BY LOU BYERLY AND SUSUMU HAGIWARA University of ...

J. Physiol. (1982), 322, pp. 503-528 503 

With 12 text-ftgures 

Printed in Great Britain 

CALCIUM CURRENTS IN INTERNALLY PERFUSED NERVE CELL 

BODIES OF LIMNEA STAGNALIS 

BY LOU BYERLY AND SUSUMU HAGIWARA 

From the Department of Biological Sciences, University of Southern California, 

Los Angeles, CA 90007, U.S.A. and the Department of Physiology, 

Ahmanson Laboratory-B.R.I., and Jerry Lewis Neuromuscular Research Center, 

University of California, Los Angeles, CA 90024, U.S.A. 

(Received 22 May 1981) 

SUMMARY 

1. When K+ is removed from both sides of the somal membrane of Limnea 

neurones, time-dependent, voltage-dependent outward currents are observed at 

positive potentials. These currents can be carried by Tris+ and tetraethylammonium 

(TEA+), as well as Cs+, but the Cs currents are several times larger. The Cs currents 

are not affected by external or internal TEA, but are strongly reduced by 4aminopyridine 

(4-AP) and all Ca blockers tried. 

2. The presence of these non-specific outward currents and their sensitivity to all 

treatments that eliminate the Ca currents prevent the complete isolation of Ca 

currents. The non-specific outward currents are most prominent at large positive 

potentials and as slow tail currents on stepping back to the holding potential. 

3. Ca currents are 'washed out' in well perfused cells. Typically the Ca current has 

decayed to less than one tenth of its original size after I h of perfusion. This wash-out 

is specific for the Ca current; Na and K currents persist for several hours. 

4. Once the Ca current has completely decayed, it is possible to study one type 

of non-specific current without overlapping inward currents. This current activates 

between 0 and + 30 mV and appears to reverse near 0 mV. 

5. In spite of the probable presence of slowly activating outward currents, the net 

inward currents measured show little apparent inactivation. In all the cells studied 

the inward current evoked at + 20 mV has never decayed by more than 50 % during 

a 60 ms pulse. So the true inactivation of these Ca currents must be quite slow, with 

time constants of the order of 100 ms and larger. 

6. The activation of the Ca current agrees with m2 kinetics. The rate of activation 

is the same for Ba currents as for Ca currents. 

7. When the membrane potential is stepped back to the holding level (-50 mV), 

the Ca current turns off quite rapidly with a time constant of about 100 /Ls (25 0C). 

The time constant for turning off the Ca current is not related to the time constant 

for turning on the Ca current at the same voltage as expected for m2 kinetics in the 

Hodgkin and Huxley model. At -30 mV the Tm for turn-on is eight times larger than 

the rm for turn-off. 

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504 

L. B YERL Y AND S. HAGIWARA 

INTRODUCTION 

Biophysical studies of voltage-dependent Ca currents have been impeded by the 

presence of overlapping K currents and the complicated geometries of the membranes 

involved. Invaginated membranes, attached axons and electrical coupling to other 

cells have prevented good control of the membrane potential. The overlapping K 

currents cannot be completely blocked by any pharmacological means and are often 

dependent on the Ca current, making complete isolation of the Ca current impossible. 

In the last few years new techniques have been developed which appear to largely 

overcome these difficulties for biophysical studies of Ca currents in the nerve cell 

bodies of snails. Krishtal & Pidoplichko (1975) first introduced techniques for voltage 

clamping and exchanging intracellular ions of isolated nerve cell bodies. The 

subsequent development and exploitation of this technique for studying Ca currents 

was reported in a series of papers (Kostyuk, Krishtal & Pidoplichko, 1975, 1977a, 

1981; Kostyuk, Krishtal & Shakhovalov, 1977b; Kostyuk & Krishtal, 1977; Doroshenko, 

Kostyuk & Tsyndrenko, 1978a, b, 1979; Doroshenko & Tsyndrenko, 1978; 

Krishtal, 1978; Kostyuk, 1980). Most of these studies were done on neurones from 

Helix pomatia. Lee, Akaike & Brown (1977, 1978) developed somewhat different 

methods for isolating, voltage clamping and internally perfusing snail nerve cell 

bodies and used them to study the Ca currents of Helix aspersa neurones (Akaike, 

Lee & Brown, 1978). 

When we began our study of the Ca current in Limnea neurones, we tried 

procedures published by both of the above laboratories. Some of the techniques never 

worked in our hands. The techniques we finally adopted, which are described below, 

are a combination of those used by the two laboratories, plus a few innovations of 

our own. This paper reports the properties of the Ca current of Limnea nerve cell 

bodies as determined using our technique for internal perfusion and voltage clamping. 

In general we find that the Ca currents and the overlapping background currents in 

Limnea neurones are much more like those of Helix pomatia than those reported for 

Helix aspersa. Preliminary reports of this work have been published (Byerly, 

Hagiwara, Masuda & Yoshii, 1979; Byerly & Hagiwara, 1981). 

METHODS 

Isolation of cells 

The circumoesophageal nerve ring is dissected out of an adult Limnea stagnalis and then soaked 

in a 0-2 % trypsin (Sigma, Type III) solution for 90 min at room temperature. The visceral, parietal 

and pedal ganglia are used. Each ganglion is opened and the neuropil with cell bodies attached is 

freed from the covering sheath, using sharpened tungsten wires and irredectomy scissors. The 

exposed neuropil and cell bodies are then transferred by pipette to a dish containing Limnea saline 

with 7 mM-glucose. Using two glass micro-electrodes, the neuropil is then torn apart until a length 

of the axon connecting a cell body to the neuropil is exposed. This axon is then severed with the 

tip of one of the micro-electrodes, while the axon is lying on the casting resin (Dow Corning, Sylgard 

184) covering the bottom of the dish. The axon is usually severed within 50,cm of the soma. The 

isolated cell body is given 2 h to recover, during which time the axon stub rounds into the soma. 

Many cells do not survive the isolation procedure; those that do can easily be identified by their 

shiny appearance and the absence of the white colour which appears in damaged cells. Usually more 

than ten healthy isolated cell bodies with diameters from 80 to 120,m are obtained from each 

animal. These cells remain in a healthy state for at least 24 h in glucose-containing saline solution. 




Ca CURRENTS IN LIMNEA NEURONES 

505 

We find this method of isolating the cells before sucking them onto the suction electrode to be 

superior to isolating the cells after they are attached to the suction electrode (Lee et al. 1978). With 

this method we routinely obtain healthy cells with strong Ca currents. In contrast, only a small 

percentage of the attached cells sucked into the suction electrode can be isolated without killing 

the cell, and those that do survive the isolation usually have too long an axon stub to allow good 

voltage control or complete perfusion. Since the membrane currents irreversibly change during the 

Fig. 1. Experimental apparatus. Drawing is not to scale. The suction electrode (SE) is 

3 mm in diameter and 4 cm long, while the cell diameter is 80-120 #sm. The plastic block 

holding the suction electrode is mounted to a micromanipulator. The arrows drawn inside 

the suction electrode indicate the flow of the internal solution. The inputs to the voltage 

follower (VF) and virtual ground (VG) amplifiers are connected to Ag/AgCl wires. The R8 

potentiometer connected to the summing amplifier (1) provides series resistance compensation. 

The three positions on the switch of the negative input of the clamp amplifier 

(A) allow for suction electrode voltage clamp (SEC), current clamp (IC), and hybrid 

voltage clamp (HC). The micro-electrodes (uE) are filled with 3 M-KCl. The identified 

signals are micro-electrode voltage (TVME), suction electrode voltage (VSE), command 

voltage (VI), and membrane current (I). 

first I h of perfusion (see below), it is important for our studies to have the cell in an otherwise stable 

state when it is sucked against the suction electrode. By the time we study the isolated cell the 

axon stub has rounded into the soma, so that relatively stable cells with nearly spherical shapes 

are obtained. In contrast, when the cell is isolated after being sucked against the suction electrode, 

the currents must be studied while the cell is recovering from the isolation procedure. 

