Mar 7, 1989 - upstroke of the action potential. During each voltage-clamp step the Na+ current activated slowly (seconds) and did not inactivate within many ...
DEVELOPMENTAL
BIOLOGY
134, fig-71 (1989)
Ionic Currents Underlying the Action Potential of Ram pipiens Oocytes LYANNE Department
of Physiology,
Medical
Sciences
C.%HLICHTER
Building, Accepted
University
of Toronto,
Toronto,
Ontario,
Canada
Ms.9 1A8
March 7, 1989
Ionic currents in immature, ovulated Rana pipiens oocytes (metaphase I) were studied using the voltage-clamp technique. At this stage of maturity the oocyte can produce action potentials in response to depolarizing current or as an “off response” to hyperpolarizing current. Reducing external Na+ to l/10 normal (choline substituted) eliminated the action potentials and both the negative-slope region and zero-crossing of the I-Vrelation. Reducing external Cl- to l/10 or l/100 normal (methanesulfonate substituted) lengthened the action potential. The outward current was reduced and a net inward current was revealed. By changing external Na+, Cl-, and K+ concentrations and using blocking agents (SITS, TEA), three voltage- and time-dependent currents were identified, I No, IK and Ic,. The Na+ current activated at about 0 mV and reversed at very positive values which decreased during maturation. Inward Na+ current produced the upstroke of the action potential. During each voltage-clamp step the Na+ current activated slowly (seconds) and did not inactivate within many minutes. The Na+ current was not blocked by TTX at micromolar concentrations. The K+ current was present only in the youngest oocytes. Because IK was superimposed on a large leakage current, it appeared to reverse at the resting potential. When leakage currents were subtracted, the reversal potential for IK was more negative than -110 mV in Ringer’s solution. 1, was outwardly rectifying and strongly activated above -50 mV. The outward K+ current produced an after hyperpolarization at the end of each action potential. Ix was blocked completely and reversibly by 20 mM external TEA. The Cl- current activated at about +lO mV and was outwardly rectifying. ICI was blocked completely and reversibly by 400 PM SITS added to the bathing medium. This current helped repolarize the membrane following an action potential in the youngest oocytes and was the only repolarizing current in more mature oocytes that had lost IK. The total leakage current had an apparently linear I-V relation and was separated into two components: a Na+ current (IN) and a smaller component carried by as yet unidentified ions. 0 1989 Academic Press, Inc. INTRODUCTION
internal Na+ activity increases four- or fivefold, probably because of Na+ influx during the action potentials. The action potentials may play a role in normal oocyte maturation since inhibiting firing delays maturation. The action potential of oocytes from the African clawed frog, Xenopus, shows some similarities to that of Rana oocytes. One difference is that a depolarizing current. must be injected into Xenopus oocytes for several seconds before the regenerative response occurs (Kado et aL, 1979). The action potential can then last many minutes because, in common with Rana, the Na+ channels do not inactivate and there is little or no repolarizing outward current (Baud et al, 1982). In this paper I use the voltage-clamp technique to investigate the ionic currents in early metaphase I oocytes from R. pitiens. At this early stage of maturation the currents are complex and the oocyte does not produce spontaneous, repetitive action potentials like those I described previously for older metaphase I oocytes (Schlichter, 1983a,b). The ionic currents were identified and separated by modifying the external ionic composition and by using drugs that block specific types of ion channels. I have thus identified three time- and voltage-dependent currents, a Na+ current,, a K+ current., and a Cl- current, and a time-independent leakage current that has a large Naf-dependent component. My
Action potentials in oocytes (eggs) were first reported for a tunicate (Miyazaki et al., 1972); however, Maeno (1959) previously showed a nonlinear I-V curve for ovarian oocytes of a toad. Since then, unfertilized eggs of numerous animal species have been found to be electrically excitable at some stage of their meiotic maturation (for review, see Hagiwara and Jaffe, 1979). In some cases, the inward current producing the action potentials is carried solely by Ca2’. The tunicate egg, however, has both Na- and Ca-dependent action potentials (Miyazaki et al., 1972) and oocytes of the frogs, Xenopus laevis (Baud et al., 1982) and Rana pipiens (Schlichter, 1982a,b), have a Na-dependent action potential. I have previously reported several features of the action potential of R. pipiens oocytes (Schlichter, 1983a,b). This is the only oocyte reported to generate spontaneous, repetitive action potentials. The peak of the action potential depends on external Na+ responding in a Nernstian manner to small changes in the external Na+ activity. Repolarization at the end of the action potential depends on external Cl-. As the oocyte matures, two notable changes take place, the action potential lengthens from a few seconds to many minutes and the 59
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Q 1989 by Academic Press, Inc. of reproduction in any form resewed.
