Brain Research, 119 (1977) 487-492
487
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Time course separation of two inward currents in molluscan neurons
JOHN A. CONNOR Department of Physiology and Biophysics, University of Illinois, Urbana, IlL 61801 and University of Washington, Friday Harbor Laboratories, Friday Harbor, Wash. 98250 (U.S.A.)
(Accepted October 4th, 1976)
This report presents studies carried out on molluscan neurons in which all but the proximal 200-300 # m of axon were removed, greatly simplifying the interpretation of voltage clamp results. These neurons display an inward current composed of two distinct phases, the slower of which shows a pronounced calcium dependence and does not appreciably inactivate during large voltage clamp steps of over 100 msec duration. Quantitative studies of membrane conductance changes in central neurons are hindered by the presence of currents arising from the axon or other neurites which are regions where necessary space clamp conditions fail. Separation of true membrane properties from artifacts then becomes a complex problem. Despite the experimental difficulties involved in making measurements, a convincing body of evidence is accumulating that the membrane of molluscan neuron somata displays a sizeable conductance to calcium ions as well as to sodiuml,a,7, s,la,14,19. Several studies reported have minimized the axon currents by ligation of the soma 2,5, focal recording of soma current 15, or by enzymatic treatment and elimination of all but a proximal length of axon12, la. In most of these studies a dependency of the inward current on both Na and Ca was observed, but with the exception of the study of Eckert and Lux 7 where detailed measurements have been made at voltages around threshold, there seemingly has not been a clear indication of a separation in time course of the current carried by each ion species. Experiments were carried out primarily on three identifiable, non-burster neurons (diam. ca. 400 #m) from the nudibranch mollusc ,4rchidoris montereyensis collected intertidally at Friday Harbor, Wash. The neurons occur in a cluster of three cells which occupies the posterior end of the left pleural ganglion. Removal of epineurium and loose connective tissue was facilitated by brief application of pronase 5. Cells to be isolated were then undercut using a pair of fine irridectomy scissors. Following some experiments, cells were injected with cobalt in order to determine the length of axon remaining. Control experiments were done where connective tissue was removed without the use of enzymatic softening to verify that the current pattern was not an effect of pronase. The record of Fig. 2C was obtained in one such experiment. Basic voltage clamp electronics were similar to that reported previously ~. Physiological saline has
488 A
B
L/
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1200nA
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-
--
I 4 0 mV
2om
. . . .
.
12°°nA -
- -
1
~
I
'
, ~
40mY
Fig. 1. Voltage clamp steps from constant holding voltage to levels which activate inward currents. Upper traces, membrane current, lower traces, membrane voltage. Initial part of trace shows zero current and holding voltage. A: cell with intact axon displaying characteristic delayed, sudden turn on of inward current at small depolarization. Holding voltage, --35 mV. B: same cell after removal of distal axon. Holding voltage, ~15 mV. Bottom frames A and B: negative going voltage step showing charging characteristics of the cell.
been described 5 and normally contained 15 m M calcium. Records were taken at 6-8 °C. Fig. 1 illustrates the change in voltage clamp records brought about by undercutting the cell body. Records are from the same neuron. In the intact cell Fig. IA indicates that the magnitude of inward current arising in non-space-clamped axon may be as large as 1/3-1/2 of the maximum total inward current. This fraction is deduced from the magnitude of the inward current which activates after a delay and is most prominent in the top records of Fig. 1A. Such obvious artifacts of poor spatial control are entirely missing where the axon has been truncated as in Fig. lB. Depending upon a number of factors, some of them hard to identify, truncation may not be as effective as the display record in all preparations; however, reductions of at least 5 × in the axon transient are generally obtainable without significant damage to the functioning of the soma. The remaining axon is in all probability not adequately space-clamped over its length but the membrane area represents only a small fraction of the total in the preparation. The current flow following a negative step (Fig. 1, bottom records) settles to a steady level faster where the axon is truncated reflecting a less extensive storage network to charge. Fig. 2A illustrates the primary experimental finding: inward current develops in two distinct phases, a fast one which begins activation in the neighborhood of---20 mV and a slower component which does not become prominent until membrane potential is more positive than 0 mV. The slower current probably activates to some degree at more negative voltages, but the total current record is dominated by the fast transient. The experiment shown is one in which an extra voltage electrode was used to demonstrate internal voltage homogeneity. The slower component is easily camou-
489
A
B
- -
r-
-~_~
C
. . . . .