In these studies no attempt has been made to identify neurones. Cells are only selected to have 

a diameter of 80-120 ,gm and to have survived the isolation procedure (this may select against cells 

with larger axons or stronger ties to connective tissues). However, the technique is suitable for 

isolating identified neurones. In preliminary trials a pre-selected neurone could be isolated with 

about a 50 % success rate. 

It is important to point out that only one type of Ca current was found, despite the variation 




506 


in size and probable variation in function of the cells studied. Although the magnitude of the Ca 

current varied remarkably from cell to cell, the voltage and time dependencies of the Ca current 

showed no significant variation. The apparent inactivation of Ca currents was somewhat faster in 

some cells, but we assume this resulted from the presence of a greater amount of overlapping 

outward current in these cells, rather than a basic difference in the Ca current. 

Apparatus 

Figure 1 shows a schematic diagram of the physical arrangement used in these studies. 

Suction electrode. The suction electrode is made from 3 mm (o.d.) Pyrex glass tubing. The glass 

tubing is pulled on an electrode puller (Narishige, PE2) in two pulls (the second at a lower 

temperature) to give a sharp taper. The tip is next broken at about 80 num (o.d.) and then fire-polished 

in a microforge to have an inner diameter at the tip of 25-30 ,sm. These tips form the best seal 

(highest resistance) to the somal membrane when they are clean. Coating the tip with various 

adhesive or sealant materials (Parafilm, oil, silicon, protamine, polylysine, etc.) does not improve 

the seal. The best seals are obtained the first few times an electrode is used. Therefore, we change 

suction electrodes frequently. The attachment for mounting the suction electrode in the plastic 

block is designed to allow easy replacement of the suction electrode. 

Internal solution flow system. The internal solution enters the suction electrode through a glass 

pipette that enters the plastic block through a vaseline-filled metal sleeve. This allows for position 

adjustment so that the tip of this inlet tube can be placed just a few hundred microns from the 

cell, in spite of the unavoidable variation in the length of the suction electrodes. With the inlet 

so positioned intracellular K+ can be completely replaced in less than 5 min (see below). 

The internal solution flows from open bottles sitting at a level about 40 cm above the suction 

electrode. The exhaust for the internal solution leads to a trap bottle in which the pressure is 

measured by a manometer. The pressure difference (relative to atmospheric) measured by the 

manometer is expected to be somewhat greater than the pressure difference at the tip of the suction 

electrode, due to the drop in pressure along the exhaust line. The trap bottle is connected to a 

vacuum pump via a second trap bottle. The pressure at the tip of the suction electrode is varied 

by means of two valves, one on the vacuum line to the trap bottle and the other on a bleeder line 

to the trap bottle. Internal solution flows through the suction electrode at a rate of about 1 ml every 

5 min when the cell is in place. 

Current measurement. Membrane current flows from the control amplifier (Biodyne AL- 1) to the 

stainless-steel sleeve through which the internal solution inlet enters. The current is then carried 

through the internal solution into the cell. In the external solution the current flows into a pipette 

filled with a 3 M-KCl solution in agar. This electrode is connected via a Ag/AgCl wire to the virtual 

ground input of a current-to-voltage converter (5 #s time constant). Even during the large 

capacitive current transients associated with voltage steps the potential in the bath varies by less 

than one millivolt. 

Potential measurement. The potential across the membrane is measured in two ways. One way 

is with a conventional micro-electrode (5-10 MCI) inserted through the membrane. The second 

measurement is from a flowing-KCl electrode in the internal solution at a considerable distance 

downstream from the cell. This is a conventional micro-electrode broken to a resistance of less than 

1 MC. The open end of the micro-electrode is connected to a reservoir of 3 M-KC1. Given the lower 

pressure in the suction electrode, a constant stream of 3 M-KCl flows from this electrode. The 

potential measured from a Ag/AgCl wire in the KCl reservoir is then corrected electronically for 

the potential drop that occurs at the tip of the suction electrode due to the flow of current across 

the tip resistance (usually about 400 KM) when current is passed through the membrane. The 

membrane current is usually less than 30 nA, even during positive potential pulses, but can reach 

several microamperes during the capacitive transients accompanying potential steps. 

Clamping techiques. When current-clamp experiments are done the output of the current-tovoltage 

converter is fed back to the control amplifier. This is superior to clamping the current put 

out by the control amplifier, since there is considerable capacitance between the tubing carrying 

the internal solution and ground. 

Voltage-clamp experiments are done using either of two feed-back signals. In 'hybrid-clamp' 

experiments the potential measured by an intracellular micro-electrode is fed back to the control 

amplifier. This type of voltage-clamp is faster and superior whenever large currents are involved 

or when large liquid junction potentials exist between different internal solutions. In the 




Ca CURRENTS IN LIMNEA NEURONES 507 

suction-electrode clamp' the corrected potential measured by the flowing-KCl electrode is fed back 

to the control amplifier. This type of voltage-clamp has the advantage of convenience, since it is 

not necessary to impale the cell with a micro-electrode. Smaller, slower currents measured by the 

suction-electrode clamp are the same as when measured by the hybrid clamp; but, without the 

micro-electrode penetrating the membrane, the cell is much more stable and external solution 

changes are easier. The series-resistance correction to the suction-electrode potential is made by 

adjusting the fraction of the current signal subtracted so as to give the fastest possible capacitive 

current transient. When this procedure is applied to a resistor-capacitor equivalent circuit, it is 

found to compensate for about 80 % of the series resistance. Therefore, the remaining effective series 

resistance is less than 100 kfl; so the true membrane potential will be less than 3 mV from the 

recorded value as long as the current is less than 30 nA. 

Data storage. All current and voltage records are photographed from the oscilloscope screen and 

stored on 35 mm film. When current or voltage records are to be subtracted, fitted or manipulated 

in some other manner, they are digitized on a 12 bit A/D converter (Datel DAS-250), sampling 

at intervals down to 10 ,us. The digital data is then stored on floppy disk and later manipulated 

by a microprocessor (North Star-Horizon). 

TABLE 1. Composition of solutions (mM) 

External solutions 

Solution Na+ K+ Cl- Ca2+ Mg2+ Tris 

Limnea saline 

Tris saline 

Internal solutions 

50 

0 

2-5 

0 

78 

74 

4 

4 

4 

4 

10 

65 

Solution K+ Cs+ Tris Aspartate HEPES EGTA 

K aspartate 

Cs aspartate 

Tris aspartate 

74 

0 

0 

0 

74 

0 

0 

0 

78 

62 

62 

59 

5 

5 

5 

5 

5 

5 

Solutions 

Table 1 shows the compositions of the main external and internal solutions used. The major anion 

in the external solutions is Cl-, while it is aspartate- in the internal solutions. When 7 mM-glucose, 

10mM-4-aminopyridine(4-AP) or 1 mM-Cd2+ are used in the external solutions, they are added 

without adjustment of the concentrations of other ions. Extracellular tetraethylammonium (TEA) 

is tested by substituting it for all the Na+ and K+ in Limnea saline. Ba currents are studied by 

replacing the Ca2+ with Ba2+ in Tris saline. When Co2+ is used as a Ca-current blocker it is 

substituted for all the Mg2+ and 3 mM-Ca2+ in Tris saline. The pH of all external solutions is 7-4. 

The pH of internal solutions is 7-3. The concentration of free Ca2+ in the internal solutions is assumed 

to be less than 10-8 M. All experiments are done at room temperature (23-27 'C). 

Procedure 

Zero potential is established by removing the flowing-KCl electrode from the plastic block and 

placing it in the external bath. When the flowing-KCl electrode is returned to its position in the 

plastic block with K aspartate solution inside the suction electrode and Limnea saline outside, a 

potential of about -15 mV (inside negative with respect to outside) is recorded due to the junction 

potential between the two solutions; the junction potential between NaCl and K aspartate solutions 

can be calculated from the Henderson equation to be - 14-5 mV. Individual isolated nerve cell 

bodies are transferred by pipette from the dish in which they are isolated to the recording chamber. 