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earlier experiments (Schlichter, 198313) suggested that the action potentials are involved in maturation and I will speculate on ways in which the ionic currents could affect ion regulation by the frog oocyte. METHODS
Oocytes. Sexually mature Northern leopard frogs, Rana fipiens, were obtained commercially and stored at 4°C until used. Female frogs were injected intraperitoneally with one or two homologous, macerated pituitaries and intramuscularly with 0.5-3.0 mg progesterone, the lower doses being used as the natural breeding season approaches. After 16-20 hr at WC, ovulated, jellyfree oocytes (1.4 to 1.6 mm in diameter) were removed from the body cavity and stored for 0 to 3 hr in amphibian Ringer’s solution. Experiments were performed at 18 to 21°C. The stage of maturity of oocytes was judged by examining their external appearance and applying criteria as previously described (Schlichter, 1983a). Electrical measurements. For recording, a single oocyte was placed on a nylon mesh mounted in a plexiglass chamber (volume N 0.5 ml) which was continuously perfused. A conventional two-microelectrode voltage clamp was used. Microelectrodes made from thinwalled glass (resistance, 2 to 10 MQ) were filled with either 3 M KC1 (current and extracellular voltage electrodes) or a 2:l mixture of 3 M KC1 and 0.6 M K2S04 (intracellular voltage electrode). Membrane potential was recorded as the differential output of the amplifier for intra- and extracellular recordings (input impedance, >101’ Q; leakage current, ~2 PA) and monitored on an oscilloscope and on a chart recorder (response frequency, >50 Hz, full scale). Membrane current was recorded by a current-to-voltage converter (feedback resistor, 10 MQ) connected to the bath by an Ag/AgCl wire in the outlet well of the perfusion chamber. Because of the high value of the total capacitance of the oocyte membrane (about 0.3-0.4 PF at this stage of maturity) the capacitive transient was usually 10 to 20 msec even with a high gain (1000-2000) from the voltage-clamp amplifier. However, this slow settling time was not a problem for recording the time-dependent currents because of their slow time courses. Solutions. Normal amphibian Ringer’s solution contained (in mM) NaCl, 111; KCl, 1.9; CaCla, 1.1; MgSO,, 0.8; Hepes, 2.5; and was adjusted to pH 7.8 with NaOH. Low Cl- solutions were made by replacing 90% (l/l0 Cl) or 99% (l/100 Cl) of the NaCl with Na-methanesulfonate and KC1 and CaClz were replaced with KN03 and Ca(NO&. For l/10 Na-Ringer’s, 90% of the NaCl was replaced with choline chloride and KOH was used for pH adjustment. The K+ concentration was increased lOfold by adding 17.1 mM KN03. The following channel blockers were used; tetrodotoxin (TTX, Sankyo), tetra-
134,1989
ethylammonium (TEA, Sigma), and 4-acetamido-4’isothiocyanostilbene-2,8-disulfonic acid (SITS, Sigma). Ringer’s solution containing the appropriate drug concentration was perfused through the chamber for a minimum of 10 min, whereas only 3 min was required for complete solution exchange. RESULTS
I have previously shown that the form of the action potential changes as the oocyte matures (Schlichter, 1983a). Although the present paper is restricted to oocytes at metaphase I there are changes in the proportions of the different currents which are pointed out under Results. The youngest ovulated oocytes I obtained do not fire spontaneously; however, Fig. la shows that an action potential can be evoked by injecting a sufficiently long and large depolarizing current and, less reliably, as an off response after hyperpolarizing current is injected. The rapidly rising phase is monotonic whether the action potential is spontaneous or evoked by injecting current. The peak potential observed here (70 to 80 mV) is typical of this stage of maturity (Schlichter, 1983a,b). The membrane potential repolarized and the undershoot at the end of each action po-
a
b
-- -__--
---
-N-v--v-l-
i\nn _
_
-
Ringer’s
--
---
s_lr’---tf
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r---------Y -----
L
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FIG. 1. (a) Action potentials in a young (metaphase I) oocyte elicited by injecting a suprathreshold depolarizing current pulse (left) or as an “off response” to a hyperpolarizing current pulse (right). In this and all subsequent figures voltage scales are in millivolts, currents in nanoamperes, and time scales in seconds. (b) Ionic dependence of action potentials (upper traces) in a different metaphase I oocyte evoked by injecting depolarizing current pulses (lower traces). Na+ was substituted by choline and Cl- was substituted by methanesulfonate. Scales indicated for Ringer’s apply to all traces in b.
LYANNE
C. SCHLICHTER
Ionic
tential was also typical of oocytes at this stage of maturity (early metaphase I). Eflects of External Na and Cl The form of the action potential depended on the external concentrations of Na+ and Cl- as shown in Fig. lb. In normal Ringer’s solution, injecting current resulted in a rapid, regenerative depolarization that reached a large positive potential but spontaneously repolarized despite the maintained current injection. Note that responses to repeated stimuli reached the same peak potential. At the end of each current pulse there was a small afterhyperpolarization and then the membrane potential returned to the resting level. Reducing the external Na+ concentration eliminated the regenerative depolarization, strongly suggesting that an inward Na+ current produces the upstroke of the action potential. The afterhyperpolarization was still present. In normal external Na but with reduced external Cl- an all-or-none regenerative depolarization was easily evoked and always outlasted the current injection. The action potentials were long (-20 set) and became longer when Cl- was further reduced. This is evidence that an influx of Cl- (outward Cl- current) produces the repolarization of these young oocytes. Note that external Cl- had no effect on the peak potential reached. Voltage clamp recordings of the total current were consistent with the interpretation that an inward Na+ current produced the upstroke and an outward Cl- current repolarized the membrane. To explain these data I describe the membrane currents produced by voltageclamp steps in Ringer’s solution (Fig. 2a) and refer to the current-versus-voltage (I-V) relations in Fig. 2b. In constructing the I-V relations in Fig. 2 the current was measured at two times during each clamp pulse, times chosen to represent early (at 2 set) and late (at 10 set) currents. Results are described separately for each ionic solution. Ringer& In normal Ringer’s, under voltage clamp (Fig. 2a), the membrane current displayed a complex time course since it represented the sum of several time-dependent currents. With small depolarizations there first appeared an outward current that increased in amplitude and rate of rise as the membrane was depolarized further. The current trace at +26 mV (closed arrow) clearly shows this early outward current (I will show later that this is a K+ current). With further depolarization the early outward current leveled off, and then a second increase in outward current began (see current at +46 mV). It is shown that this late outward current is carried by Cl-. At slightly more positive potentials the total outward current decreased slightly before rising dramatically. This transient de-
Currents
in
Frog
00&e
61