-
lOOr
-]
A
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Fig. 2. A: voltage clamp records employingtwo internal voltage electrodes. Zero current is marked by the initial portion of the top trace. Holding voltage, --30 mV. For small depolarizations (top frame) a standard inward current time course results. As step size is increased (descending order) the initial transient becomes faster but a slow inward phase develops during its decay time course. B: effect of different holding voltages on total current. Approximately equal test voltages are paired in left and right columns. Holding voltage is ---40 mV (left) and - - 30mV (right). There is little or no evidence of slow inward current in left hand records because of the large outward current flow. C: records from the same cell as in B. 50 mM of TEA applied to bathing solution. The slow inward current is more pronounced. Holding voltage, --30 mV. raged, if the voltage is too far negative, by outward currents which develop over much the same time course. Holding voltages between --35 and - - 2 5 mV are optimal for displaying inward current in this preparation. In this range transient outward current, In, is near complete inactivation6,15 and delayed outward current is also partially inactivated 5. Fig. 2B illustrates this decrease in apparent slow inward current due to more negative holding potential and Fig. 2C shows the apparent increase in the slow current due to partial blockage of outward current by tetraethylammonium ion (50 mM). Presumably the slow inward current is the same in all cases but outward currents are radically different. Experiments on intact cells have shown that the axonal outward current also may cancel the slow inward current. In spite of the relatively small axon area associated with a truncated neuron, it is possible that the complex inward current records of Fig. 2 result from the summation of at least two simpler transients, one arising in the soma and the other from the axon. Differences in time course and reversal potential, in critical levels for activation and in different pharmacological specificities of the two components, might then be explained on geometrical grounds. To examine this question, external recordings of current were made at the soma membrane using a differential recording scheme similar to one described by Kado n. Two sample records are shown in Fig. 3. Fig. 3A is from an intact neuron and is provided to show the degree of suppression of the axon spike in the differential record (top frame). The fast and slow phases are still present, however. Fig. 3B was recorded from a truncated neuron using the same electrode configuration.
490
A
[]
2'0 ms
mV
Fig. 3. S i m u l t a n e o u s recordings of s o m a current (top traces), total current injected into cell (middle trace) a n d m e m b r a n e voltage. A: cell with axon. B: truncated cell (not the same neuron).
The slow inward current is decreased by cobaltous ions, internally or externally applied, and by lowering external calcium ion concentration in the same way that transmembrane calcium current is altered in muscle tissuetT, 19 and in other molluscan neurons. The transition from net inward current to net outward current in the records of Fig. 2 represents not a decrease of the slow inward current, but an increase in outward flowing (potassium) current with time. Analysis of tail currents indicates that the slow inward current, once activated, remains on during voltage clamp steps of up to 100 msec. Fig. 4A illustrates an experiment in which a large activating pulse ( + 3 5
soo
6~
A,
e~ Q o
10o
o A o Q
TIME (msec) 5
10
A
20ms
B
Fig. 4. A: analysis o f tail currents following an activating pule. Ordinate displays time m e a s u r e d after c l a m p of m e m b r a n e voltage to EK. Inset shows tracings o f a n experimental record: heavy trace, total time course of c l a m p ; light trace, negative-going step at 10 × e x p a n d e d sweep. Open square, Io = - - 2 9 0 n A , t = 16 m s e c ; triangle with dot inside, I: = - - 4 7 nA, t = 42 msec; open circle with dot, Io = + 2 9 4 nA, t = 65 msec. B: m e m b r a n e current in calcium (transient inward) a n d b a r i u m (steady inward) saline.
