Calcium of A-currents in perfused Aplysia neurons

Brain Research, 346 (1985) 294-300

294

Elsevier BRE 11102

Calcium Dependence of A-Currents in Perfused Aplysia Neurons DOUGLAS JUNGE

School of Dentistry and Department of Physiology and Brain Research Institute, University of California, Los Angeles, CA 90024 (U.S.A.) (Accepted January 21st, 1985)

Key words: Aplysia neuron - - transient outward current - - A-current - - potassium current - - calcium - - cobalt

Transient outward currents were studied in neurons in the visceral ganglion of Aplysia californica, using intracetlular perfusion and voltage-clamp techniques. The early outward currents in response to depolarizations from holding potentials near -90 mV were activated in the range -60 to -20 mV, below the threshold for the delayedoutward current. Resting inactivation of the early outward currents was removed by prehyperpolarizations in the range -130 to -70 mV. A-currents produced in this manner were blocked by external application of CoC12 and augmented by increasing external Ca-concentration. They were also blocked by treatment with 4-aminopyridine. The currents were reduced by treatment with verapamil hydrochloride, further suggesting a role for calcium in the currentgenerating mechanism. A model with a fourth-power activation process and first-power inactivation process could fit the early outward currents reasonably well. The effect of application of Ca-free, cobalt-containing solution was modeled as a decrease in peak conductance and an increase in the time constants of activation and inactivation.

INTRODUCTION

potentials near the potassium equilibrium potential 9A4,18,30,33,34. Thus, it is usually listed among potas-

The transient outward current which occurs in voltage-clamped neurons following a conditioning hyperpolarization and return to a normally subthreshold potential was first reported by Hagiwara et a1.17

sium currents 2. This current is blocked by external application of 4-aminopyridine (4-AP) at concentrations which do not affect the delayed rectifier, or classical outward current mechanism16.18,33.34. Be-

in Onchidium neurons. It was then studied by Gola and Romey 14 and Neher30 in Helix, and was first

cause of this pharmacological difference and the more negative threshold potential for activation, the

called IA by C o n n o r and Stevens 9, who found it in

A-currents are thought to flow through a different set of channels than the delayed rectifier currents. The question of sensitivity of A-currents to external calcium concentration has received limited attention in the literature. Thompson34 showed that these currents were reduced 40% by external application of 30 mM cobaltous ions, which block inward calcium currents. He felt that this result was due to a shift of the steady-state activation and inactivation parameters in the positive direction along the voltage axis, just as these parameters are shifted for the delayed rectifier current by Ca 2+ ions 12. Connor7 found that A-currents were present in E G T A - i n j e c t e d cell bodies, indicating that they were i n d e p e n d e n t of the in-

Anisodoris. This current was finally n a m e d A-current by Thompson34, in a study of its pharmacological properties in Tritonia neurons. Thus, the current is found widely in molluscan nerve cell bodies; it has also been seen in arthropod axons 6 and cell bodies 31, rat sympathetic n e u r o n s 13, rat sensory n e u r o n s z4, hippocampal slices16, cultured hippocampal and spinal neurons33 and in coelenterate eggs 18. It has been suggested that this current acts to slow the rate of depolarization in repetitively firing neurons, to permit firing at very slow rates ( < l/s) 8. In every case where it has been studied with twostep voltage commands, the A-current reverses at

Correspondence: D. Junge, School of Dentistry and Department of Physiology, University of California, Los Angeles, CA 90024. U.S.A. 0006-8993/85/$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)

295 ternal Ca-concentration. MacDermott and Weight 26 have described a calcium-sensitive transient outward current in bullfrog sympathetic neurons, and Premack et al. 31 have also seen one in barnacle motor neurons. The results in the present report suggest that A-currents in perfused Aplysia neurons are dependent in part on Ca-ions entering from the external solution. MATERIALS AND METHODS Adult sea hares 15-20 cm in length were relaxed by injecting 50 ml of 0.5 M MgCI2 into the body cavity and waiting about 15 min. The visceral ganglion was then removed and pinned in the 0.5 ml recording chamber. A solution of 1% pronase (B-grade, Calbiochem) in normal saline was applied to the ganglion for 20 min at room temperature to soften the connective tissue capsule. A pair of iris scissors was used to cut around the circumference of the ganglion, and the top of the capsule was removed. The exposed neuron cell bodies were then treated with 0.2% trypsin (bovine, Calbiochem) for 15 rain at room temperature, a necessary step to ensure good adhesion of the cell to the perfusion pipette. The cells studied had diameters of 100-200 #m, and most were located in the caudal region of the ganglion, near the midline. The perfusion and voltage-clamping technique was essentially that of Lee et al. 25, which is a modification of the method of Kostyuk et al. 23. The cell body was held onto the tip of the pipette (diameter 25 Hm) by negative pressure, and a stream of internal solution was directed against the patch of membrane inside the tip. When the membrane inside the pipette broke, the intracellular salt concentrations could be altered, and a low access resistance was provided between the shaft of the pipette and the cell interior. Potentials were recorded either with a calomel cell in the pipette or a microelectrode in the cell body. The external reference electrode was a calomel half-cell. Series resistance compensation was effected by adding a small amount of the measured membrane current to the voltage command signal. Because of the low resistance of the internal and external solutions used with Aplysia cells, it was possible to adjust the compensation so that potentials recorded with the microelectrode were within 2% of those in the pipette shaft (equal to the command potential). Cur-