crease is caused by a superimposed inward Na+ current as shown below. I-V relations constructed from these voltage-clamp data are consistent with the existence of the regenerative depolarization observed in normal Ringer’s in Figs. la and lb. When measured at 2 set (Fig. 2b, circles), the I- Vrelation had a negative-slope region and crossed the zero-current axis between +60 and 65 mV. However, at 10 set (circles) there was no negative-slope region or zero-crossing and since both are needed for a regenerative response to occur, the action potential would not be expected to last as long as 10 set in Ringers. This is consistent with Figs. la and lb (Ringer’s) in which each action potential reached a very positive peak potential and then began to repolarize in 1 to 3 set as the late outward current increased. At the end of each action potential the afterhyperpolarization probably resulted from the large outward tail currents observed in Fig. 2a (left panel). The tail current lasted several seconds after the repolarization and is discussed further under K+ Current. Low sodium. Reducing the external Na+ concentration to l/10 normal eliminated the regenerative response to depolarizing current (Fig. lb). During each current pulse the membrane depolarized approximately like a passive resistance. With the largest current pulses a sharp partial repolarization was observed as though an outward (repolarizing) current was rapidly activated. The voltage-clamp currents (not shown) and I-V relations (Fig. 2b) are consistent with this interpretation. I have previously shown (Schlichter, 1983a,b) that the peak of the action potential is close to the equilibrium potential for Na” (ENa) and responds to changes in external Na+ as predicted by the Nernst equation. In l/10 Na+ solution ENa should be about f20 mV. A fast component of outward current was seen at potentials more positive than +20 mV in addition to the delayed outward current that strongly activated at about +24 mV. The I-Vrelations no longer displayed a negative-slope region or a zero-crossing either at an early time (Fig. 2b, 2 set) or later (10 set); therefore, no action potential would be expected to occur. The presence of action potentials, inward current, a negativeslope region and zero-crossing in the I-V relation depends on external Na. This is consistent with a Nat current, henceforth called I,,, producing the upstroke of the action potential. Low chloride. The duration of each action potential depended on the external Cl- concentration (Fig. lb); that is, reducing external Cll from 115 mM (Ringer’s) to 11.5 mM (l/10 Cl) and to 1.1 mM (l/100 Cl) increased the duration of each action potential but had little effect on the peak potential or the rate of rise. This suggests that the outward current repolarizing the mem-
DEVELOPMENTALBIOLOGY
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VOLUME 134,1989
Ringer’s
a
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0
-10
-
I
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2 set
bW, 0 r * 0
115 11.5
115 115
ICII,
225
115 115
200
11.5 1.15 175
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at
10 set
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50
FIG. 2. Effects of external Cl- and Na’ on membrane currents from the oocyte in Fig. lb. (a) Normal Ringer’s; Na, 115 mM; Cl, 115 mM, V,, -32 mV. In this and all subsequent voltage-clamp records the current pulses are 10 set long except as indicated. Leakage currents were not subtracted. Voltage steps are at approximately IO-mV intervals; exact values can be read from the current-versus-voltage relations in b. Closed arrow indicating current at +26 mV marks the threshold for the late outward current (ICI). Arrow at +46 mV shows inward current (Ina). (b) Current-versus-voltage (Z-V) relations for the currents from the oocyte in Fig. lb. (Top) Current was measured at 2 set after the beginning of the voltage-clamp pulse. (Bottom) Current was measured at the end of each lo-see-long voltage-clamp pulse. Solutions were the same as for Fig. lb. Normal Ringer’s (0); l/10 Na, choline substituted (*); l/10 Cl, methanesulfonate substituted (A); l/100 Cl (0). In this and all subsequent I-Vcurves the symbols are larger than the actual uncertainty in the measurements and the curves were fitted by eye.
brane is carried mainly by Cl- influx. This interpretation is supported by voltage-clamp analysis of the current. Reducing external Cl- to l/10 normal exposed
an inward current (IN,) and there existed a net inward current between +38 and 73 mV. The delayed outward current first appeared at a more positive voltage than
LYANNE C. SCHLICHTER
Ionic Currents in Frog Oocyte
in Ringer’s or l/10 Na solution. This is expected for a Cl- current since the driving force for Cl- influx will be much reduced in l/10 Cl. At short times (2 set, Fig. 2b) the I- Vcurve in l/10 Cl (triangles) had a negative slope between +22 and 64 mV, which appears to correspond with an inflection in each action potential at about +25 mV. When the I-Vcurve for hyperpolarized potentials was extrapolated linearly it crossed the curve at +76 mV, a value that agreed well with the peak of the action potential (+75 mV) and was close to the reversal potential for the Na+ current (+78 mV). Further reducing external Cl- (l/100 Cl-Ringer’s) decreased Cl- influx (outward Cl- current), hence the net inward current was larger and I& was evident at a less positive potential (-1 mV). The I-V curve (Fig. 2b, squares) had a negative slope between +lO and +58 mV, about the same potential as the inflection in the rising phase of the action potential (about +lO mV, Fig. lb). The inward Na+ current again reversed at +75 mV; about the same as the peak of the action potential (Fig. lb) and the potential at which a linear extrapolation of the leakage current crossed the I-Vcurve (Fig. 2b, squares). Results similar to those in Figs. 1 and 2 were observed in over 30 young oocytes, although there were quantitative differences in the proportions of each current present. These differences corresponded with a loss of the K+ current early in maturation, followed by loss of the Cl- current as previously described (Schlichter, 1982a,b, 1983a,b). Overall, reducing the external Cl- concentration reduced the outward current and dramatically increased the duration of the action potential. This is evidence that Cl- entry (an outward Cl- current, ICI) helps to repolarize the action potential. In Fig. 2 the effects of changing external Na+ and Cl- were seen as changes in complex current-versus-voltage relations and the qualitative dependence of the currents on Na+ and Cl- are apparent. For further analysis of the currents it was necessary to pharmacologically separate the various types of ionic currents. To separate the currents, each oocyte must act as its own control, since basal ionic currents vary considerably between oocytes of different ages. The holding potential was constant throughout experiments on each oocyte so that currents at each test potential could be directly compared. Holding potentials were chosen to minimize the amount of continuously injected current (ions) to reduce changes in cytoplasmic ions during the long experiments required. Separate experiments to isolate each current type were necessary because of the drug combinations required and typical results from individual oocytes will be presented. Because of developmental differences and different drug combinations, quantitative comparisons between different oocytes should not be made.