491 mV) was terminated at different times by a return step to the potassium current reversal potential (E~). Net current was inward, approximately zero, and outward. The tail currents for the three situations overlay indicating that the inward component is constant over the time range of the tests. Passive currents were negligible in the time period shown. Return steps to voltages other than EK demonstrated the development of potassium conductance over this time range. Replacement of all the calcium in the bathing solution (15 m M ) with an equal concentration of barium results in a situation where net current is inward for clamp voltages up to + 6 0 mV and undergoes only a 1 0 - 2 0 ~ decline over test periods as long as 5 sec. As the fast sweep record of Fig. 4B shows, there is no significant change in the activation time course or magnitude of the current upon going from Ca to Ba. Although the data presented here have been taken primarily from preparations composed of three specific neurons, the inward current pattern, with some variation in relative magnitudes, has been observed in most of the Archidoris neurons tested. In a closely akin dorid mollusc, Anisodoris, the slow inward current is not at all pronounced in voltage clamp current records of most neurons even at low values of holding potential. Partial blockage of outward current by TEA reveals a strong component of this current is present, however. What may be of most general interest is the strong similarity of the inward current pattern presented here and that of such diverse cell types as cardiac muscle 4A8, eggs of starfish 1° and tunicate 16, preparations in which both Na and Ca ions have been shown to be involved in the generation of excitation. Supported by GB 39946 from the National Science Foundation.
1 Adams, D. J. andGage, P. W., Gating currents associated with sodium and calcium currents in an Aplysia neuron, Science, 192 (1976) 783-784. 2 Alving, B., Differences between pacemaker and nonpacemaker neurons of Aplysia on voltageclamping, J. gen. Physiol., 54 (1969) 512-531. 3 Chamberlain, S. G. and Kerkut, G. A., Voltage clamp analysis of the sodium and calcium inward current in snail neurons, Comp. Biochem. Physiol., 28 (1969) 787-801. 4 Connor, J. A., Barr, L. and Jakobsson, E., Electrical characteristics of frog atrial trabeculae in the double sucrose gap, Biophys. J., 15 (1975) 1047-1067. 5 Connor, J. A. and Stevens, C. F., Inward and delayed outward membrane currents in isolated neural somata under voltage clamp, J. Physiol. (Lond.), 213 (1971) 1-20. 6 Connor, J. A. and Stevens, C. F., Voltage clamp studies of a transient outward current in gastropod neural somata, J. Physiol. (Lond.), 213 (1971) 21-30. 7 Eckert, R. and Lux, H. D., A voltage sensitive persistent calcium conductance in neural somata of Helix, J. Physiol. (Lond.), 254 (1976) 129-151. 8 Geduldig, D. and Gruener, R., Voltage clamp of the Aplysia giant neurone: early sodium and calcium currents, J. Physiol. (Lond.), 211 (1970) 217-244. 9 Hagiwara, S., Ca spike, Advanc. Biophys., 4 (1973) 71-102. 10 Hagiwara, S., Ozawa, S. and Sand, O., Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish, J. gen. Physiol., 65 (1975) 617-644. 11 Kado, R., dplysia giant cell: soma-axon voltage clamp current differences, Science, 182 (1973) 843-845. 12 Kostenko, M. A., Geletyuk, V. I. and Veprintser, B. N., Completely isolated neurons in the mollusk, Lymnae stagnalis. A new objective for nerve cell biology investigation, Comp. Biochem. PhysioL, 49A (1974) 89-100. 13 Kostyuk, P. G., Krishtal, O. A. and Doroshenko, P. A., Calcium currents in snail neurons. 1I.
492
14 15 16 17 18
19
Effects of calcium concentration on the calcium inward current, Pfliigers Arch. ges. Physiol., 348 (1974) 95-104. Krishtal, O. A. and Magura, I. S., Calcium ions as inward current carriers in mollusk neurones, Comp. Biochem. PhysioL, 35 (1970) 857-866. Neher, E., Two fast transient current components during voltage clamp on snail neurones, J. gen. Physiol., 58 (1971) 36-53. Okamoto, H., Takahashi, K. and Yoshii, M., Two components of the calcium current in the egg cell membrane of the tunicate, J. Physiol. (Lond.), 255 (1976) 527-561. Reuter, H., Divalent cations as charge carriers in excitable membranes, Progr. Biophys. molec. BioL, 26 (1973) 1-43. Rougier, O., Vassort, G., Garnier, D., Gargouil, Y. M. and Coraboeuf, E., Existence and role of a slow inward current during frog atrial action potential, Pfliigers Arch. ges. PhysioL, 308 (1969) 91. Standen, N. B., Voltage-clamp studies of the calcium inward current in an identified snail neurone: Comparison with the sodium inward current, J. Physiol. (Lond.), 249 (1975) 253-268.