The cell is gently sucked against the tip of the suction electrode and then the pressure in the trap 

bottle is reduced to about 50 cm of water lower than atmospheric pressure. Constant-current pulses 

show a large (about 100-fold) increase in resistance as the cell seals against the glass. When the 

internal solution inlet is brought close to the cell, the potential drops and shows attenuated action 

potentials as the patch of membrane across the tip breaks down, due to the very low concentration 

of Ca2+ on the extracellular side of the membrane. A constant current is applied to hold the 




508 

L. BYERLY AND S. HAGIWARA 

membrane potential near -50 mV. If an instantaneous jump in potential occurs at the beginning 

of the square-current pulse, the rupture of the membrane is not complete; so the pressure in the 

suction electrode is reduced further for a few seconds to completely break the membrane patch. 

Once this membrane is properly broken, the resistance through the tip of the suction electrode (the 

resistance in series with the membrane) is increased by less than 50% from the value it had before 

the cell was present. 

Shunt resistance 

When isolated nerve cell bodies are studied with a single micro-electrode, they have membrane 

resistances of 40-400 MCI and time constants from 50 to several hundred milliseconds. When the 

same parameters are measured in cells sealed to the suction electrode by passing hyperpolarizing 

current pulses through the suction electrode, apparent valves of 20-200 MCI and 10-100 ms are 

obtained. Since the time constant and resistance measured when a cell is sealed to the suction 

electrode are somewhat less than the values measured by the micro-electrode impalement, we 

conclude that the shunt resistance (the resistance to flow of current between the membrane and 

the glass), is, in general, of about the same magnitude as the membrane resistance. In some 

preparations the shunt resistance is probably considerably less than the membrane resistance, so 

the total resistance is roughly equal to the shunt resistance. In other cases the shunt resistance 

is considerably larger than the membrane resistance, possibly exceeding a gigaohm, as judged by 

the nearly equal resistances (and time constants) measured first by micro-electrode and then by 

suction electrode. The size of the shunt resistance relative to the resting membrane resistance is 

of little importance in this study of the Ca current. Cells sealed to the suction electrode are only 

used if they generate fast, all-or-nothing, overshooting (+ 20 to + 40 mV) action potentials in 

response to small pulses of outward current. So clearly the inward membrane currents are 

considerably larger than shunt currents in all cells studied. 

Space clamp 

When a negative-voltage pulse is applied to the membrane, the capacitive current transients 

(Fig. 2 A and B) settle in less than1 ms for both suction-electrode and hybrid clamps. The membrane 

capacitance is given by the ratio of the area under the capactive current transient and the 

magnitude of the voltage step. The specific membrane capacitance is calculated by dividing the 

membrane capacitance by the surface area of the cell, assuming the cell to be a sphere. (No 

correction is made for the area of membrane eliminated by the suction electrode; thus, the specific 

capacitance is underestimated by about 10 %o.) The specific capacitances calculated for thirty-eight 

cells have a mean value of 1-5 puF/cm2 (S.D. = 05 #sF/cm2). This value is close to that expected for 

a simple membrane, suggesting that there is little invagination of this nerve cell body membrane. 

Therefore, the space clamp should be good, i.e. there should be little variation of membrane 

potential from one region to another. 

A much smaller, slow displacement-type current is also observed in these cell-suction-electrode 

preparations. This current decays over several milliseconds and is symmetrical for negative or 

positive voltage pulses. It does not result from error in potential measurement, since it appears 

the same in suction-electrode clamp and hybrid clamp of the same cell. Since this slow current has 

not been seen in the few two-micro-electrode voltage-clamp studies we have done on isolated nerve 

cell bodies, we suspect that it is associated with the glass-membrane seal. If portions of the 

membrane pressed against the glass slowly charge due to current flowing between the membrane 

and glass, such a prolonged displacement current would result. The charge carried by this slow 

current is always less than one tenth of the charge carried by the fast capacitive transient, which 

implies an area of membrane is involved that is consistent with the area of the cell in contact with 

the glass. Since this slow current is symmetrical, its presence can be overcome by adding the 

currents from equal negative and positive voltage steps. However, if this 'covered' portion of 

membrane is active, the quality of the clamp is clearly degraded. 

Clamp speed 

The most reliable indication of the speed of a voltage-clamp is the duration of the transient 

capacitive currents. We adjust the clamp amplifier so that the response is slightly underdamped, 

so that the current reverses sign after the main current surge (see Fig. 2A and B). It is convenient 

to measure the speed of the clamp by the duration of this main current surge. After this time the 





membrane potential is close to the command value, and the remaining capacitive transient can be 

eliminated by adding currents for equal positive and negative voltage steps. When the membrane 

potential is clamped using the signal recorded from the flowing-KCl electrode (suction-electrode 

clamp), the main surge lasts 150-600 ts (Fig 2A), the slower clamps resulting from larger cell 

capacitance and larger suction-electrode resistance. When the same preparation is clamped using 

the signal recorded from an intracellular micro-electrode (hybrid clamp) the speed of the clamp 

is about doubled (Fig. 2B). The main surge of capacitive current for hybrid clamps lasts 80-400 Is, 

A B 

V 

1OnALj 

SEC HC 

20 nAL__ 

1 ms 1 ms 

7 C¢ O~~min Hlo_ 

1 mnI5 / 

V~~~ ~ ~~~~~~~~~ andI4 min 

nA 

150 mV 

10 ms 

Fig. 2. Speed of voltage clamp and intracellular ion exchange. Capacitive current 

transients associated with 10 mV hyperpolarizing voltage pulses during suction electrode 

(A) and hybrid (B) voltage clamps of the same cell. Note change in current scale. Diameter 

of cell is 90 ,um; opening of suction electrode is 24 pm. Holding potential is -50 mV. C, 

exchange of intracellular K+ with Cs+. Currents are recorded at 1 min intervals following 

the transition from K aspartate to Cs aspartate in the suction electrode. External solution 

is Tris saline. Holding potenial is -50 mV; pulse is to + 20 mV. Cell diameter is 100 ptm; 

suction electrode opening is 26,um. SEC, suction electrode voltage clamp; HC, hybrid 

voltage clamp. 

depending on micro-electrode resistance as well as suction-etectrode resistance and cell capacitance. 

Typically, the main current surge lasted about is 150 for the studies reported in this paper. The 

brevity of the capacitive current confirms the good space clamp obtained with this preparation. 

The hybrid clamp is always used when currents are to be studied within 1 ms of a potential step. 

Exchange of intracellular ions 

Using suction electrodes with an inner diameter of 25-30 pm and cell bodies about 100 pam in 

diameter, the intracellular K+ can be replaced with Cs+ or Na+ in less than 5 min. This conclusion 

is based upon the times required for the membrane current to reach new steady-state levels 

following changes of the internal solution. When the internal solution is switched from K aspartate 




510 L. B YERL Y AND S. HAGIWARA 

to Cs aspartate, the currents measured during steps to + 20 mV reach a new steady state in 3-4 min 

(see Fig. 2C). The return of the K currents follows approximately the same time course when the 

internal solution is switched back to K aspartate. A similar time course of exchange is observed 

when replacing intracellular K+ with Na+. Five minutes after the internal solution is switched from 

K aspartate to Na aspartate (Na+ substituted for K+) the Na current reverses at a potential near 

the value expected for complete replacement of intracellular K+ with Na+. Thus, it appears that 

monovalent cations can be exchanged completely and rapidly. However, we have not yet 

determined the extent to which we can control more highly regulated intracellular ions, such as 

Ca2+ and H+. 

+80 * 60 ms 

10 nA 

/(nA) 

0 Peak 20 

10 Ms -100 100 

-10 V (mV) 

+20 

Fig. 3. Currents in absence of K+. On the left, tracings of the currents elicited by stepping 

the membrane potential to values (in MV) indicated on each trace. On the right, I- V plots 

of the peak current (0) and the current at 60 ms from the pulse beginning (0). Peak 

current is measured when the current reaches its most negative (inward) value. External 

solution is Tris saline; internal solution is Cs aspartate, Holding potential is -50 mV. 

Suction electrode voltage clamp used. 

RESULTS 

This study of the Ca current in Limnea neurones is complicated by two problems. 