Separating
63
the Ion Currents
In the previous section evidence was presented for two currents underlying the action potential, I&, and ICI. In this section I show that a third time- and voltage-dependent current, IK, is present. Then, by selectively blocking Icl or Ix, each current (LJ, Ix, INa) could be separated from the time-independent leakage current (I,). Cl- current. By reducing external Cl- it was shown that a large part of the outward current is carried by Cl-; therefore, the effect of a drug known to block Cltransport in some cells was investigated. SITS is a well-established inhibitor of anion exchange in many tissues, including red blood cells; however, SITS also blocks a voltage-dependent Cl- channel in eel electroplax membranes (White and Miller, 1979). Figure 3 shows the effect of SITS on the late outward current (ICI). This oocyte was older than those in Figs. 1 and 2 and it showed the characteristic loss during maturation of the low-threshold outward current (shown below to be a K+ current). Hence in Fig. 3, voltage steps below the resting (zero-current) potential or to slightly depolarized potentials did not evoke time-dependent currents. In normal Ringer’s (Fig. 3a) INa first appeared at about +lO mV (closed arrow) and was seen as an early, inwardly directed current. Icl also activated at about +lO mV (open arrow) producing a delayed outward current. The corresponding I- Vrelation measured at 10 set (closed triangles, Fig. 3d) shows a strongly increasing outward current above about +30 mV and no negative-slope region. However, at shorter times (not shown) there was a negative-slope region and zerocrossing in the I-V relation and an action potential briefer than 10 set, reaching a peak of about +60 mV could be easily evoked by each depolarizing current pulse. Adding SITS (100 PLM, Fig. 3b; 200 PM, Fig. 3~) greatly reduced the Cl current (ICJ in a dose-dependent manner. SITS treatment exposed the inward (Nat) current beginning at about 10 mV and increasing with voltage, then decreasing, and reversing at +58 to 59 mV (see arrows). This value of the Na+ reversal potential (I&,) which was less positive than that for the younger oocyte in Fig. 2 is expected, since I have previously shown that ENa decreases as the oocyte matures (Schlichter, 1983b). The I-V curves in 100 and 200 PM SITS showed net inward current from around +20 to 50 mV and strong negative-slope regions, hence action potentials occurred in the presence of SITS. Under open circuit, in 200 PM SITS, the oocyte spontaneously depolarized to about +58 mV and did not repolarize during the 3-min recording period. The effect of SITS was fully reversible; washing for 10 to 15 min in normal Ringer’s restored
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a
b
FIG. 3. SITS blocks the late outward current (I&. SITS (4-acetamido-4’-isothiocyanostilbene-2,2nic acid) was added to normal Ringer’s solution. This oocyte was older than those in Figs. l-3 and lacked the initial outward K+ current. Holding potential (V,,) was the resting potential of -20 mV throughout. (a) Normal Ringer’s. Both outward current (1~1, open arrow) and inward current (INa, closed arrow) activated at about +lO mV in this oocyte. (b) 100 &f SITS added to normal Ringer%. Arrow at +lO mV indicates net inward Na+ current. Outward Cl- current is indicated by open arrow at +50 mV. (c) 200 &f SITS. Inward current was larger since ICLwas further reduced. Arrow at +53.5 mV indicates a small remaining 1~1. For complete elimination of 1~~. 400 to 500 & SITS was used in subsequent experiments. Closed arrow at +58 mV shows the reversal potential of the Na+ current (Exa). (d) I- Vrelations in Ringer’s (v), 100 j&f SITS (0), or 200 &fSITS (A) measured at the end of each lo-set-long current.
ICI. A similar block by SITS was observed in at least 40 cells. To directly isolate the Cl- current it would be necessary to block both I, and INa and I show below that Iz could be blocked by 20 mM TEA added to the bath. However, INa could not be easily eliminated since TTX did not block it and substituting a nonpermeant cation for Na+ (e.g., choline) eliminated the inward current but left a large outward cation current through the Na+ channel. Therefore, as shown in Fig. 4, to separate Icl, first 1, was blocked with TEA and the remaining current (INa + I,, + I]) was recorded. Then 400 @f SITS was added to completely block 1~~and the remaining current (&a + 1,) was subtracted on a point-by-point basis to reveal ICI. The general features of Ic, are that the threshold voltage was about 0 mV; Icl was always outward above threshold, indicating that the Nernst potential for Cl- is negative (- -24 mV for mature oocytes, Jaffe and Schlichter, 1935); and that Icl increased slowly with time and showed no sign of inactivating during a lo-set-long pulse. The calculated tail current was very small at the holding potential of -40 mV fur-
ther showing that the reversal potential of this current is close to the Nernst potential for Cl-. The I-Vrelation for I,1 in this cell (Fig. 4b) shows an outwardly rectifying current that activates at about 0 mV and increases with voltage. K+ Current
This section describes the voltage-dependent outward K+ current which is observed when Icl is blocked with SITS and which is superimposed on the Na+ current. Similar results were seen in at least 30 cells. Figure 5a shows currents in normal Ringer’s which contains 1.9 mM K. With depolarization the amplitude and the rate of rise of the time-dependent outward current increased up to about +20 mV. At the end of each voltage-clamp pulse there was a long tail of current (marked by stars) that slowly decayed toward zero. The initial amplitude and the rate of decay of the tail current increased with voltage between -40 mV and -10 mV, suggesting a voltage-dependent conductance increase over this potential range. At about +20 mV the
LYANNE
Ionic Currents in Frog Ooc@e
C. SCHLICHTER
65
a 20 1
2
loo32 50-
1601401201006060-
b
FIG. 4. Cl- current isolated by blocking Is and subtracting I Na. (a) Currents were recorded during lo-set-long voltage-clamp pulses to the voltages indicated above each set of traces. All solutions contained 20 mM tetraethylammonium (TEA) to block the K+ current. The heavy curves are currents in normal Ringer’s + TEA and represent IN, + Ic, + Il. The light curves are currents obtained after Ici was blocked with 400 pMSITS and represent INa + I,. The dotted curves were obtained from point-by-point subtractions of the light curves from the heavy curves. (b) Current-versus-voltage relation for ICI constructed from the dotted curves in part a, measured at the end of each lo-set-long pulse.