487
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Time course separation of two inward currents in molluscan neurons
JOHN A. CONNOR Department of Physiology and Biophysics, University of Illinois, Urbana, IlL 61801 and University of Washington, Friday Harbor Laboratories, Friday Harbor, Wash. 98250 (U.S.A.)
(Accepted October 4th, 1976)
This report presents studies carried out on molluscan neurons in which all but the proximal 200-300 # m of axon were removed, greatly simplifying the interpretation of voltage clamp results. These neurons display an inward current composed of two distinct phases, the slower of which shows a pronounced calcium dependence and does not appreciably inactivate during large voltage clamp steps of over 100 msec duration. Quantitative studies of membrane conductance changes in central neurons are hindered by the presence of currents arising from the axon or other neurites which are regions where necessary space clamp conditions fail. Separation of true membrane properties from artifacts then becomes a complex problem. Despite the experimental difficulties involved in making measurements, a convincing body of evidence is accumulating that the membrane of molluscan neuron somata displays a sizeable conductance to calcium ions as well as to sodiuml,a,7, s,la,14,19. Several studies reported have minimized the axon currents by ligation of the soma 2,5, focal recording of soma current 15, or by enzymatic treatment and elimination of all but a proximal length of axon12, la. In most of these studies a dependency of the inward current on both Na and Ca was observed, but with the exception of the study of Eckert and Lux 7 where detailed measurements have been made at voltages around threshold, there seemingly has not been a clear indication of a separation in time course of the current carried by each ion species. Experiments were carried out primarily on three identifiable, non-burster neurons (diam. ca. 400 #m) from the nudibranch mollusc ,4rchidoris montereyensis collected intertidally at Friday Harbor, Wash. The neurons occur in a cluster of three cells which occupies the posterior end of the left pleural ganglion. Removal of epineurium and loose connective tissue was facilitated by brief application of pronase 5. Cells to be isolated were then undercut using a pair of fine irridectomy scissors. Following some experiments, cells were injected with cobalt in order to determine the length of axon remaining. Control experiments were done where connective tissue was removed without the use of enzymatic softening to verify that the current pattern was not an effect of pronase. The record of Fig. 2C was obtained in one such experiment. Basic voltage clamp electronics were similar to that reported previously ~. Physiological saline has
488 A
B
L/
1400nA _~.,~/.~,=~
1200nA
~
-
--
I 4 0 mV
2om
. . . .
.
12°°nA -
- -
1
~
I
'
, ~
40mY
Fig. 1. Voltage clamp steps from constant holding voltage to levels which activate inward currents. Upper traces, membrane current, lower traces, membrane voltage. Initial part of trace shows zero current and holding voltage. A: cell with intact axon displaying characteristic delayed, sudden turn on of inward current at small depolarization. Holding voltage, --35 mV. B: same cell after removal of distal axon. Holding voltage, ~15 mV. Bottom frames A and B: negative going voltage step showing charging characteristics of the cell.
been described 5 and normally contained 15 m M calcium. Records were taken at 6-8 °C. Fig. 1 illustrates the change in voltage clamp records brought about by undercutting the cell body. Records are from the same neuron. In the intact cell Fig. IA indicates that the magnitude of inward current arising in non-space-clamped axon may be as large as 1/3-1/2 of the maximum total inward current. This fraction is deduced from the magnitude of the inward current which activates after a delay and is most prominent in the top records of Fig. 1A. Such obvious artifacts of poor spatial control are entirely missing where the axon has been truncated as in Fig. lB. Depending upon a number of factors, some of them hard to identify, truncation may not be as effective as the display record in all preparations; however, reductions of at least 5 × in the axon transient are generally obtainable without significant damage to the functioning of the soma. The remaining axon is in all probability not adequately space-clamped over its length but the membrane area represents only a small fraction of the total in the preparation. The current flow following a negative step (Fig. 1, bottom records) settles to a steady level faster where the axon is truncated reflecting a less extensive storage network to charge. Fig. 2A illustrates the primary experimental finding: inward current develops in two distinct phases, a fast one which begins activation in the neighborhood of---20 mV and a slower component which does not become prominent until membrane potential is more positive than 0 mV. The slower current probably activates to some degree at more negative voltages, but the total current record is dominated by the fast transient. The experiment shown is one in which an extra voltage electrode was used to demonstrate internal voltage homogeneity. The slower component is easily camou-
489
A
B
- -
r-
-~_~
C
. . . . .