(1) The Ca current is not stable under internal perfusion. Within half an hour of the 

beginning of the exchange of cytoplasm for saline solution the Ca current has almost 

completely disappeared. (2) Although the background currents have been greatly 

reduced by the replacement of intracellular K+ with less permeant ions, non-specific, 

time- and voltage-dependent currents are still present. Since these currents are 

sensitive to all the treatments that change the Ca current, a complete isolation of 

the Ca current is not possible. However, in spite of these problems, a number of the 

properties of the Ca current can be studied. We will first discuss these two problems, 

before turning to the properties of the Ca current. 

I. Labile nature of Ca currents 

When the neurone is internally perfused with Cs aspartate and bathed externally 

in Tris saline, prolonged inward currents are elicited by stepping the membrane 



by guest on January 20, 2013 

-20

-100 

s C p L- 

IL) 

Cu a) 


-10 

-20 

A 

B 

20 60 

Before decay 

* 60ms 

o Peak 

After decay 

x 60ms 

6 Peak 

-100 

C 

Time (min) 

V (mV) 

Fig. 4. Wash-out of Ca current. A, dependence of magnitude of Ca current on time. The 

peak current evoked by a step to + 20 mV is plotted. The size of the opening of the suction 

electrode used in each ofthe four experiments is given near the data; all cells were 90-100 yam 

in diameter. Times are measured from the completion of the replacement of intracellular 

K+ with Cs+. B and C, I-V curves for two different cells before (0O peak; @, 60 ms) and 

after (A, peak; x, 60 ms) Ca current decay. External solution is Tris saline and internal 

solution is Cs aspartate. Suction electrode voltage-clamp used. 

potential to values between -20 and + 50 mV (see Fig. 3). These inward currents 

are assumed to be carried by Ca2+, since they disappear when external Ca2+ is replaced 

by Mg2+ and are blocked by Ca blockers such as Co2+, La3+ and Cd2+. These Ca 

currents are not stable. If the membrane potential is stepped to + 20 mV for 60 ms 

at intervals of 1 or 2 min, the magnitude of the inward current decreases each time, 

until after 20-30 min there is no longer a net inward current (see Fig. 4A, records 

for 24 and 25 ,tm). This is not a general deterioration of the cell, because the current 

,-10 




511


asymptotically approaches a small positive value of about the size expected for the 

leakage current. The I-V relation measured for cells after the current reaches this 

new steady state no longer contains a negative-slope region (Fig. 4B and C), indicating 

that the Ca current has disappeared. As illustrated in Fig. 4B and C, the resistance 

of the cell can increase or decrease during this decay of the Ca current. The 

capacitance of the cell does not change. This is a selective loss of the Ca current; the 

Na and K currents last for much longer times. If after the Ca current has decayed 

the internal solution is changed to K aspartate, the K currents return. After complete 

decay of the Ca current cell bodies which originally had a combined Na and Ca spike 

still have a fast overshooting Na spike. 

The loss of the Ca current is due to the exchange of intracellular solution. We found 

that as we improved the intracellular perfusion, increasing the speed with which 

intracellular ions could be exchanged, the Ca current decayed more rapidly. When 

intracellular exchange is retarded by using suction electrodes with small openings, 

the Ca current decays much more slowly. Figure 4A shows that the Ca currents 

measured in 100 jzm cells sealed to suction electrodes with 11-12 jam openings 

decayed only slightly over a period of 1 h. It is not Cs+ that causes the decay of the 

Ca current. If a cell is perfused with K aspartate for 30 min and then switched to Cs 

aspartate to eliminate the outward currents, the Ca current is already very small, 

presumably due to decay during the perfusion period. 

We use suction electrodes with openings of about 25 gm and measure the Ca current 

during the first 15-20 min following the beginning of intracellular perfusion. Since 

the magnitude of the Ca current is continually declining during this period, the Ca 

current in test solutions is always compared to the Ca currents in control solutions 

before and after the test (when possible). We do not use suction electrodes with 

smaller openings, which greatly reduces the decline of the Ca current, because the 

rate of intracellular perfusion is so reduced that the ability to control intracellular 

ionic concentrations in the presence of large membrane currents is questionable. The 

exchange of intracellular K+ for Cs+ requires 15-30 min using suction electrodes with 

10 jtm openings and 80-120 #am cells. 

II. Non-specific outward current 

The currents measured when internal K+ is replaced with Cs+ and external Na+ 

and K+ are replaced with Tris+ (Fig. 3) are not carried by Ca2+ alone. Given the very 

low concentration of free Ca2+ in the cell, Ca2+ can carry almost no outward current. 

The large time-dependent outward currents measured at potentials above + 60 mV 

can only be carried by an efflux of Cs+ or an influx of Cl-. These outward currents 

show very little change when external Cl- is replaced by methane sulphonate or when 

80 % of the external Tris+Cl- is replaced with glucose. Therefore, it seems Cs+ must 

carry this outward current. The replacement of intracellular Cs+ with Tris+, TEA+ 

or arginine+ reversibly reduced the outward currents at large positive voltages by 

factors of two to four. The fact that these large cations can carry outward current 

demonstrates that this conductance is not very selective. The inward Ca current was 

also reversibly reduced by each of these replacements. (These experiments were done 

using a hybrid clamp, due to the increase in suction-electrode resistance caused by 

the low mobility of these ions.) Since intracellular Cs+ gives the largest Ca current 




. ~~~~~~~~~G 


and a low suction-electrode resistance, it is used to replace K+ in all the studies 

reported here. 

Since the substitution of Tris+, TEA+, or arginine+ for Cs+ affected the Ca current as well as the 

outward current, it is not clear if the reduction of outward currents is due to the lower permeability 

of these large cations compared to Cs+, or if this is a pharmacological effect of certain intracellular 

/O -/L 

(nA) 

80 

10 

60 

20 ..x 50 100 

V (MV) 

