solution from the patch-pipette causes a shift in the IK activation curve to more negative potentials as has been reported for both Na ÷ currents 11 and for certain K ÷ currents such as IM21. One observation which suggests that ISA and I K are separate currents is that ISA is potentiated by muscarine in Rana pipiens B and C cells whereas, at least in bullfrog B neurones at potentials negative to -20 mV, I K is probably muscarine insensitive 2. Despite this, it is difficult to unequivocally distinguish IK from ISA. This is because, in their original work on K ÷ channels in bullfrog ganglia, Adams and his coworkers 2 noted that muscarinic agonists increased the outward current seen at -20 to -10 mV when the holding potential was stepped from -50 mV. There are two

242 interpretations of this result, either I K t~ potentiated by muscarine, in which case there would be no basis for distinction between ISA and I K, or the voltage protocol used by Adams and his coworkers was effective in activating both IK and ISA and the increase in outward current which they reported 2 resulted from ISA potentiation as described in the present paper. This latter possibility is feasible because the activation curve for ISA is strongly voltage dependent (Fig. 3A) such that the amount of activation seen at -10 to -20 mV could overcome the steady-state inactivation established by holding at -50 mV. Since these two possibilities are presently indistinguishable, it is not possible to clearly distinguish ISA from IK. Even the results with T E A are difficult to interpret because the weak blocking effect (cf ref. 2) seen in some cells could imply that ISA is contaminiated with, or is identical to, IK. As mentioned above, the effect of 4-AP also cannot be used to distinguish between ISA and IK12. A related question is whether ISA potentiation by muscarine (in B cells) simply results from more effective space-clamp following I M suppression such that the voltage at the cell membrane is moved into the range for IK activation. This argument is countered by the fact that Ba 2+, which also suppresses IM2, failed to induce ISAFurthermore, muscarine could still induce /SA when I M was blocked with Ba 2+. It might also be argued that the muscarine-induced ISA (in B cells) results from the voltage-dependent removal of muscarine-induced I M block during the hyperpolarising voltage command so that the ISA transient results from the re-blocking of I M by muscarine when the potential is returned to -30 mV at the end of the command. This is unlikely because muscarine also induced ISA in sympathetic C cells where I M is insensitive to muscarine 14'21. The sensitivity of/SA to 4-AP altered the time course of decay of current at -30 mV from a single exponential to a double exponential. The appearance of a fast r value is not due to the unmasking of an I A transient because the experiments were carried out on C cells which lack I A. The initial attenuation of the peak ISA and the initial rapid rate of decay of the current seen in the presence of 4-AP (Fig. 4A) probably resulted from open-channel block as suggested for blockade of I A in molluscan REFERENCES 1 Adams, P.R., Brown, D.A. and Constanti, A., M-currents and other potassium currents in bullfrog sympathetic neurones, J. Physiol. (Lond.), 330 (1982) 537-572. 2 Adams, P.R., Brown, D.A. and Constanti, A., Pharmacological inhibition of the M-current, J. PhysioL (Lond.), 332 (1982) 223-262. 3 Adams, P.R., Jones, S.W., Pennefather, P.S., Brown, D.A.,

neurones 27. The subsequent slow decay of ISA may represent another component of aminopyridine block which increases in strength during hyperpolarisation. The slow decay of current reflects the slow unblocking of the channels at -30 mV. Again this type of block has been shown to contribute to the effect of aminopyridines on IA in molluscan neurones 27. Other currents with characteristics similar to /SA have been recorded in nodose ganglion neurones 2° and hippocampal neurones 24'28. These slow currents are sensitive to high micromolar concentrations of 4-AP; 500 nM almost completely blocked the current described by Weight and Zbicz 2s whereas the present ISA was reduced by less than 50% by 2 mM 4-AP. The current recorded in hippocampus 24 has been shown to have profound effects on the encoding properties of these neurones whereas the role of/SA in governing repetitive discharge in autonomic neurones is more difficult to define. Since ISA is not normally detected in 'healthy' microelectrodevoltage-clamped frog sympathetic neurones 1, it is unlikely that this current plays any role in determining the repetitive discharge characteristics of these neurones under normal conditions. Indeed, it could be argued that the appearence of /SA is an artifact of the 'wash-out phenomenon' which occurs during whole-cell patchclamp recording. Despite this, the important observation in the present work is that, even in freshly patched cells, the current is reversibly affected by muscarinic agonists. In most autonomic cells, the current is potentiated, but occasionally inhibition is observed (Table II). Whether or not the slow, aminopyridine-sensitive, transient outward current induced at -30 mV following a step from negative potentials simply represents activation of IK, or the activation of a separate current which we have termed ISA, effects of muscarinic agonists on slow, transient outward currents must be considered when attempting to explain cholinergic mechanisms in autonomic ganglia (see ref. 2).