-
lOOr
-]
A
40mY
Fig. 2. A: voltage clamp records employingtwo internal voltage electrodes. Zero current is marked by the initial portion of the top trace. Holding voltage, --30 mV. For small depolarizations (top frame) a standard inward current time course results. As step size is increased (descending order) the initial transient becomes faster but a slow inward phase develops during its decay time course. B: effect of different holding voltages on total current. Approximately equal test voltages are paired in left and right columns. Holding voltage is ---40 mV (left) and - - 30mV (right). There is little or no evidence of slow inward current in left hand records because of the large outward current flow. C: records from the same cell as in B. 50 mM of TEA applied to bathing solution. The slow inward current is more pronounced. Holding voltage, --30 mV. raged, if the voltage is too far negative, by outward currents which develop over much the same time course. Holding voltages between --35 and - - 2 5 mV are optimal for displaying inward current in this preparation. In this range transient outward current, In, is near complete inactivation6,15 and delayed outward current is also partially inactivated 5. Fig. 2B illustrates this decrease in apparent slow inward current due to more negative holding potential and Fig. 2C shows the apparent increase in the slow current due to partial blockage of outward current by tetraethylammonium ion (50 mM). Presumably the slow inward current is the same in all cases but outward currents are radically different. Experiments on intact cells have shown that the axonal outward current also may cancel the slow inward current. In spite of the relatively small axon area associated with a truncated neuron, it is possible that the complex inward current records of Fig. 2 result from the summation of at least two simpler transients, one arising in the soma and the other from the axon. Differences in time course and reversal potential, in critical levels for activation and in different pharmacological specificities of the two components, might then be explained on geometrical grounds. To examine this question, external recordings of current were made at the soma membrane using a differential recording scheme similar to one described by Kado n. Two sample records are shown in Fig. 3. Fig. 3A is from an intact neuron and is provided to show the degree of suppression of the axon spike in the differential record (top frame). The fast and slow phases are still present, however. Fig. 3B was recorded from a truncated neuron using the same electrode configuration.
490
A
[]
2'0 ms
mV
Fig. 3. S i m u l t a n e o u s recordings of s o m a current (top traces), total current injected into cell (middle trace) a n d m e m b r a n e voltage. A: cell with axon. B: truncated cell (not the same neuron).
The slow inward current is decreased by cobaltous ions, internally or externally applied, and by lowering external calcium ion concentration in the same way that transmembrane calcium current is altered in muscle tissuetT, 19 and in other molluscan neurons. The transition from net inward current to net outward current in the records of Fig. 2 represents not a decrease of the slow inward current, but an increase in outward flowing (potassium) current with time. Analysis of tail currents indicates that the slow inward current, once activated, remains on during voltage clamp steps of up to 100 msec. Fig. 4A illustrates an experiment in which a large activating pulse ( + 3 5
soo
6~
A,
e~ Q o
10o
o A o Q
TIME (msec) 5
10
A
20ms
B
Fig. 4. A: analysis o f tail currents following an activating pule. Ordinate displays time m e a s u r e d after c l a m p of m e m b r a n e voltage to EK. Inset shows tracings o f a n experimental record: heavy trace, total time course of c l a m p ; light trace, negative-going step at 10 × e x p a n d e d sweep. Open square, Io = - - 2 9 0 n A , t = 16 m s e c ; triangle with dot inside, I: = - - 4 7 nA, t = 42 msec; open circle with dot, Io = + 2 9 4 nA, t = 65 msec. B: m e m b r a n e current in calcium (transient inward) a n d b a r i u m (steady inward) saline.