inward IN, appeared and increased with voltage, obscuring the K+ current and decreasing the tail current amplitude. The inward current reversed at +60 to 65 mV. These voltage-dependent changes in current are reflected in the I-V relation (Fig. 5d) which shows an outward voltage-dependent current activating above the zero-current potential and a negative-slope region owing to the activation of the voltage-dependent Na+
current. The net current reversed at about +60 mV and action potentials evoked by current injection peaked at about +60 mV in this bathing solution. Currents below the resting potential are explained in more detail under Leakage Current. High external K+ (19 mM) decreased the amplitude of the outward currents and tail currents (data not shown), owing to the smaller driving force on the K+
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FIG. 5. Effect of external K+ and tetraethylammonium (TEA) on the K+ current in a young oocyte. All solutions contained 500 n&f SITS to block Icl. Holding potential was -40 mV in all solutions. Arrows labeled -90 are explained in the text. Other arrows show the threshold of the inward Na+ current (20, -10 mV) and reversal potential for the Na+ current (62,55 mV). Tail currents (stars) are explained in text. (a) Normal Ringer’s solution. (b) Normal Ringer’s with 20 m&f tetraethylammonium (TEA). (c) I-V relations measured at the end of each IO-set-long voltage-clamp pulse. (d) Current-versus-voltage relation for Ix constructed by point-by-point subtraction of (I,, + 4) from (I,, + I, + Ix) as explained in the text.
ions (Nernst potential less negative). Thus, &a was less obscured by IK and the inward current was larger (see I-V relation in Fig. 5~). This is evidence that the out-
ward voltage-dependent current is carried by K+. TEA blocks some, but not all, K+ currents in a wide variety of tissues (Thompson and Aldrich, 1980). Therefore, I
LYANNE C. SCHLICHTER
Ionic
tested TEA on the K+ current in the oocyte. Adding 20 mM TEA to the bathing solution eliminated the early outward current and the tail current (Fig. 5b). By blocking IK, TEA exposed INa at a more negative potential than in normal Ringer’s (-10 mV compared with +20 mV). Above this activation level the tail currents were always inward and were probably carried by Na+. The effects of external K+ and TEA can be seen in the I-V relations measured at the end of each lo-set-long voltage-clamp pulse (Fig. 5~). In normal Ringer’s (closed triangles) the I-Vrelation showed outward rectification between the holding potential (-40 mV) and +lO mV because of the large increase in IK. With 19 mM K+ (open circles) the outward current was smaller above the resting potential and the net inward current appeared larger. In TEA-Ringer’s (open triangles) the outward K+ current disappeared and there was no outward current below +53 mV. Because the net current was inward at all voltages below +53 mV there was no stable negative resting potential: under open circuit the membrane potential went spontaneously to +55 mV. This is expected since there were no outward currents to repolarize the membrane. At voltages below the resting potential the effects of external K+ and TEA indicate two separate currents: A TEA-insensitive increase in inward current with time appeared at -90 mV and below (Fig. 5a). Both the activation voltage and magnitude of this current were unaffected by a lo-fold increase in external K+. It will be shown below that the TEA-insensitive current is the sum of two components that are both time independent: a Na+ current and an uncharacterized leakage current. The K+ current that was blocked by external TEA produced the early outward current and long tail currents which were reduced by increasing the external K concentration and blocked by TEA. To isolate IK during each voltage-clamp pulse it was necessary to use a subtraction procedure like that used for Ic, in Fig. 4. (Individual current traces will not be shown.) The cell was held at -40 mV between test pulses and the holding current was zero. All solutions contained 400 pM SITS to block ICI. Currents were first recorded in Ringer’s and represent INa + I, + I,. Then 20 mM TEA was added to block IK and the remaining current represents INa + 1,. Finally, point-by-point subtraction of (INa + 1,) from (1x + I& + 1,) was performed to yield IK in isolation at each voltage. Figure 5d shows the I-V relation for the isolated K+ current obtained from this subtraction. Both outward rectification and a decrease in slope conductance at highly positive voltages are apparent. Above the holding potential of -40 mV the K+ current activated strongly, increasing in amplitude and rate of rise with voltage, as is typical for a voltage-dependent outwardly rectifying K’ current.
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The tail currents were outward and increased with voltage to about +40 mV and then decreased, suggesting a decrease in Kf conductance at very positive potentials. Leakage Currents
Figure 6 shows currents at voltages below the activation voltages for INa (about -10 mV) and for Icl (about 0 mV). In this early metaphase I oocyte the resting (zerocurrent) potential was about -43 mV in normal Ringer’s. In Ringer’s (Fig. 6a) small voltage excursions (values indicated at the right) elicited time-dependent inward and outward currents accompanied by long tail currents. Compared with normal Ringer’s the time-dependent currents and tail currents were somewhat reduced by raising external K+ (Fig. 6b) showing they are carried in part by K’. This is further demonstrated by the complete block of the time-dependent currents at -90 mV and above by 20 mM TEA (Fig. 6~). The I-V relation for this K+ current was obtained (Fig. Sf) by a subtraction procedure; that is, the total current was recorded at each voltage (stars) and then IK was blocked with 20 mM TEA and the remaining current (dots) was subtracted from the total current to leave only Ix (open circles). When this was done, IK was outward at all voltages above -110 mV, the most negative voltage tested. This is consistent with the previously observed reversal of the K+ current at -70 mV in 10 times normal external K+, indicating an & of about -130 mV in normal Ringer’s. Therefore, Ix should be outward at all voltages above about -130 mV, as observed. The K+ current strongly activated at about -50 mV and showed outward rectification. When all Ix was blocked by TEA (Fig. 6c) there remained large inward currents both during and after the pulse. The current was independent of time except at very negative potentials (see -110 mV). This remaining inward current was greatly reduced by replacing external Na+ with choline (Fig. 6d), suggesting it is carried mainly by Na+. The Na-dependent leak (IN) was separated from the remaining leak (1,) in the I-Vrelations in Fig. 6e. Both leakage currents had a fairly linear I-V relation but 1, reversed at a negative potential and IN reversed at a very positive potential (probably close to the Nernst potential for Na+). Therefore, IN represents only the Na+ influx component of leak and I, represents the remaining component of leak not including the Na+ influx component. At the resting potential (about -43 mV), Ix was nearly equal to IN. In normal Ringer’s EK was about -130 mV and ENa was about +55 mV; therefore, the resting potential was about halfway between EK and ENa. This suggests that the permeabilities to Na+ (&a) and to K+ (&) are about equal at rest (from the Goldman-Hodgkin-Katz equation).