Acknowledgements. Supported by MRC (Canada) and the Alberta Heritage Foundation for Medical Research (AHFMR). A.A.S. was an AHFMR visiting scientist, P.A.S. is an AHFMR scholar and J.A.Z. is in receipt of an MRC studentship. We thank Drs. S. Ikeda and G. Schofield for advice on experimental procedures, Dr. P.S. Pennefather for his comments on the manuscript and H. Chen and B. Jassar for their help with some of the experiments. Koch, C. and Lancaster, B., Slow synaptic transmission in frog sympathetic ganglia, J. Exp. Biol., 124 (1986) 259-285. 4 Aghajanian, G.K., Modulation of a transient outward current in serotonergic neurones by al-adrenoceptor , Nature (Lond.), 315 (1985) 501-503. 5 Beiluzzi, O., Sacchi, O. and Wanke, E., A fast transient outward current in the rat sympathetic neurone studied under voltage clamp conditions, J. Physiol. (Lond.), 358 (1985) 91-108.

243 6 Cassell, J.E and McLachlan, E.M., Muscarinic agonists block five different potassium conductances in guinea-pig sympathetic neurones, Br. J. Pharmacol., 91 (1987) 259-261. 7 Connor, J.A. and Stevens, C.E, Voltage clamp studies of a transient outward membrane current in gastropod neural somata, J. Physiol. (Lond)., 213 (1971) 21-30. 8 Connor, J.A. and Stevens, C.E, Prediction of repetitive firing behaviour from voltage clamp data on an isolated neurone soma, J. Physiol. (Lond.), 213 (1971) 31-53. 9 Dodd, J. and Horn, J.E, A reclassification of B- and C-neurons in the ninth and tenth paravertebral sympathetic ganglion of the bullfrog, J. Physiol. (Lond.), 334 (1983) 255-269. 10 Dodd, J. and Horn, J.E, Muscarinic inhibition of sympathetic C-cells in the bullfrog, J. Physiol. (Lond.), 334 (1983) 255-269. 11 Fernandez, J.M., Fox, A.E and Krasne, S., Membrane patches and whole cell membrane: a comparison of electrical properties in rat clonal pituitary (GH3) cells, J. Physiol. (Lond.), 356, (1984) 565-585 12 Goh, J.W., Kelly, M.E.M. and Pennefather, ES., Electrophysiological function of the delayed rectifier (IK) in bullfrog sympathetic ganglion neurones, Pflagers Arch., 413 (1989) 482-486. 13 Goh, J.W. and Pennefather, ES., Pharmacological and physiological properties of the after-hyperpolarization current of bullfrog ganglion neurones, J. Physiol. (Lond.), 394 (1987) 315-330. 14 Jones, S.W., A muscarine-resistant M-current in C-cells of bullfrog sympathetic ganglion neurones, Neurosci. Left., 74 (1987) 309-314. 15 Krishtal, O.A. and Pidoplitchko, V.I., A receptor for protons in the nerve cell membrane, Neuroscience, 5 (1980) 2325-2327. 16 Lancaster, B. and Pennefather, E S., Potassium currents evoked by brief depolarizations in bull-frog sympathetic ganglion cells, J. Physiol. (Lond.), 387 (1987) 519-548.

17 Neher, E., Two fast transient current components during voltage clamp on snail neurones, J. Gen. Physiol., 58 (1971) 36-53. 18 Numann, R.E., Wadman, W.J. and Wong, R.K.S., Outward currents of single hippocampal cells obtained from the adult guinea pig, J. Physiol. (Lond.), 393 (1987) 331-353. 19 Pfaffinger, P., Muscarine and t-LHRH suppress M-current by activating an IAP-insensitive G-protein, J. Neurosci., 8 (1988) 3343-3353. 20 Rudy, B., Diversity and ubiquity of K+ channels, Neuroscience, 25 (1988) 729-749. 21 Selyanko, A.A., Smith, P.A. and Zidichouski, J.A., Effects of muscarine and adrenaline on neurones from Rana Pipiens sympathetic ganglia, J. Physiol. (Lond.), in press. 22 Selyanko, A.A., Zidichouski, J.A. and Smith, P.A., M-currents in patch-clamped frog parasympathetic neurones: effects of muscarine and adrenaline, Soc. Neurosci. Abstr., 15 (1989) 527. 23 Stansfeld, C.E., Marsh, S.J., Halliwell, D.V. and Brown, D.A., 4-Aminopyridine and dendrotoxin induce repetitive firing in rat visceral sensory neurones by blocking a slowly inactivating outward current, Neurosci. Left., 64 (1986) 299-304. 24 Storm, J.E, Temporal integration by a slowly inactivating K÷ current in hippocampal neurons, Nature (Lond.), 336 (1988) 379-381. 25 Taxi, J., Morphology of the autonomic nervous system. In R. Llinas and W. Precht (Eds.), Frog Neurobiology: A Handbook, Springer, Berlin, 1976, pp. 93-150. 26 Thompson, S.H., Three pharmacologically distinct potassium conductances in molluscan neurones, J. Physiol. (Lond.), 265 (1977) 465-488. 27 Thompson, S.H., Aminopyridine block of transient potassium current, J. Gen. Physiol., 80 (1982) 1-18. 28 Weight, EE and Zbicz, K.L., Transient voltage and calciumdependent outward currents in hippocampal CA3 pyramidal neurones, J. Neurophysiol., 53 (1985) 1038-1058.