-= -40 

-40 

~~~~~~~~~~~~(,umho) 

--0-2 

5 nA 

10 ms 

/ox~X~ 510 

x 

100 

V (mV) 

Fig. 5. Residual currents left after Ca current decay. On the left, tracings of currents 

evoked by depolarizing pulses. Numbers on traces give potentials (in mV) reached by 

pulses. External solution is Tris saline; internal solution is Cs aspartate. Suction electrode 

clamp used. On the upper right, plot of steady-state outward current (I.) against 

membrane potential. A linear leakage current (IL) has been subtracted from the data. Data 

are plotted both for currents (0) shown at left and for currents ( x ) from a second cell. 

On the lower right, conductance (0) is plotted against voltage for the currents plotted 

above. Conductance is calculated from G = (IO- IL)/(V- Vr), where Tr is the potential 

at which the straight line drawn through the currents intersects the voltage axis. 

cations that affects all currents. The Ca current was similarly reduced when the internal solution 

was changed from Cs aspartate to a mixture of one part Cs aspartate and four parts isotonic glucose, 

suggesting that the inward Ca current may be promoted by intracellular alkaline cations (Cs+, Na+ 

and K+). (Replacing intracellular Cs+ with Na+ produced little change in the inward Ca current 

or the outward current.) Almers & Palade (1981) reported a similar effect on the Ca current of the 

frog muscle membrane; replacing intracellular K+ with TEA+ reduced the Ca current. The 

interaction of intracellular cations with the Ca currents is not clear. 

Residual current 

The time course and voltage dependence of the non-specific current in the presence 

of the Ca current is not known. As will be discussed below, all treatments that block 

the Ca current also change (usually reduce) the magnitude of the non-specific current 

and quite possibly also change its time course and voltage dependence. However, the 

17 




PRHY 322

514 L. BV7ERLY AND S. HAGIWARA 

current remaining after the Ca current has completely decayed provides a description 

of at least one component of the non-specific current. The voltage and time 

dependencies of this residual current are shown in Fig. 5. This conductance is turned 

on over a range of about 30 mV, somewhere between - 10 and +40 mV. The 

activation of these currents becomes more and more rapid as the membrane potential 

is stepped to more positive levels; there is no inactivation. The linear regions of the 

Tris saline 

o Peak 

* 60Oms 

Tris saline+ 1 mm-Cd 2 

+2 10 mAs j 

10msL 

/ (nA) 

40 

Fig. 6. Effects of 1 mm-Cd2+ on currents. I-V relations before (0, peak; 0, 60 msec) and 

after ( x, 60 ins) the addition of 1 mm-Cd2+ to external solution. Inset shows currents 

before and after addition of Cd2+, at + 20 and + 90 mV. External solution is Tris saline 

(with and without Cd2+) ;internal solution is Cs aspartate. Suction electrode clamp is used. 

I- V curves for the steady-state values of this current project to zero current near 

the origin, suggesting that this current would reverse around 0 mV. This reversal 

potential is supported by the fact that inward tail currents are seen when the potential 

is returned to -50 mV following positive steps which activate this current (see Fig. 

12 C). Given the highly asymmetric nature of the solutions on the two sides of the 

membrane (Cs+Asp- inside and Tris+Clh outside), this reversal potential implies that 

the conductance mechanism responsible for the residual current is fairly non-selective. 

Sensitivity to Ca blockers 

The Ca current could be isolated from the background currents that persist with 

external Tris saline and internal Cs aspartate if some treatment were available which 

selectively reduced or eliminated the Ca current. Unfortunately, we find the outward 

currents at large positive voltages are always changed (usually reduced) when the 

Ca current is reduced, regardless of the treatment used to reduce the Ca current. (1) 

The Ca current can be eliminated by replacing Ca2+ with Mg2+. This causes the cells 





to rapidly become leaky. (2) The current can be blocked by a number of polyvalent 

cations and organic blockers. La3+, Cd2+, Co2+, Ni2+ and Verapamil are effective in 

blocking the Ca current, but they all also reduced the outward current. Cd2+ was 

chosen as the preferred blocker, because it eliminates the Ca current at 1 mm, a 

concentration which should have little effect on the surface potential. Figure 6 

illustrates the action of 1 mM-.Cd2 . It has little effect on the resting resistance, 

eliminates the inward Ca current, but also greatly reduces the outward current. (3) 

Ca current spontaneously decays with internal perfusion, but the outward current 

also changes in the process (Fig. 4), either increasing or decreasing. (4) Ca current 

can be quickly eliminated by adding 10 mM-Mg2+ to the internal solution. This always 

also reduces the outward currents, leaving a residual current of the type characterized 

in Fig. 5. 

One possible explanation for the reduction in outward currents that usually 

accompanies elimination of the Ca current is that part of the outward current is 

passing through the Ca-activated K conductance (Meech, 1974). This does not seem 

likely for several reasons. First, there is 5 mM-EGTA inside the cell, which strongly 

limits accumulation of free Ca2+ at the inner surface of the membrane. Secondly, 

Woolum & Gorman (1981) have shown that the Ca-activated K conductance is quite 

selective, having a permeability for Cs+ that is only 0-03 that for K+. Thirdly, when 

the Ca2+ in Tris saline is replaced with Ba2+, the outward currents at large positive 

voltages get larger. The increase is probably due to a 15 mV shift of the I-V curves 

to the left, resulting from the weaker binding of Ba2+ to the membrane surface charge. 

However, the fact that the outward current is not reduced argues against a 

contribution from a Ca-activated K conductance, since Gormon & Hermann (1979) 

demonstrated that Ba2+ is much less effective than Ca2+ in activating this 

conductance. 

Another explanation for the reduction of outward current when the Ca current is 

blocked is that the Ca conductance is not very selective, so that Cs+ could flow 

outward through the Ca conductance. This explanation is not supported by the 

details of the reversal of the current. When the membrane is stepped to potentials 

around +60 mV, the current is initially inward and then becomes outward later. 

Unless we are willing to accept the idea of a conductance with a time-dependent 

selectivity, the switching of current direction at one voltage must indicate the 

involvement of more than one type of conductance. If only the Ca conductance were 

involved, there should be one voltage at which the membrane current is zero (ignoring 

leakage) at all times, which is clearly not the case (see Figs. 3 or 6). 

Sensitivity to K blockers 

Since molluscan neurones are known to have a number ofK conductances that are 

activated by depolarization, it is reasonable to question if these outward Cs currents 

might be blocked by K blockers. (1) Ba2+ has been found to be quite effective in 

blocking some K currents (Hagiwara, Fukuda & Eaton, 1974; Hagiwara, Miyazaki, 

Moody & Patlak, 1978; Gorman & Hermann, 1979). However, as discussed above, 

replacing the Ca2+ in Tris saline with Ba2+ does not reduce the outward current. (2) 

When the Tris in Tris saline is replaced with TEA, outward K currents are greatly 

reduced (but are still larger than the outward currents obtained when intracellular 




17-2


K+ is replaced with Cs+). However, when the internal solution is Cs aspartate, 

switching from Tris saline to the TEA saline does not reduce the outward Cs currents. 

Also, adding 10 mM-TEA+ to the internal solution does not reduce the outward Cs+ 

current. (3) The K blocker 4-AP is found to have some effect on the outward Cs current. 

It does not completely block the outward current, but at a particular membrane 

potential it slows down the activation of the outward current and reduces its 

steady-state magnitude. This action of 4-AP could be interpreted as a shifting of the 

voltage dependence of the outward current to the right. Since 4-AP reduces the 

magnitude of the outward Cs current, the inward current becomes larger and more 

prolonged. Therefore, 10 mM-4-AP has been added to all external solutions in the 

following experiments. 

Cd Co 

- ~~~90 2 nA -90 

10 ms 

+100' 

+10 +80 

0 |nA 5 

f 

Fig. 7. Difference between currents measured before and after Ca blockers. Numbers of 

current records indicate the potentials (in mV) reached by the pulses. Holding potential 

is -50 mV. External solution is Tris saline with 10 mM-4-AP (with and without Ca 

blocker); internal solution is Cs aspartate. Suction electrode clamp is used. On the left, 

difference currents using Cd2+ for Ca blocker. 1 mM-Cd2+ is added to the Tris saline. On 

the right, difference currents using Co2+ for Ca blocker. 7 mMCo2+ is substituted for 

3 mm-Ca2+ and 4 mM-Mg2+ in Tris saline. 

III. Ca Current 

Isolation 

The closest approach we can make to isolating the Ca current is to subtract the 

current recorded after a Ca blocker is applied from the current recorded before the 

blocker application; all currents are measured (1) with internal Cs aspartate and 

external Tris saline, (2) with 10 mM-4-AP in external solutions and (3) within 15 min 

after replacing internal K+ with Cs+. These difference currents will show all the 

blocker-sensitive currents. Figure 7 shows typical difference currents for Cd2+ and 

for Co2+. A higher concentration of Co2+ is necessary to block the Ca current. At this 





concentration (7 mM) Co2+ clearly reduces the leakage current, as well as outward 

currents at large positive potentials. The Cdd2+ has very little effect on the leakage 

current, but still blocks some outward current. The amount of outward current 

appearing in these difference currents varies from cell to cell. Figure 8 shows the best 

cell studied with respect to amount of blocker-sensitive outward current. For this cell 

the difference currents reverse above + 80 mV. Presumably the currents shown in Fig. 

8, at least up to + 50 mV, are almost pure Ca currents. Note that the I- V relations 

2 nA 

-90 0 

_ -100 100 

X-% ~~V(mV) 

10 ms 

100 60rns -10 

oPeak 

-20 x 1 ms tail 

InA 

T 

(nA) 

20 -20tS 

Fig. 8. Difference currents with smallest non-specific current component. On the left, 

difference of currents recorded before and after 1 mM-Cd2+. Numbers indicate the 

potentials (in mV) reached by the pulses. Holding potential is -50 mV. External solution 

is Tris saline with 10 mM-4-AP (with and without Cd2+); internal solution is Cs aspartate. 

Suction electrode clamp is used. On the right, I- V relations for the peak current recorded 

during the pulse (0), the current 60 ms from beginning of pulse (@), and the current 1 

ms after stepping back to the holding potential (x). 

at large positive potentials have a negative curvature, as should be expected from 

the very low concentration of Ca2+ inside the membrane. Since there is too little 

intracellular Ca2+ to carry appreciable outward current, true Ca I-V relations must 

approach the voltage axis at a very small angle. These inward currents show little 

decay (inactivation) during the 60 ms pulses used. Similar difference currents for 

voltage pulses of 1 s duration show that the Ca current decays with two time constants 

at + 10 mV, one of about 70 ms, the other almost 3 s. 

