Nagasaki 852, Japan. (Na+, Li+) cations can pass through the Ca channels to generate action potentials. performed using single glass micro-electrodes.

Transcription

1 J. Physiol. (1983), 339, pp With 8 text-figures Printed in G0reat Britain PERMEATION OF DIVALENT AND MONOVALENT CATIONS THROUGH THE OVARIAN OOCYTE MEMBRANE OF THE MOUSE BY SHIGERU YOSHIDA From the Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan (Received 14 September 1982) SUMMARY 1. Ovarian oocytes were isolated from adult mice and intracellular recording was performed using single glass micro-electrodes. 2. The resting potential was mv in standard solution, and the oocyte showed a regenerative response at the cessation of hyperpolarizing current pulse. 3. Ca spikes were observed under Na+-free conditions. The overshoot of the spike increased 28 mv for a 10-fold increase in [Ca2+]0 and showed saturation as [Ca2+]0 was elevated. The spike was insensitive to tetrodotoxin (TTX) and was blocked by polyvalent cations such as Co2+, Cd2+, Mn2+ and La3+. Sr2+ or Ba2+ substituted for Ca2+ in generating action potentials. 4. Na spikes were observed under Ca2+-free conditions. The overshoot of the spike showed the slope of 39 mv for a 10-fold increase in [Na+]o and a saturation was detected when [Na+]0 was raised. The spike was resistant to TTX and was blocked by Ca antagonists such as Co2+, Cd2+, Mn2+ or La3+. Li+ substituted for Na+ in producing spikes, while Rb+ did not. The overshoot and maximum rate of rise of the Na spike became smaller when Ca2+ was present in the bathing solution, indicating a competition between Na+ and Ca Mn2+ acted not only as a Ca blocker but also as a charge carrier during excitation. Mn spikes were detected in Na+-, Ca2+-free solutions and were blocked by Ca antagonists. 6. The resting membrane is permeable to not only Na+ but also to some extent to K+. 7. It is suggested that the ovarian oocyte membrane of the mouse has voltagedependent Ca channels, and both divalent (Ca2+, Sr2+, Ba2+, Mn2+) and monovalent (Na+, Li+) cations can pass through the Ca channels to generate action potentials. INTRODUCTION Electrical properties of the egg cell membrane have been studied in marine animals, amphibians, insects and mammals (see reviews: Hagiwara & Miyazaki, 1977; Hagiwara & Jaffe, 1979; Hagiwara & Byerly, 1981). Since Takahashi and his colleagues showed that the tunicate eggs were excitable (Takahashi, Miyazaki & Kidokoro, 1971), action potentials have been reported in various kinds of eggs and

2 632 such action potentials were found to be produced by divalent cations passing through Ca channels. In contrast, Na channels were described only in tunicate eggs (Miyazaki, Takahashi & Tsuda, 1974) and in Xenopus oocytes (Baud, Kado & Marcher, 1982). Thus, it has been generally considered that the Ca channels but not Na channels are widely distributed in eggs. As for mammalian preparations, electrophysiological study has been performed in oocytes of the mouse (Powers & Tupper, 1974; Okamoto, Takahashi & Yamashita, 1977; Yamashita, 1982; Powers, 1982). It was shown that the unfertilized oocytes could generate Ca-dependent action potentials as off-responses at the cessation of hyperpolarization, and the excitability was retained by substituting Sr2+ or Ba2+ for Ca2+ (Okamoto et al. 1977). They concluded that the inward current is attributable to Ca current through Ca channels and the contribution of Na ions to the current was negligible. In the present study, electrophysiological experiments were carried out on isolated ovarian oocytes of the mouse. A preliminary report has been published (Yoshida, 1982). TABLE 1. S. YOSHIDA Ionic compositions of solutions (mm) NaCl KCl CaCl2 MnCl2 MgCl2 TMA-Cl PIPES (1) Standard (2)0Na,OCa (3)0Na,80Ca (4)ONa, 320Ca (5) 0 Na, 0Ca,80Mn (6) XNa, 0Ca X All solutions contained1 mg/ml. glucose and were adjusted to ph 7*4 using 5 mm-pipes. Solutions of desired ionic concentration were obtained by mixing these media in an appropriate ratio. X indictes that X mm (between 150 and 750 mm) NaCl is present in the solution (6). In solutions lacking Ca2+, 2 mm-egta was added to chelate residual CA2+. TMA-Cl is tetramethylammonium chloride. For other solutions see text. METHODS Material&. Ovaries were dissected from sexually mature (older than 8 weeks) female mice of ICR strain. The ovaries were cleared of the adipose and the connective tissues covering the surface of them with surgical knives and the large follicles were broken with shap hypodermal needles under a dissecting microscope in the standard solution containing 0-2 % hyaluronidase (Type I-S, Sigma) at room temperature (20-23 C). The follicular cells surrounding the oocytes were dispersed by hyaluronidase in a few minutes and the isolated ovarian oocytes were collected and rinsed with standard solution (Iwamatsu & Chung, 1972). The diameter of the isolated oocytes was between 40 and 60 #m. Solutions. Table 1 lists the compositions of the solutions used in the present study. All solutions contained 1 mg/ml. glucose and the ph was adjusted to 7-4 using piperazine-n-n'-bis(2- ethanesulphonic acid) (PIPES) as a buffer. In the standard solution, PIPES was titrated with NaOH to adjust the ph. In Na+-free solutions, NaCl was replaced with tetramethylammonium chloride (TMA-Cl; Nakarai Chemicals, Japan) and the PIPES was titrated with tetramethylammonium hydroxide (TMA-OH). Sr2+ or Ba21 solution was prepared by substituting SrCl2 or BaCl2 for CaCl2. Similarly, Li+ or Rb+ solution was made by substituting LiCl or RbCl for NaCl, and the PIPES was titrated with NaOH in Na+ solutions and with TMA-OH in Li+ and Rb+ solutions to adjust the ph. 2 mm-ethylene glycol bis (fl-aminoethylether)-n,n'-tetraacetic acid (EGTA) was added to these Ca2+-free, monovalent-cation solutions to chelate residual Ca2+. A medium having a desired concentration of ions was obtained by mixing these solutions in an appropriate ratio. Tetrodotoxin (TTX; Sankyo, Japan), CoC12, CdCl2 and LaCl3 were applied to test solutions when necessary.