rents were measured with a feedback circuit between the bath and circuit ground, and were passed through a 1 kHz low-pass filter before being recorded. The current and voltage traces were digitized using a Northwest Systems Model 85a Scope, and stored in binary form on diskettes. The method of compensation for leak currents is shown in Fig. 1. The top trace is the total outward current measured upon depolarizing from a holding potential o f - 9 0 to -22 mV. The bottom trace is the leak current seen upon stepping from -90 to -158 mV, an equal and opposite step to that above. The center trace is the net active current, obtained by digital addition of the first two traces. Little inward rectification, or increase in conductance at more negative potentials, was seen in the range -60 to -160 mV. The soh~tions used in these experiments had the composition shown in Table I. The internal solution was prepared by adding DL-aspartic acid, HEPES and E G T A to water, and titrating with 1 N KOH. 4Aminopyridine and verapamil hydrochloride were added directly to normal saline. All experiments were done at 13-15 °C, which improved cell longevity over that seen at room temperature. RESULTS An example of an early outward current seen after depolarizing from a holding potential o f - 9 0 to -21 mV is shown in the inset of Fig. 2. The peak current, Ip, is

.\

2O ?

nA

I

.2s

Fig. 1. Digital method of leak compensation. Top trace: current measured upon depolarizing from -90 to -22 inV. Bottom trace: leak current upon hyperpolarizingfrom -90 to -158 mV. Middle trace: sum of top and bottom traces, or active outward current.

296 TABLE 1 Composition o f external and internal solutions

Concentrations shown in raM. Asp, aspartate+ Solution

NaCI

KCI

K-Asp

CaCI2

CoCI2

MgCI2

Tris.HCl

HEPES

EGTA

pH

Normal ext. High-Ca Cobalt Internal

450 450 450 -

10 10 10 -

500

11 30 .

-

49 30 30

10 10 10

-

-

5

3

7.7 7.7 7.7 7.3

30 .

.

plotted vs c o m m a n d potential, E, in the graph. In these cells, usually no early inward current was seen with c o m m a n d s b e l o w - 6 0 mV, and above - 2 0 m V the delayed rectifier currents, lasting several seconds, began to overlap the early current. H e n c e , in this study the range of c o m m a n d voltages was kept more negative than - 2 0 mV. Similar linear c u r r e n t voltage relations for activation of this current have been seen in other preparations9, 30. The effect of application of 30 m M cobaltous ions in the external solution is shown in Fig. 3. The inset shows current and voltage traces in normal, Co-containing (and Ca-free), and n o r m a l solutions. In the graph the p e a k outward current, Ip, is plotted vs the conditioning potential, E c. This illustrates the point that inactivation of A-currents was r e m o v e d by hyperpolarizations down t o - 130 m V and below. Application of 30 m M cobalt in Ca-free solution reduced

.

the p e a k outward current almost 50% at all potentials tested. The blockage of p e a k transient current in the different cells tested varied from 30 to almost 100%. This suggests that the A-currents are calciumd e p e n d e n t , since cobalt is known to interfere with Ca-entry in a variety of excitable m e m b r a n e s 32. It was next decided to increase the external calcium concentration, and d e t e r m i n e the effects on outward currents; the result is shown in Fig. 4. In the insets, recorded with conditioning c o m m a n d s of about

NS

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-°--100/ mVl__ :is

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• NS o Ca0 Co30 ~'~ N S

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-11o

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40

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Ec , m V

-60

-50

-40

-30

-20

-10

E, mV Fig. 2. Current-voltage relation for A-currents. lnset shows sample current (upper) and voltage (lower) traces. Graph shows peak currents (Ip) obtained upon depolarizing from -90 mV to the potentials plotted as E.

Fig. 3. Effect of cobalt applied in Ca-free solution. Insets show current (upper) and voltage (lower) records when voltage was stepped from -30 mV to more hyperpolarized levels and back again. (Current during hyperpolarizing steps not shown.) Records obtained in normal saline (NS), Co 30 mM Ca-free solution and in NS afterwards. Graph shows peak currents recorded upon stepping from conditioning levels, Ec, to -30 mV, in normal (solid line) and cobalt-containing solution (dashed line).