491 mV) was terminated at different times by a return step to the potassium current reversal potential (E~). Net current was inward, approximately zero, and outward. The tail currents for the three situations overlay indicating that the inward component is constant over the time range of the tests. Passive currents were negligible in the time period shown. Return steps to voltages other than EK demonstrated the development of potassium conductance over this time range. Replacement of all the calcium in the bathing solution (15 m M ) with an equal concentration of barium results in a situation where net current is inward for clamp voltages up to + 6 0 mV and undergoes only a 1 0 - 2 0 ~ decline over test periods as long as 5 sec. As the fast sweep record of Fig. 4B shows, there is no significant change in the activation time course or magnitude of the current upon going from Ca to Ba. Although the data presented here have been taken primarily from preparations composed of three specific neurons, the inward current pattern, with some variation in relative magnitudes, has been observed in most of the Archidoris neurons tested. In a closely akin dorid mollusc, Anisodoris, the slow inward current is not at all pronounced in voltage clamp current records of most neurons even at low values of holding potential. Partial blockage of outward current by TEA reveals a strong component of this current is present, however. What may be of most general interest is the strong similarity of the inward current pattern presented here and that of such diverse cell types as cardiac muscle 4A8, eggs of starfish 1° and tunicate 16, preparations in which both Na and Ca ions have been shown to be involved in the generation of excitation. Supported by GB 39946 from the National Science Foundation.
1 Adams, D. J. andGage, P. W., Gating currents associated with sodium and calcium currents in an Aplysia neuron, Science, 192 (1976) 783-784. 2 Alving, B., Differences between pacemaker and nonpacemaker neurons of Aplysia on voltageclamping, J. gen. Physiol., 54 (1969) 512-531. 3 Chamberlain, S. G. and Kerkut, G. A., Voltage clamp analysis of the sodium and calcium inward current in snail neurons, Comp. Biochem. Physiol., 28 (1969) 787-801. 4 Connor, J. A., Barr, L. and Jakobsson, E., Electrical characteristics of frog atrial trabeculae in the double sucrose gap, Biophys. J., 15 (1975) 1047-1067. 5 Connor, J. A. and Stevens, C. F., Inward and delayed outward membrane currents in isolated neural somata under voltage clamp, J. Physiol. (Lond.), 213 (1971) 1-20. 6 Connor, J. A. and Stevens, C. F., Voltage clamp studies of a transient outward current in gastropod neural somata, J. Physiol. (Lond.), 213 (1971) 21-30. 7 Eckert, R. and Lux, H. D., A voltage sensitive persistent calcium conductance in neural somata of Helix, J. Physiol. (Lond.), 254 (1976) 129-151. 8 Geduldig, D. and Gruener, R., Voltage clamp of the Aplysia giant neurone: early sodium and calcium currents, J. Physiol. (Lond.), 211 (1970) 217-244. 9 Hagiwara, S., Ca spike, Advanc. Biophys., 4 (1973) 71-102. 10 Hagiwara, S., Ozawa, S. and Sand, O., Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish, J. gen. Physiol., 65 (1975) 617-644. 11 Kado, R., dplysia giant cell: soma-axon voltage clamp current differences, Science, 182 (1973) 843-845. 12 Kostenko, M. A., Geletyuk, V. I. and Veprintser, B. N., Completely isolated neurons in the mollusk, Lymnae stagnalis. A new objective for nerve cell biology investigation, Comp. Biochem. PhysioL, 49A (1974) 89-100. 13 Kostyuk, P. G., Krishtal, O. A. and Doroshenko, P. A., Calcium currents in snail neurons. 1I.
492
14 15 16 17 18
19
Effects of calcium concentration on the calcium inward current, Pfliigers Arch. ges. Physiol., 348 (1974) 95-104. Krishtal, O. A. and Magura, I. S., Calcium ions as inward current carriers in mollusk neurones, Comp. Biochem. PhysioL, 35 (1970) 857-866. Neher, E., Two fast transient current components during voltage clamp on snail neurones, J. gen. Physiol., 58 (1971) 36-53. Okamoto, H., Takahashi, K. and Yoshii, M., Two components of the calcium current in the egg cell membrane of the tunicate, J. Physiol. (Lond.), 255 (1976) 527-561. Reuter, H., Divalent cations as charge carriers in excitable membranes, Progr. Biophys. molec. BioL, 26 (1973) 1-43. Rougier, O., Vassort, G., Garnier, D., Gargouil, Y. M. and Coraboeuf, E., Existence and role of a slow inward current during frog atrial action potential, Pfliigers Arch. ges. PhysioL, 308 (1969) 91. Standen, N. B., Voltage-clamp studies of the calcium inward current in an identified snail neurone: Comparison with the sodium inward current, J. Physiol. (Lond.), 249 (1975) 253-268.