Activation kinetics 

It seems reasonable to assume that the background currents that contaminate the 

Ca currents are small and activate slowly for potentials below + 30 mV, somewhat 

like the residual currents of Fig. 5. Therefore, the activation of Ca currents at 

potentials below + 30 mV can probably be studied without complication from the 




518 

Ca 


-10 

WIIA*~~~~~~~~~~~~ 

V 

(mV) 

a_ +10 

Ca 

+50 

+30 \ 

Fig. 9. Activation of Ca and Ba currents. Upper part of the Figure, the sum of currents 

recorded during equal-amplitude positive and negative pulses. The records are identified 

by the membrane potential reached by the positive pulse. Holding potential is -50 mV. 

Both Ca (left) and Ba (right) currents are recorded from the same cell. Internal solution 

is Cs aspartate; external solution is Tris saline with 10 mM-4-AP for Ca currents. For Ba 

currents the Ca2+ in Tris saline is replaced by Ba2+. Hybrid voltage clamp is used. Lower 

part of the figure, peak current and half-activation time (T.) are plotted against membrane 

potential. The curve drawn through the Ba half-activation times is the same curve drawn 

through the Ca data, but shifted to the left by 15 mV. 




Ba 

2 ms


519 

background current. Figure 9 shows the activation of Ca and Ba currents in the same 

cell. Each current record is the sum of the currents recorded during equal-amplitude 

positive and negative pulses from the holding potential of -50 mV; the trace is 

labelled with the potential reached by the positive pulse. Summing the current 

50 

,20 

=12 ms1 0=O Ms 

2-0 10 1V m 

T=12 ms 

5-5 ms11 ms 

10 'I1n 

T ~~~+20- -50 mV 

j5 nA IlonA 

Fig. 10. Time course of tail currents. Lower part of Figure, two examples (from different 

cells) of the current recorded before and after stepping the membrane potential from 

+ 20 mV to -50 mV. Current records start with the Ca current already activated; the 

duration of the pulse to + 20 mV was 60 ms for the cell on the left, 10 ms for the cell on 

the right. Capacitive transients have been minimized (on the left) by subtracting the 

currents recorded from an identical pulse after application of 1 mM-Cd2+ and (on the right) 

by addition of the current recorded during a step from -120 mV to -50 mV. Suction 

electrode voltage-clamp was used for data on left, and a hybrid voltage-clamp was used 

for data on right. External solution is Tris saline with 10 mM-4-AP; internal solution is 

Cs aspartate. Upper part of Figure, semi-log plots of currents given below. Filled circles 

represent experimental data. Straight lines show the exponential components (of time 

constants given in the Figure) that add to give the continuous curves drawn through the 

data. 

_~~~~~~~~~~~~~~~~~~ - 

records eliminates most of the capacitive and linear leakage currents without having 

to apply Cd2+. The outward currents recorded immediately after the capacitive 

transient for more positive potentials probably result from the non-linear nature of 

the leakage current. The shape of the early Ca current best fits an m2 form where 

m = [1-exp (-t/7rm)], as was found by Kostyuk et at. (1977 b). When the fit is 

constrained to agree with the data at half-maximum current, the m shape rises too 

rapidly at early times and too slowly later, while the m3 shape rises too slowly at early 

times and too rapidly after passing the half-maximum value. The time required for 

the Ca current to reach one half of its maximum value, which is 1-23 Tm, decreases 

from about 3 ms at -30 mV to 1 ms at + 30 mV (Fig. 9). 

When the external Ca2+ is replaced by Ba2+, the inward current becomes larger 





and reaches its maximum value at a potential 15 mV more negative than the potential 

where the Ca current was maximal (see Fig. 9). This 15 mV shift probably results 

from a change in surface potential. Allowing for this surface potential shift, the time 

course of the Ba currents is almost identical to that of the Ca currents. The times 

required for half-activation of the Ba currents fit reasonably well the curve fitted to 

the Ca current activation times, shifted to the left by 15 mV (Fig. 9). This argues 

that the activation kinetics of this channel do not depend on the species ofion carrying 

the current. 

Thus, the Ca conductance activates with m2 kinetics following a positive voltage 

step, and the rate of activation is the same when Ba2+ carries the current as it is 

when Ca2+ is the current carrier. Once activated the Ca conductance inactivates very 

slowly under these conditions, which include EGTA inside the cell. 

IV. Tail currents 

When the membrane potential is stepped to a level that activates the Ca current 

and then stepped back to the holding potential (-50 mV) after the Ca current is 

turned on, inward tail currents are recorded. These tail currents are quite complicated, 

even after linear capacitive and leakage currents have been subtracted. Figure 10 

presents two examples of these tail currents, along with semilog plots of the same 

currents. It can be seen that the tail currents have at least three components, a fast 

component with a time constant of about 100 ,us and slower components with time 

constants of about 1 and 10 ms. The driving force on Ca2+ is greater at -50 mV than 

at + 20 mV; therefore, the fact that the magnitude of the inward current drops to 

a small fraction of its value at + 20 mV within 500 ,ts of the return to -50 mV 

indicates that the Ca current is turning off with the smallest time constant. We will 

argue below that the slower components to the tail current are not related to the Ca 

current. Since our clamp requires at least 100 Its to make a voltage step, accurate 

measurement of the magnitude of the Ca tail currents is impossible at room 

temperature. 

Ca tail currents 

We studied the voltage dependence of the time constant for change of the Ca 

current by stepping the membrane potential to + 20 mV long enough to turn on the 

Ca current and then stepping to potentials above as well as below + 20 mV (Fig. 11). 

Immediately after positive steps (allowing 200 ,us for the voltage change), the inward 

current was smaller, as expected, due to the reduced driving force. The inward current 

then grew larger, presumably due to the opening of more Ca channels. The inward 

current 200 ,us after negative steps was larger, reflecting the larger driving force on 

Ca2+; the inward current then rapidly fell as Ca channels closed. The time constant 

of the Ca tail current was 350 ,ts at +10 mV and dropped with more negative 

potentials to about 100 /ts at -40 mV. These Ca tail currents are surprisingly fast 

and suggest that the Ca channel cannot be described by a simple two-state model 

such as Hodgkin & Huxley (1952) applied to the Na channel (see Discussion). 





Slow tail currents 

The slower components of the tail current appear not to be related to the Ca 

current. This conclusion was reached with some difficulty because the slow tail 

currents have two properties expected of Ca tail currents. They are blocked by Ca 

blockers like Cd2+ and Co2+, as is demonstrated by their presence in the difference 

currents of Figs. 7 and 8. Secondly, the magnitude of the tail current at 1 ms, which 

5 nA 

1=0 

From +20 mV 

t ~~~~~A 

, 

; . W 

+10 0 -10 -20 -30 -40 

1 Ms 

t '(M) 

V 

200 Ad 

J a' t I I I I~~~ I I I | 

+30 +40 +50 -30 0 30 

V (mV) 

Fig. 11. Transient Ca currents. Currents recorded on stepping from + 20 mV to the 

potentials indicated in the Figure. Each current record starts a few milliseconds after 

stepping to + 20 mV, when the Ca current is fully activated. The zero-current level is 

indicated for all current records. Capacitive transients are minimized by adding the 

currents recorded during equal-amplitude, opposite-polarity pulses. Hybrid voltage-clamp 

used. External solution is Tris saline with 10 mM-4-AP; internal solution is Cs aspartate. 

Inset, voltage dependence of Ca current time constant. Time constant r is determined by 

fitting an exponential to the data between 250 and 500 jes after the beginning of the voltage 

step. 

includes only the slower components, increases with the potential of the positive pulse 

in roughly the same voltage range as that in which the Ca permeability activates (Fig. 

8). However, both of these properties would also be expected if the slow tail currents 

are associated with the non-specific current. Note that the slow tail currents are larger 

in Fig. 7, where the outward currents during the pulse are larger, than in Fig. 8, even 

though the Ca currents during the pulse are larger in Fig. 8. Figure 12 illustrates 

several other lines of evidence that argue against the identification of the slow tail 





currents as Ca tail currents. When external Ca2+ is replaced with Ba2+, the inward 

currents are considerably increased, but the slow tail currents do not increase (Fig. 