3 EXCITABILITY OF OVARIAN OOCYTES Recordings. Intracellular recordings were performing by penetrating the isolated ovarian oocytes with a single glass micro-electrode filled with 3 M-KCl (30-60 MO resistance). The penetration was performed by applying an oscillating current through the electrode by transiently increasing the negative capacitance of the pre-amplifier. A bridge circuit was used for simultaneous current injection and membrane potential recording. The measurement of the membrane potential was performed by recording the potential difference between an intracellular electrode and a reference electrode. The intensity of the current injected through the electrode was monitored by the potential drop across a 109 Q resistor. Differentiation of the membrane potential was carried out using an electrical differentiator. All experiments were performed at room temperature (20-23 TC). A mV - 2nA 05 sec Fig. 1. Intracellular recordings of potential responses obtained from the isolated ovarian oocyte of the mouse in standard solution. A, regenerative response elicited at the cessation of hyperpolarizing current pulse. B, action potentials revealed by two-step current stimulation. Dashed line: reference potential level. Voltage, current and time scales apply to both records. (Part of action potentials is retouched.) RESULTS Action potentials in standard solution The resting potential of the isolated mouse ovarian oocytes was mv (mean + S.D.) (n = 8) in standard solution. The membrane resistance was between 11-4 and MQ (average, 39-0 Me). The present study shows for the first time that the mammalian oocyte membrane is excitable even before ovulation. Fig. 1 illustrates responses of the ovarian oocyte having the resting potential of -7 mv in standard solution. The dashed line indicates the reference or zero potential level. The membrane showed only a passive electrotonic response when hyperpolarized with a small current. However, a regenerative response was elicited at the cessation of the current pulse when the membrane was hyperpolarized approximately below -70 mv (Fig. 1 A). The spike reached a voltage of +4 mv. Two-step current stimulation was applied to the oocyte to reveal action potentials more clearly (Fig. 1 B). A hyperpolarizing pre-pulse was used to remove inactivation of the ionic channels, and a test pulse, triggered on the end of the pre-pulse, evoked the action potentials. The time course and the threshold membrane potential of the spike can be clearly seen using this technique. The action potentials are slow in time course, and the duration of the spike is 0-22 sec at the critical level in this case.