297 NS

Ca30

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o

a~ " ~ ' ~ A ,,,,,~

4

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2

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Ec , m Y

Fig. 4. Effect of increasing external Ca-concentration. Insets show current (upper) and voltage (lower) records when voltage was stepped from -30 mV to more hyperpolarized levels and back again. (Currents during hyperpolarizing steps not shown.) Records obtained in normal saline (NS), Ca 30 mM, and NS afterwards. Graph shows peak currents recorded upon stepping from conditioning levels, Ec, to -30 mV, in normal (solid line) and high-Ca solution (dashed line).

-100 mV, increasing Ca-concentration about 3-fold did increase the amplitude of the A-currents. As shown in the graph, this effect was potential-dependent, a smaller or opposite effect being seen at potentials more positive than - 8 0 mV. The reason for this complex behavior is not known. However, over most of the range of potentials tested, the A-current amplitude increased with increasing external Ca-concentration. In studying the effects of calcium ions and calcium blockers on these outward currents, it was important to exclude the possible confounding effects on known Ca-sensitive outward currents, or C-currents28,34. However, this current rises much more slowly than the A-current, and is only activated at potentials above 0 mW 11,20,34. Thus, it should have contributed little if any to the currents measured in the present study. The reversal potential for the A-current was measured using a double-pulse experiment (not shown). First, the potential was stepped from the holding lev-

el o f - 9 0 mV to + 2 m V for 0.15 s; this activated the transient outward current. Then the potential was stepped to a second level between - 1 0 0 and - 2 3 mV, and the current jumped to a new level and either increased with time (at more negative potentials) or decreased with time (at more positive potentials). At a test potential of about - 9 0 mV, the outward current did not change with time following the second step; this was taken as the reversal potential. (In another cell similarly tested, the reversal potential for the Acurrent was - 8 5 mV.) This was fairly close to the theoretical potassium equilibrium potential o f - 1 0 0 mV for the internal and external solutions used. It was possible that the internal K-concentration may have been less than that in the internal perfusion fluid, due to poor circulation (especially near the inner side of the cell membrane). The proximity of the reversal potential to the K-equilibrium potential is supportive evidence that the A-current is carried by potassium ions. T h o m p s o n 34 has shown that A-currents in Tritonia neurons are blocked by low-miUimolar concentrations of 4-aminopyridine, which is also known to block delayed outward currents in other preparations 29,35. The experiment shown in the top row of Fig. 5 shows that A-currents in Aplysia neurons are also sensitive to 4-AP. Two traces are shown in each part, obtained with slightly different c o m m a n d voltages. The effect of 4-AP on the currents was reversiNS

:. NS

_-----_

4-AP

. NS

CaO C o 3 0

_-

NS

_

-JNL .28

Fig. 5. Effects of 4-aminopyridine and cobalt-containing (Cafree) solutions. Top row: current (upper) and voltage (lower) traces recorded at two different potential steps from -90 mV. NS, normal saline. 4 mM 4-AP in external solution blocked Acurrent, which recovered in NS afterwards. Bottom row: currents recorded with 3 different potential steps from -90 inV. 30. mM cobalt ions in Ca-free solution blocked A-current, which recovered in NS afterwards.

298 ble. However, the drug m a d e control of the cell potential m o r e difficult, as seen by the active inward current in the third trace, and this was not reversible for at least 1 h. The b o t t o m part of Fig. 5 shows the effect of application of cobalt ions (in Ca-free solution) in a different cell from that of Fig. 3. The A - c u r r e n t was almost completely blocked in this case, and a recovery was seen upon washing the p r e p a r a t i o n with normal saline. Since the transient outward current in these neurons was evidently affected by entry of Ca-ions, it was also of interest to examine the effects of externally applied verapamil hydrochloride, which is known to block Ca-currents in other p r e p a r a tions3, 21,22. It has been o b s e r v e d with Helix neurons that 10 -5 to 10 -4 M verapamil increased the rate of inactivation of A-currents 22. The result of application of 10 -4 M verapamil to the perfused Aplysia neuron is shown in Fig. 6. The p e a k A - c u r r e n t was reduced to 78% of the control value, and recovered upon washing to 108% of control. No significant effect on the rate of inactivation was seen. R e d u c t i o n of p e a k A current to 85% of control was also seen in another cell, upon application of 10 -4 M verapamil. In o r d e r to describe the effects of calcium ions on the A-currents, an exponential model of activation and inactivation was used 9. The current, IA, was given by