DEVELOPMENTAL BIOLOGY
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0
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VOLUME 134,1989
--_-----
--
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-90
-~ c
-20
3
0
e
___-_--
. r
IN
o
IN
-20 I
--------
--------
----
--
-110
aI, L- II
-
FIG. 6. Separating the K+ current (Iz) from the leakage currents. All solutions contained 400 j&SITS to block Icl. Holding potential was -40 mV throughout. The voltage-clamp potentials are indicated at the right of each set of currents. Currents at -30 mV were elicited by lo-set-long depolarizing steps. All other currents are in response to hyperpolarizing steps. All voltages are more negative than the thresholds for activating Na+ and Cl- currents. (a) Ringer’s; 1.9 mMK; resting potential (V,) -43 mV. (b) High-K Ringer’s; 19 mMK, V,, -45 mV. (c) Ringer’s with 20 mM TEA; 1.9 mM K; V,, +55 mV. (d) Na-free choline Ringer’s; 1.9 n&f K, V,, -40 mV. (e) I-V relations measured at 10 set; in TEA solution (dots), called [I, + It] which represents the Na-dependent leak and remaining leak, respectively. Currents in Na-free solution (stars) represent I,; and the difference (open circles) represents IN, the Na-dependent leak. (f) I- Vrelations; in Ringer’s (stars) represents [Iz + IN + I;]. Currents in TEA solution (dots) include [IN + Id and the difference, (open circles) represents Ik.
The results in Fig. 6 show that 1k is an outward current that decreases with time at voltages below the resting potential, but is superimposed on a large inward leakage current. Therefore the net current in Ringer’s (Fig. 6a) was inward, increasing with time. Above the resting potential, & was outward, increasing with time, and was superimposed on an outward I, and inward IN. Therefore, the net current during the pulse was outward, increasing with time. Tail currents in Figs. 6a and 6b can also be explained in this way. After a hyperpolarizing pulse (see -50, -90, -110) the tail current was inward because I, was reduced by the hyperpolarizing pulse. Then, as outward IK increased at the more positive potential, with time the total tail current decayed toward zero. After a depolarizing pulse to -30 mV the tail current was initially large and outward because of the voltage-dependent activation seen in Fig. 6f. Returning to the more negative resting potential then caused K+ channels to close and the tail current decayed toward zero. Similar results were seen in at least 15 cells. Naf Current The Na+ current could be isolated in the presence of SITS (to block ICI) and TEA (to block Iz). Figure 7a shows the isolated Na+ currents in normal Ringer’s. These currents are very similar to those in Fig. 5c for a different oocyte bathed in the same solution. Na+ cur-
rent was first evident at +lO mV, increased in amplitude with further depolarization, then decreased, and reversed at +55 mV in Fig. 5c and about +62 mV in Fig. 7a. I-Vrelations measured at 10 set (Fig. 7b, open triangles) were linear below the resting potential in the presence of SITS and TEA and, when extrapolated, crossed at the observed reversal potential for INa. Therefore, it can be assumed that the entire I-V relation for the leakage currents is linear. Next, 6 pM tetrodotoxin (TTX) was added. TTX is a specific toxin that blocks most types of voltage-dependent Na+ channels with Ki’s in the nanomolar to micromolar range. However, in the frog oocyte even very high concentrations of TTX had little or no effect on the Na+ current. There was no change in the resting potential (-12 mV), in the activation potential for INa (- +8 mV), or in its reversal potential (61 to 62 mV). I-Vrelations measured at 10 set (Fig. 7c, closed circles) coincided through most of the voltage range, with at most a 9% decrease in the peak inward current with TTX. The lack of effect of micromolar TTX concentrations was confirmed in eight cells in which INa was isolated from Ix and Ic,. Figure 7a shows no sign of Na+ current inactivation during a lo-set long pulse. Figure 7c shows that the action potential in normal Ringer’s containing SITS and TEA remained close to ENa for many minutes, which shows that INa did not inactivate during this time. This conclusion was borne out by a long voltage-
LYANNE
Ionic Currents in Frog Ooc&
C. SCHLICHTER
a
-,o3
-100
-75
-50
-25 (mV)
FIG. 7. Isolated Na+ currents (INa) and the effects of TTX. Ringer’s solutions contained 500 pM SITS to block 1~1, and 20 mMTEA to block 1,. The holding potential was the resting potential (-12 mV) throughout. Voltages are indicated for the threshold of INa (solid arrow) and the reversal potential (ENa, open arrow). (a) Ringer’s solution. (b) I-V relations measured at the end of each lo-set-long pulse in Ringer’s (V) and TTX (0). (c) Under current clamp a prolonged action potential was evoked by a brief depolarizing current pulse. (d) At a voltage clamp potential of f38 mV a prolonged inward current was elicited, which did not inactivate even during this 2.3min-long pulse.