12A). The slow tail current appears to be related to a conductance that activates 

much more slowly than the Ca conductance. Figure 12B shows an experiment where 

the membrane potential was returned to the holding level at various times following 

a step to + 20 mV. The magnitude of the slow tail current increases with the duration 

of the positive pulse up to about 30 ms, while the Ca current is fully activated in 5 ms. 

A 

/ (nA) 

Ba 

B 

C 

I,- I 

_ 

10 ms 

I 

10 ms 

; 50mV 

Fig. 12. Slow tail currents are unrelated to Ca currents. A, I-V plots for peak current 

(@) during the pulse and tail currents ( x ) 1 ms after the end of the pulse. Data for Ca 

are on the left, those for Ba on the right. B, dependence of slow tail currents on duration 

of pulse to + 20 mV. Arrowheads indicate the current at 2 ms after the return to -50 mV. 

C, currents evoked by a pulse to + 20 mV before and after the decay of the Ca currents. 

Holding potential is -50 mV. Internal solution is Cs aspartate; external solution is Tris 

saline, except for the Ba currents in A, where Ba2+ replaces Ca2+ in Tris saline. The external 

solution has 10 mM-4-AP added in A and B. A hybrid voltage clamp is used in A; 

suction-electrode voltage-clamp is used in B and C. 

Finally, the spontaneous decay of Ca currents with perfusion provides another chance 

to separate the slow tail currents from the Ca current. After the Ca current measured 

during the positive pulse has decayed, the slow inward tail persists, sometimes even 

increasing in magnitude (Fig. 12C). The magnitude of the slow tail current changes 

during the decay of the Ca current in a way that reflects the change in the magnitude 

of the outward currents measured at large positive potentials. Thus, several lines of 

evidence suggest that the slow inward tail currents and the non-specific outward 

currents are flowing through the same conductance mechanisms. 





DISCUSSION 

Non-specific currents in K-free cells 

Even when K+ has been removed from both sides of the snail neuronal membrane, 

outward currents are activated by depolarization. Their presence is obvious at large 

positive voltages (> + 50 mV), since the total current becomes outward. However, 

they are presumably also activated at lower potentials. Kostyuk et al. (1977b) first 

reported these non-specific outward currents in Helix neurones, in which intracellular 

K+ had been replaced by Tris+. These non-specific currents had a time course 

proportional to (1- exp (- t/r)), where r decreased with potential, being about 5 ms 

at +50 mV. The residual currents we recorded after the Ca currents have decayed 

(Fig. 5) seem similar, although they activate somewhat faster. The faster time course 

may result from our using Cs+, instead of Tris+, to replace K+. When we switch from 

Cs+ to Tris+ inside the cell, the outward currents at positive voltages become smaller 

and activate more slowly. The non-specific outward current reported by Kostyuk and 

co-workers showed similar sensitivities to blockers as does the outward current 

reported here. It was depressed by Cd2+, but fairly resistant to TEA. This current 

was not described by Akaike et al. (1978). 

We find that these non-specific currents are sensitive to every treatment that 

changes the Ca currents. In particular, they are strongly suppressed by Ca blockers. 

This sensitivity of the non-specific currents to Ca blockers prevents the complete 

isolation of Ca currents by subtracting the currents before and after application of 

Ca blockers. However, some background currents are eliminated by this procedure, 

and the background is even further reduced when pharmacological agents are 

included in all solutions to suppress the non-specific current. We use 10 mM-4-AP in 

all external solutions, which appears to slow down the activation and reduce the 

steady-state magnitude of the outward current. Doroshenko et al. (1978b) reported 

a similar effect produced by reducing the external pH to 5*1. (We find lowering 

external pH has a similar effect on Limnea neurones, but have not tried combining 

the two treatments.) The closest we have approached to the isolation of Ca currents 

is the subtraction of currents measured before and after the addition of 1 mM-Cd2+ 

to the external solution, with 10 mM-4-AP in both external solutions (Figs. 7 and 8). 

There is probably a substantial background current in the difference currents for all 

voltages above + 50 mV. Even at lower potentials the apparent inactivation of the 

Ca current may be partially due to slowly activating non-specific currents. 

The residual currents recorded after the Ca current has decayed (Fig. 5) illustrate 

one component of the background currents. However, the component of the 

background current that is lost when the Ca current decays might have different time 

and voltage dependencies. So it is not possible to make any strong arguments as to 

the amount of background current in the difference currents of Figs. 7 and 8. 

There are other studies where the outward currents have been found to be sensitive 

to treatments that change the Ca current, but where the outward current does not 

seem to be activated by intracellular Ca2+. Kass & Tsien (1975) found that the slow 

outward current of cardiac Purkinje fibres was decreased by Mn2+, La3+, D600, and 

also elevated [Ca2+]O, even though elevating [Ca2+]0 increased the Ca current. 

Likewise, Ca-current blockers reduced the slow outward current in frog muscle fibres, 

even with high levels of intracellular EGTA (Palade & Almers, 1981). 




523

524 


Time course of Ca currents 

The Ca current activation that we measure in Limnea neurones (Fig. 9) agrees with 

that reported by Kostyuk and co-workers (Kostyuk et al. 1977 b; Kostyuk et al. 1981). 

The inward current has m2 kinetics and turns on faster at more positive potentials. 

Kostyuk et al. (1977b) found a Tm of 2-4 ±10 ms at 0 mV and 19-20 'C, while our 

data gives a Tm of 09 + 03 ms at 0 mV and about 25 'C. Accepting the value of 2-6 

for the Q10 of Tm (Kostyuk et al. 1981), these measured time constants are compatible. 

We find that Ba currents activate with the same time course as Ca currents, allowing 

for the surface potential shift caused by replacing external Ca2+ with Ba2+. This 

disagrees with the results reported for many nerve and muscle preparations where 

Ba currents appear to activate either faster or slower than Ca currents. To the best 

of our knowledge, this is the first study to find that Ba and Ca currents activate with 

the same time course, except for a study of a Na-modified Ca current in starfish egg 

membrane, where the same result was obtained (Hagiwara, Ozawa & Sand, 1975). 

The Ca current we record in Limnea neurones decays only a small amount during 

the typical 60 ms positive pulses used (Fig. 8). Longer pulses show that the decay 

of the Ca current has two components: at + 10 mV one time constant is about 70 ms 

and the other nearly 3 s. Doroshenko et al. (1978b) reported that the inactivation of 

Ca currents in Helix pomatia had two components at -8 mV with time constants 

(at 22-24 IC) of about 90 and 660 ms in pH 7.5 external solution and about 160 and 

720 ms in pH 5-1 external solution, where non-specific currents are suppressed. 

Tail currents 

Our result that the Ca current turns off with a time constant of about 100 ,Ps at 

-50 mV is rather surprising, considering the relatively slow Ca tail currents that are 

measured in various molluscan neurones on stepping back to the K+ reversal potential 

(Connor, 1977; Adams & Gage, 1979; Eckert & Ewald, 1981). These inward currents 

decay with time constants of several milliseconds. Some of the difference is due to 

the temperature difference; our time constants for snail (measured around 25 °C) 

would be expected to be 3-5 times shorter than time constants measured at 10-15 'C. 

It may be that Limnea Ca channels turn off faster, or it is possible that for Aplysia 

and Archidoris neurones there is sufficient resistance in series with the membranes 

conducting the Ca current to considerably extend the time required to bring these 

membranes to the intended potential. When we first looked at Ca tail currents with 

suction-electrode voltage clamps, where there is still some uncompensated series 

resistance, the tail currents decayed considerably slower (with time constants of up 

to 500 Its at -50 mV). However, on switching to hybrid voltage-clamps the Ca tail 

currents become much faster, as reported above. It is difficult to think of complications 

that could make a measured tail current turn off faster than the inherent turn-off 

of the channel. The rapid turn-off of the Ca current is not an artifact of the subtraction 

of the capacitive currents, since essentially the same turn-off rates can be determined 

from the unsubtracted currents. The usual slightly underdamped condition of the 

clamp does not distort appreciably the time course of the tail currents (capacitive 

currents subtracted), since similar time constants are measured when the clamp is 

adjusted to critical damping. 