4 634 S. YOSHIDA In order to check the ionic mechanisms underlying the generation of action potentials, excitability of the oocyte membrane was examined both under Na+-free and Ca2+-free conditions. Action potentials dependent on divalent cations For checking responses of the ovarian oocyte under Na+-free conditions, external Ca2+ concentration ([Ca2+]0) of the bathing medium was chosen to be 10, 20, 40, 80, 160 or 320 mm. Na+ was replaced with tetramethylammonium+ (TMA+) to compensate the osmolarity of the solution when [Ca2+]o was less than 80 mm (see Table 1.) A 0 Na, 20 mm-ca B 0 Na, 80 mm-ca C 0 Na, 320 mm-ca - - -N mv v...j... L10 V/sec IlnA 0-5sec Fig. 2. Effects ofexternal Ca2+ concentration on the overshoot of the action potential under Na+-free conditions. Dashed line: zero potential level. Top traces: membrane potential. Middle traces: differentiated record of the membrane potential. Bottom traces: current pulses used for stimulation. Spikes in A and B are generated by hyperpolarizing current pulse in the form of off-responses, while the spike in C is produced by a depolarizing current pulse because the resting potential is large in Na+-free, 320 mm-ca2+ solution. Voltage, differential, current and time calibrations apply to all records. The resting potential increased with log [Ca2+]O approximately in a linear fashion (Fig. 3A, Er). The ovarian oocytes generated action potentials as off-responses when the external Ca2+ concentration was below 160 mm. However, at 320 mm-ca2+, action potentials were elicited by a short depolarizing pulse as displayed in Fig. 2C because the resting potential was large enough to produce spikes in such a way. The overshoot of the spike increased directly with [Ca2+]o (Fig. 2). The values of the peak potential are plotted against log [Ca2+]o (Fig. 3A, E.). The peak value became larger in amplitude and saturated as the magnitude of the membrane hyperpolarization was increased. Thus, the saturated peak values are plotted. The overshoot increased by 28 mv per 10-fold change in [Ca2+]o when [Ca2+]o was changed between 10 and 80 mm. The dashed line in Fig. 3A shows the expected Nernstian slope of 29 mv for a 10-fold increase in [Ca2+]O. A saturation phenomenon was found when [Ca2+]o was greater than 80 mm (Hagiwara, 1973). Fig. 3B shows that the maximum rate of rise (m.r.r.) of the spike, indicating the amount of inward Ca current, was approximately 1 V/sec at 10 mm-ca2+, and it

5 EXCITABILITY OF OVARIAN OOCYTES 635 increased in a linear fashion up to 6 V/sec at 80 mm-ca2+. However, it showed a saturation when [Ca2+]. was more than 80 mm, and it was close to 7 V/sec at 320 mm-ca2. The critical level or the threshold membrane potential of the spike became more positive with the increase of [Ca2+]o (Fig. 3A, En). The critical membrane potential was obtained by examing the spike elicited by two-step current stimulation. This stabilizing action of Ca2+ has been thought to reflect the surface potential of the membrane (Hagiwara, 1973). 40 A - E _ 0' CL (U~~~~~~~~~~~~~E E -50 Er 8 M LB 5e Ca2+ concentration (mm) Fig. 3. A, effects of [Ca2+]0 on the overshoot potential (E8, O), the threshold or critical membrane potential (E,, A) and the resting potential (Er, El) of the spikes under Na+-free conditions. Bars indicate standard deviation. The dashed lines shows the Nernstian slope of 29 mv for a 10-fold increase in [Ca2+]0. B, effects of [Ca2+]o on the maximum rate of rise (m.r.r.) of the spikes. The concentration of Ca2+ is indicated in logarithmic scale (mm). Thus the action potential under Na+-free conditions appears to be dependent on Ca2+. The Ca spikes (Fig. 4A) were insensitive to high concentration of tetrodotoxin (10-5 g/ml. TTX) (Fig. 4B), and were totally blocked by 5 mm-cd2+ added to the bathing medium (Fig. 4C). Other 'Ca blockers' such as 5 mm-co2+, 10 mm-mn2+ and 1 mm-la3+ were also effective in suppressing the Ca spikes (not illustrated). The spikes persisted when external Ca2+ was replaced by Sr2+ (Fig. 4D) or by Ba2+ (Fig. 4E). This evidence indicates that the ovarian oocyte membrane of the mouse has voltage-dependent Ca channels. Action potentials dependent on monovalent cations Possibility that monovalent cations could maintain the action potential was examined in Ca2+-free solutions (2 mm-egta). The oocytes became very leaky under