clamp pulse of several minutes (Fig. 7d) which showed INa developing smoothly with time over several seconds without signs of inactivation during the entire voltageclamp pulse. DISCUSSION
Results of the present study show that there are at least three voltage- and time-dependent currents underlying the action potential mechanism of metaphase I oocytes from the frog, R. pipiens. They are a Na+ current (INa) that is insensitive to TTX, an outward K+ current (IK) sensitive to externally applied TEA, and an outward Cl- current (ICI) sensitive to SITS. Until recently the Na+ channel was considered to be a highly conserved protein that differed little in its selectivity and basic kinetics of activation and inactivation from tissue to tissue and species to species. Variations among cell types in the voltage dependence and sensitivity to TTX have been described (Cahalan, 1980). However, from results represented here and in my previous papers (Schlichter 1983a,b) it seems the Na+ channel of the Rana oocyte differs from most other voltage-dependent Na+ channels in several respects. (1) The Na+ current in the oocyte is not sensitive to micromolar concentrations (6 &f) of TTX, suggesting that the toxin receptor is absent or modified in the channel
69
protein. TTX-insensitive Naf channels have been found in other cell types including tunicate eggs (Okamoto et ah, 1976) and immature oocytes of the frog, Xenopus Zaevis (Kado et al, 1979). (2) The Na+ channel in Rana oocytes does not inactivate. As the oocyte matures, both I, and I, disappear and the action potential can then last at least 30 min (Schlichter, 1983a). Lack of Na+ current inactivation may be common to frog oocytes. Mature oocytes from Rana clamitans (my unpublished results) and immature oocytes from X Zaevti (Kado et aZ., 1979; Baud et al., 1982) can also produce Na+ action potentials that last many minutes because the Na+ channels do not inactivate. (3) The kinetics of activation are extremely slow. Time constants for Na+ activation (7,) in other excitable cells are of the order of milliseconds, whereas T,,, is of the order of seconds in Rana oocytes. (4) The Na+ current depends on voltage in the usual manner, but the I-V relation is shifted to more positive potentials. The threshold for INa is near 0 mV and the peak inward current occurs at +40 to 50 mV. This is 10 to 20 mV more negative than the Na+ current in oocytes of X laevis (Baud et aZ., 1982) but almost 75 mV more positive than for the Na+ current in tunicate eggs (Okamoto et aZ., 1976) and in nerve and muscle cells. The voltage-dependent Cl- channel in immature R. p@iens oocytes appears to be a novel mechanism for repolarizing the membrane to end an action potential, since most excitable membranes repolarize because the inward-current channels (Na+ or Ca’+) inactivate and because of an increase in outward K+ current. The most similar Cl- channel appears to be that in electroplax from marine skate or ray. Torpedo electroplax Clchannels incorporated into planar lipid bilayers are blocked by SITS (inhibition constant about 100 PM) in a reversible manner (White and Miller, 1979) much like the channels in the oocyte. Cl- currents have been reported for amphibian oocytes. Immature Xenopus oocytes have a standing electrical current that is blocked by treatments presumed to block Ca2+ entry (Robinson, 1979) and voltage-clamp experiments have shown directly the presence of a Ca2+-dependent Cl- current (Barish, 1983). Mature oocytes of toads and frogs respond to fertilization or activation with a Cl-dependent depolarization (Maeno, 1959; Ito, 1972; Cross, 1981; Grey et ak, 1982; Charbonneau et aL, 1983). This Cl- permeability was not thought to be voltage dependent because it was not activated simply by changing membrane potential (Cross and Elinson, 1980; Schlichter and Elinson, 1981) but was evoked by treatments expected to increase internal Ca’+; i.e., Ca2+ injection, Ca ionophore (A23187), and sperm entry (Cross, 1981). However, once this Cl- conductance is evoked, it shows voltage dependence (Jaffe
70
DEVELOPMENTAL BIOLOGY
and Schlichter, 1985). After egg activation, the Clchannels appear to open in a wave-like fashion around the oocyte surface beginning in the animal hemisphere (Jaffe et al., 1985), perhaps following the propagating wave of high free Ca” activity that passes across the egg (Busa and Nuccitelli, 1985). A voltage-dependent Cl- channel has been described in immature oocytes of Rana esculenta (Taglietti et al., 1984) and X. Zaevis (Peres and Bernarclini, 1983) but that Cl- current is only activated by hyperpolarization below about -80 mV. Hence, its voltage dependence is opposite to that in R. p&ens oocytes. Effects of SITS were not reported for R. esculenta or Xerwpus oocytes and neither species has been reported to produce action potentials like those of R. pipiens. The voltage-sensitive K+ channel of R. pipiens oocytes contributes about half of the conductance at the resting potential of the youngest (metaphase I) cells examined (the other half is mainly a voltage-independent Na+ conductance). I, was outward at all potentials used in the present study but the time-dependent net current was complicated by the large leakage current on which IK is superimposed. This caused the large “on” currents and “tail” currents to appear to reverse at the resting potential rather than at EK. IK was completely and reversibly blocked by 20 mM TEA added to the bathing medium. This is similar to the block of the K+ current in node of Ranvier from R. pipiens (Armstrong and Hille, 1972) in which, however, TEA blocks from the outside or the inside of the cell. When I, was observed at voltages below those that activated I,, and ICI, a small amount of 1, was present even at very negative potentials (e.g., -100 mV). The amount of current (K+ conductance) strongly increased above -50 mV and the current was outwardly rectifying. During each voltageclamp pulse 1, activated slowly (seconds), did not appear to inactivate, and decayed slowly at the end of each voltage step. The R. pipiens oocyte is a dynamic cell in which maturation continues even during the course of an electrical recording. Maturation produces several changes that can complicate the study of the voltage-dependent Na+, K+, and Cl- currents, since the currents change continuously as the oocyte matures. First the Kf current, then the Cl- current decrease and disappear before metaphase II (Schlichter, 1983~). The Na+ conductance increases with maturation and then decreases when the oocyte is mature and ready to be fertilized (Schlichter, 1982a,b; Jaffe and Schlichter, 1985). Furthermore, the Nernst potential for Na+ (ENa) decreases with maturation, corresponding to an increase in internal Na+ activity that appears to result from the spontaneous action potentials. The action potentials may play a role in maturation since preventing firing delayed maturation
VOLUME 134,1989
(Schlichter, 198313). How ion fluxes through the ion channels described in this paper could affect oocyte maturation is not known; however, there are several interesting possibilities. (1) Na+ accumulation might transiently activate the Na+/K+ATPase to pump K+ into and Na+ out of the oocyte thus accumulating K+. (2) A rise in internal Na+ might reduce Na+/H+ exchange across the plasma membrane or organelle membranes by decreasing the gradient for Na+. (3) If there is a Naf/Ca2+ exchange mechanism in the membrane of the cell or an organelle, an increased Na+ concentration could cause Ca2+ to be released into the cytoplasm. A transient rise in Ca2+ has been observed following progesterone treatment of Xenps oocytes (Wasserman et ab, 1980). It may be important to charge up Ca2+ stores inside the cell or to increase the resting Ca2’ levels, for example, to promote the fertilization wave of Ca2+ that passes across the frog oocyte (Busa and Nuccitelli, 1985) and opens Cl- and K+ channels (Jaffe et al., 1985). (4) Clwill enter the oocyte during the action potential repolarization phase and might accumulate in the cell. One possibility is that Cl- accumulation stimulates Cl-1 HCO, exchange which could increase internal pH such as observed for Xenopus oocytes during maturation (Houle and Wasserman, 1983). (5) A small Ca2’ current may be present though undetected in my voltage-clamp experiments. If it is voltage sensitive then the action potentials could increase Ca2+ influx and either charge up intracellular stores or increase the resting Ca2+ levels. (6) The changes in membrane potential during maturation and during action potentials might directly regulate some process during oocyte maturation, While all of these possibilities are speculative, the R. pipiens oocyte is an extremely rich system in which to study regulation of ion-channel activity and to ask whether factors controlling oocyte maturation also regulate the ion channels. It is a pleasure to thank Drs. J. Dainty, R. P. Elinson, and L. A. Jaf?e for stimulating discussions. This work was supported by the Natural Sciences and Engineering Research Council (Canada) and the Medical Research Council (Canada). REFERENCES ARMSTRONG, C. M., and HUE, B. (1972). The inner quaternary ammonium ion receptor in potassium channels of the node of Ranvier. J. Gen, Physiol.