525 

In comparing our tail currents with those measured by Kostyuk and co-workers, 

it must be pointed out that we are ignoring the possible presence of gating currents. 

We have made no effort to look for gating currents and their existence is not obvious 

in the data we have collected. The current records in Fig. 9, which are the sums of 

currents from equal amplitude positive and negative voltage pulses, should show 

gating currents as net outward currents immediately following the beginning of the 

pulse. However, even on stepping to + 30 mV, there is no significant outward current 

before the inward Ca current activates. (The net outward current seen at more 

positive potentials, especially for Ba2+ where there is a 15 mV shift of surface 

potential, is a result of the outward rectification of the time-independent leakage 

current.) Therefore, we assume gating currents are small enough to be ignored in our 

study of tail currents. Our Ca tail currents are somewhat faster than those measured 

by Kostyuk et al. (1981). They found that the Ca currents turned off with a time 

constant of about 1-3 ms at -40 mV and 10 0C (see their Fig. 14). This corresponds 

to about 300 pss at 25 0C, about three times larger than our value. Kostyuk and 

co-workers have not mentioned the slow inward tail currents that we report here; 

however, similar slow tail currents have appeared in Figures published by that 

laboratory (see Fig. 4 in Doroshenko et al. 1978b). 

Our data and interpretation of tail currents differ in one fundamental way from 

that of Kostyuk et at. (1981). They find that the Ca current shows no 'instantaneous' 

change in magnitude on stepping between potentials in the range from +25 to 

-40 mV and interpret this to indicate that the electromotive force is high and 

essentially unchanged by these steps in potential. In contrast, we see clear 'instantaneous' 

changes in the Ca current for even 10 mV steps up or down from + 20 mV 

(Fig. 11). The instantaneous change in Ca current for a 10 mV potential step is about 

20 % of the total current, as though the reversal potential for Ca2+ were near + 50 mV, 

assuming linear I-V relations for open Ca channels. However, the Ca2+ reversal 

potential should be above + 150 mV; so our data indicate a non-linearity of the I-V 

relation for open Ca channels, which presumably accounts for the curvature seen in 

the I-V relation of Fig. 8 for potentials above + 30 mV. This sort of non-linearity 

is to be expected from the very asymmetrical distribution of Ca2+ on the two sides 

of the membrane. Hagiwara & Byerly (1981) present the I-V relation that is 

predicted for an open Ca channel by the constant-field equation, to give a rough idea 

of the extent of non-linearity that might be expected. In this example the slope 

conductance at + 30 mV projects to a reversal potential at + 50 mV even though the 

true reversal potential is near + 150 mV. 

One of the basic assumptions of the two-state conductance model used by Hodgkin 

& Huxley (1952) to describe the Na and K channels of axon is that the rate constants 

for the movement of gating particles across the membrane depend only on the 

instantaneous voltage. Thus, Tm. the time constant for m to obtain a new value, 

depends only on the potential to which the membrane is stepped and is independent 

of the previous value of m. This implies that the time constant for the decay of tail 

currents at a particular voltage is related to the rate of activation of current at that 

same voltage. The turning on and turning off of the Na conductance does satisfy this 

relation (see Fig. 1 of Keynes & Kimura, 1978). However, the Ca tail currents 

reported here decay too fast to fit the relation expected for m2 kinetics. Assuming 




L. BYERLY AND S. HAGIWARA 

m2 kinetics, Tm for activation of Ca currents is 2-3 ms at -30 mV and 0 9 ms at 

+10 mV. If m is near one at +10 mV, the tail current would decay with a time 

constant of 0 9 ms(Tm) at +10 mV and1 1 ms (Tm/2) at -30 mV. The measured tail 

current time constants are only 0-35 ms at +10 mV and 0 15 ms at -30 mV. Even 

the sign of the voltage dependence of the tail current time constants is wrong. This 

disagreement of the behaviour of these Ca tail currents with the Hodgkin-Huxley 

model is further illustrated by the small value ofTm obtained by stepping from 

+ 20 mV to +30 mV (Fig. 11; Tm - T) compared to the Tm obtained by stepping to 

+30 mV from -50 mV 526 

(Fig. 9; Tm =Ta/123). Kostyuk et al. (1981) also found that 

the time constants from Ca current relaxation due to small shifts of the membrane 

potential were considerably smaller than the Tm obtained when activating the Ca 

current by large positive steps from the holding potential (see their Fig. 15). 

Labile nature of Ca currents 

The disappearance of the Ca current with internal perfusion is both a frustrating 

and an interesting result. It prevents experiments that require stable currents, but 

it may also indicate that Ca channels cannot be maintained in a functional state when 

bathed by a simple salt solution on the intracellular side. Kostyuk et al. (1981) 

reported that their Ca current rapidly decayed to a level between one half and one 

tenth of its initial value, after replacing the cytoplasm with saline solution. The 

remaining Ca current required 3-4 h to decay at 5°C, but this decay was strongly 

temperature dependent. In our experiments (done near 25°C) the entire Ca current 

seemed to decay at one rate (Fig. 4A). The decay has been observed to occur 

unchanged in a number of completely different internal-perfusion systems, none of 

which allows the solution to contact metal. No changes in the composition of the 

intracellular solution have been found to slow the rate of Ca current decay. 

Furthermore, the intracellular EGTA should bind any toxic metal ions that might 

be present. Consequently, it seems more plausible that the decay is due to the wash-out 

of some intracellular factor rather than the influx of a toxic substance. 

This hypothesis is especially attractive because if has already been suggested that 

the voltage-dependent Ca channel is controlled by levels of intracellular messengers. 

Epinephrine and norepinephrine produce increases in cardiac Ca currents, without 

changing the voltage dependence, activation rate constants, or apparent reversal 

potential of the Ca current (Reuter, 1974; Reuter & Scholz, 1977). This suggests that 

these catcholamines are increasing the number of Ca channels that are available for 

activation by depolarization. Since this effect involves adrenergic fi receptors and can 

be mimicked by intracellular injection of cyclic AMP (Tsien, 1973) or extracellular 

dibutyryl cyclic AMP (Reuter, 1974), it is plausible that levels of intracellular cyclic 

AMP control the number of functional Ca channels. Intracellular levels of cyclic AMP, 

as well as other soluble elements of the chain of reactions between the binding to the 

receptor and activation of the Ca channel, are probably quickly depleted in the cell 

bodies being studied by the intracellular perfusion technique. Therefore, if Ca 

channels in snail neurones require certain soluble, biological intracellular molecules 

in order to remain in a functional state, then Ca currents would be expected to decay 

as these molecules are washed out of the cell. Extracellular neurotransmitters appear 

to reduce the number of functional Ca channels in dorsal root ganglion cells (Dunlap 





527 

& Fischbach, 1978, 1979). While there is no evidence yet for the involvement of cyclic 

AMP in this effect, it supports the idea that the functioning of neuronal Ca channels 

may require soluble intracellular molecules. 

In preliminary experiments suggested by the results cited above we have watched 

the rate ofCa current decay while adding to the internal solution various combinations 

of cyclic AMP, ATP, phosphate, Mg2+ and Na+. Certain internal solutions containing 

ATP have caused a temporary halt and actual reversal of the Ca current decay, 

when first applied; but the effect is variable and only temporary, as the Ca current 

soon continues its seemingly inevitable decay. No internal solution has been found 

which can maintain the Ca current. This is not surprising, since the perfusion 

probably also depletes the levels of the kinases that are thought to be involved in 

the action of intracellular cyclic AMP. 

We wish to acknowledge the participation of Drs Masako Masuda and Mitsunobu Yoshii in the 

early stages of this work. We are grateful to Mr Joseph Stimers for his role in developing the 

microprocessor system used to digitize and manipulate the current records, and to Dr William Moody 

for help with the manuscript. This work was supported by a U.S.P.H.S. grant (NS09012) to Dr 

Hagiwara and a Muscular Dystrophy Association Fellowship and U.S.P.H.S grant (NS15341) to 

Dr Byerly. 

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BY LOU BYERLY AND SUSUMU HAGIWARA University of ...

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