NS

VERAP

\

NS

---

-lOOl mV[-.2s

Fig. 6. Effect of verapamil. Current (upper) and voltage (lower) records when voltage was stepped to levels near -140 mV and back to -40 mV. (Current during hyperpolarizing steps not shown.) NS, normal saline. 10-4 M verapamil reduced peak Acurrent, which recovered in NS afterwards. Fig. 7. In all current traces, the dots are the observed outward currents and the solid lines are fits of Eqn. 1 to the data. The time constants r A and r B were 0.008 and 0.17 s in normal (Ca 11 m M ) solution, respectively. W h e n a solution with no calcium and 30 m M cobalt was applied, the p e a k conductance fell (assuming no change in E o r E r e v ) , and the time constants increased to 0.15 and 0.23 s. W h e n the preparation was washed with normal saline the p e a k conductance increased, and the time constants became 0.008 and 0.31 s. If the effect of the cobalt solution was to prevent the normal entry of Ca-ions into the ceil, then this result suggests that calcium has at least two functions in connection with the A-currents: to make more channels available, and to increase the rates of activation and inactivation of channels. DISCUSSION

I A = K[1 - exp(-t/rA)]4exp(-t/rB)

(1)

where K is the scale factor, r A is the time constant of activation and r B is the time constant of inactivation. Note that K = ~ ( E - Erev)

(2)

where ~, is the p e a k conductance, E is the m e m b r a n e potential and Ere v is the reversal potential. Application of this m o d e l to A - c u r r e n t data was complicated in many cases by the presence of slowly rising currents which p r e v e n t e d the net outward current from returning to zero during a depolarizing test pulse. (Presumably this was due to activation of voltage-dep e n d e n t delayed rectifier currents.) In a few cases, however, the prolonged current was small enough that the A - c u r r e n t could be fitted by the model, as in

The principal finding in this r e p o r t is that transient outward currents in perfused Aplysia neurons are sensitive to extracellular calcium and Ca-blockers. Since this diminishes the neatness of categories into which outward currents can be placed, it is important to exclude the possibility that one has been studying C-current channels and not A - c u r r e n t channels.

CaOCo30

N ~

. . . . .

.

.

.

n

.

.

.

.

.

.

lOO| mVL-.2s

Fig. 7. Fits of model to A-currents recorded in normal saline (NS), in Co 30 raM, Ca-free, and in NS afterwards. Dots are observed outward currents and solid lines are fits of Eqn. 1 to data. See text for details.

299 First, as m e n t i o n e d in the Results, C-currents are app a r e n t l y not activated at potentials b e l o w 0 m V 15,2°,34. Second, all of the currents in the p r e s e n t r e p o r t reached their p e a k values in less than 50 ms, usually in 20 ms. C-currents increase m o r e or less linearly t h r o u g h o u t long depolarizing c o m m a n d s lasting up to 0.5 s 5,15,20,34. Finally, C-currents are not blocked by 4-aminopyridine at concentrations of 4 m M and belowl9, 34. P r o b a b l y some voltage-sensitive d e l a y e d rectifier current was activated by conditioning hyperpolarizations, as in Figs. 3 and 4, since this current is partially inactivated at the resting potential 9. This m a y have caused the non-zero final values of o u t w a r d current which complicated m o d e l i n g efforts. But it cannot account for the early peaks, as even inactivating delayed currents in Aplysia and dorid neurons do not peak before 200-300 msl, 4. The possibility that cobalt ions reduce A - c u r r e n t s by moving the conductance-voltage relation positively along the voltage axis was m e n t i o n e d by T h o m p son 34. T h e mechanism of this effect would presumably be screening of fixed negative charges on the cell m e m b r a n e , increasing the effective t r a n s m e m b r a n e electric field 27. O n e feature of this mechanism is that all divalent cations should be equally effective, since

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cation binding energy is irrelevanO0,27. H o w e v e r , in the present study, in high-calcium solutions with the same divalent concentration as the Co-containing solutions, the A - c u r r e n t was a u g m e n t e d over the amount in n o r m a l - C a , Co-free solutions. Thus, calcium ions themselves a p p e a r to be i m p o r t a n t in the production of early o u t w a r d currents, and their action is o p p o s e d by cobalt and verapamil, two calcium blockers. The effects of increased external Ca-concentration on the m o d e l p a r a m e t e r s (increased m a x i m u m conductance, decreased activation and inactivation time constants) indicated a complex action of the divalent ions. Decreasing the time constants could be m o d eled as lowering the energy barriers for entering and leaving the open state of the A - c u r r e n t channels. The increased m a x i m u m conductance might reflect mobilization of additional channels, p e r h a p s by uncovering of m e m b r a n e areas in infolded regions. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by U S P H S G r a n t RR05304. I thank M . E . Barish for reading the manuscript.

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300

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27

28

29

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31

32 33

34

35

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