59,388-400.
BARISH, M. E. (1933). A transient calcium-dependent chloride current in the immature Xenopus oocyte. J. Physiol. 342,309-325. BAUD, C., KADO, R. T., and MARCHER, K. (1982). Sodium channels induced by depolarization of the Xen-s laevis oocyte. Proc. Natl. Acud
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BUSA, W. B., and NUCCITELLI, R. (1985). An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frogxenqpus laevis. J. Cell Bid 100,1325-1329. CAHALAN, M. (1980). Molecular properties of sodium channels in ex-
LYANNE C. SCHLICHTER
Ionic
citable membranes. In “The Cell Surface and Neuronal Function” (C. W. Cotman, G. Poste, and G. L. Nicolson, Eds.), Vo16, pp. l-47. Elsevier/North-Holland, Amsterdam. CHARBONNEAU, M., MOREAU, M., PICHERAL, B., VILAIN, J. P., and GUERRIER, P. (1983). Fertilization of amphibian eggs: A comparison of electrical responses between anurans and urodeles. Dev. BioL 98, 304-318. CROSS,N. L. (1981). Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog. Rana pifiens. Dev. BioL
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CROSS,N. L., and ELINSON, R. P. (1980). A fast block to polyspermy in frogs mediated by changes in the membrane potential. Dew. BioL 75, 187-198.
GREY, R. D., BASTIANI, M. J., WEBB, D. J., and SCHERTEL, E. R. (1982). An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis. Dev. BioL 89,475-484. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rex Biophys. Bioeng. 8,385-416. HOULE, J. G., and WASSERMAN, W. J. (1983). Intracellular pH plays a role in regulating protein synthesis in Xenopus oocytes. Da? BioL 97,302-312. ITO, S. (1972). Effects of media of different ionic composition on the activation potential of anuran egg cells. Dev. Growth Difer. 14, 217-227. JAFFE, L. A., and SCHLICHTER, L. C. (1985). Fertilization-induced ionic conductances in eggs of the frog, Rana p&ens. J. PhysioL 358, 299-319. JAFFE, L. A., KADO, R. T., and MUNCY, L. (1985). Propagating potassium and chloride conductances during activation and fertilization of the egg of the frog, Rana pipiens. J. PhysioL 368,227-242. KADO, R. T., MARCHER, K., and OZON, R. (1979). Mise en evidence dune depolarisation de longue durite dans l’ovocyte de Xenopus laevis. C. R. Acud. Sci. Paris Ser. D 288,1187-1189. KAO, C. Y. (1966). Tetrodotoxin, saxitoxin and their significance in the study of excitation phenomena. PharmacoL Rev. 18,997-1049. MAENO, T. (1959). Electrical characteristics and activation potential of Bufo eggs. J. Gen. PhysioL 43,139-157.
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MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1972). Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane. Science 176,1441-1443. OKAMOTO, H., TAKAHASHI, K., and YOSHII, M. (1976). Membrane currents of the tunicate egg under the voltage-clamp condition. J. PhysioL
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ROBINSON, K. R. (1979). Electrical currents through full-grown and maturing Xenopus oocytes. Proc. NatL Acad Sti USA 76,837-841. SCHLICHTER, L. C. (1982a). Recurrent sodium action potentials in frog oocytes. Biophys. J. 37,324a. SCHLICHTER, L. C. (1982b). A novel action potential mechanism: Na and Cl channels lacking inactivation. J Cell BioL 95,139a. SCHLICHTER, L. C. (1983a). Spontaneous action potentials in maturing Rana pipiens oocytes. Dev. BioL S&47-59. SCHLICHTER, L. C. (1983b). A role for action potentials in maturing Rana pipiens oocytes. Dev. BioL S&60-69. SCHLICHTER, L. C. (1983c). Developmental changes in excitability in the frog oocyte. J. Gen PhysioL 82,31a. SCHLICHTER, L. C., and ELINSON, R. P. (1981). Electrical responses of immature and mature Rana pipiens oocytes to sperm and other activating stimuli. Dev. BioL 83, 33-41. TAGLIETTI, V., TANZI, F., ROMERO, R., and SIMONCINI, L. (1984). Maturation involves suppression of voltage-gated currents in the frog oocyte. J. Cell PhysioL 121, 576-588. THOMPSON, S. H., and ALDRICH, R. W. (1980). Membrane potassium channels. In “The Cell Surface and Neuronal Function” (C. W. Cotman, G. Poste, and G. L. Nicolson, Eds.), Vol. 6, pp. 49-85. Elsevier/ North-Holland, Amsterdam. WASSERMAN, W. J., PINTO, L. H., O’CONNOR, C. M., and SMITH, L. D. (1980). Progesterone induces a rapid increase in [Ca2+li, of Xenopus Levis oocytes. Proc. NatL Acud Sci. USA 77.1534-1536. WHITE, M. M., and MILLER, C. (1979). A voltage-gated anion channel from the electric organ of Torpedo califiica. J. Biol. Chem. 254, 10,161-10,166.