6 636 S. YOSHIDA such conditions, and a large current was necessary to evoke action potentials. In addition, it was very difficult to obtain a stable recording from the oocytes in such solutions, for example, some oocytes showed regenerative responses immediately after the penetration of the micro-electrode and became inexcitable within a few minutes. On the contrary, when the penetration was performed very successfully, a stable recording was obtained for more than 30 min. When Mn2+ was used to substitute for Ca2+, as was done in the case of ovulated mouse oocytes by Okamoto and others (Okamoto et at. 1977), it was found that the Mn2+ prevented the oocyte 0 Na, 40 mm-ca O'Na, 40 mm-ca A 0 Na, 40 mm-ca B 10-5 g/ml. TTX C 10- g/ml. TTX, 5 mm-cd D ONa, 0Ca, 40mM-Sr E 0Na, OCa, 40mM-Ba 1S~~~~ X g S~~~~50mV _ f'rna 0-5 sec Fig. 4. Divalent cation-dependent action potentials recorded under Na+-free conditions. Ca spikes (A) are insensitive to a high concentration of tetrodotoxin (10-5 g/ml. TTX) (B), and were blocked by 5 mm-cd2+ (C). Sr21 (D) or Ba2+ (E) substitutes for Ca2+ in generating action potentials. Voltage, current and time calibrations are the same in all records. membrane from becoming leaky. However, Mn2+ was not used for substitution in the present study since it suppresses action potentials dependent on monovalent cations as will be stated below. The resting membrane potential decreased with a small slope when [Na+]o was increased. It was mv (n = 3) at 150 mm-na+, mv (n = 7) at 300 mm-na+, -68±+ 1-9 mv (n = 5) at 450 mm-na+, mv (n = 5) at 600 mm-na+ and mv (n = 5) at 750 mm-na+ (Fig. 6A, Er). Fig. 5A-C and Fig. 6A (ES)-B show that the overshoot and the maximum rate of rise (m.r.r.) of the spike varied with [Na+]O. The overshoot showed the slope of 39 mv for a 10-fold increase in [Na+]. (dashed line in Fig. 6A) (Yamamoto & Washio, 1979). It shows a saturation phenomenon when [Na+]o was raised more than 450 mm. The m.r.r. was 3-6 V/sec at 150 mm-na+, 16-3 V/sec at 300 mm-na+, 22-1 V/sec at

7 EXCITABILITY OF OVARIAN OOCYTES 637 A 150 mm-na, 0 Ca B 300 mm-na, 0 Ca C 450 mm-na, 0 Ca D 150 mm-na, 5 mm-ca E 300 mm-na, 5 mm-ca F 450 mm-na, 5 mm-ca I 50mV.>h V/sec 0-2 sec Fig. 5. A-C, effect of [Na+]o on the overshoot and the maximum rate of rise of the spikes under Ca2+-free conditions. D-F, competition phenomenon observed between Na+ and Ca2+. The concentration of Ca2+ was held constant at 5 mm while that of Na+ was changed. Dashed line: reference potential level. Voltage, differential and time scales apply to all records. 30 _, ne _ 0 5 _ 0 0 _ 0 CL (U~~~~~~~~~E E _ Na' concentration (mm) Fig. 6. A, effects of [Na+]o on the overshoot potential (E., 0), the critical membrane potential (Er, A) and the resting potential (Er, El) of the spikes under Ca2+-free conditions. The dashed line indicates the slope of 39 mv for a 10-fold increase in concentration. 0 show the spike overshoot when 5 mm-ca2+ was present in the bathing solution. B, effects of [Na+]o on the maximum rate of rise (m.r.r.) of the spikes. The m.r.r. remains constant when 5 mm-ca2+ was added to the solutions (0). The Na+ concentration is indicated in logarithmic scale (mm).

8 638 S. YOSHIDA 450 mm-na+, 23-7 V/sec at 600 mm-na+ and 25-2 V/sec at 750 mm-na+. A saturation is seen when [Na+]. is high (Fig. 6B). These values of m.r.r. are larger than those of Ca spikes (Fig. 3B). The critical membrane potential of the spike (Fig. 6A, Ej) did not substantially change with [Na+]O. Thus it can be said that the spikes are dependent on Na+, i.e. Na spikes. The Na spikes (Fig. 7A) were resistant to 10-5 g/ml. TTX (Fig. 7B) and were completely blocked by Ca antagonists such as 5 mm-co2+, 5 mm-cd2+, 10 mm-mn2+ and 1 mm-la3+ (Fig. 7 C). This kind of blocking suggests that the Na+ goes through 300 mm-na, 0 Ca 300 mm-na, 0 Ca A 300 mm-na, 0 Ca B 10-5 g/ml. TTX -C105 g/ml. TTX, 1 mm-la 1w1 _4cCl1-_ D 300mM-Li,ONa,OCa E 600mM-Rb,ONa,OCa i= ½1I50mV. 2 na II 05 sec Fig. 7. Monovalent cation-dependent spikes recorded under CaW+-free conditions. The Na spikes (A) are resistant to 10-5 g/ml. tetrodotoxin (TTX) and are blocked by 1 mm-la3+ (C) Li+ substituted for Na+ in producing an action potential (D) while Rb+ did not (E). Voltage, current and time scales apply to all records. the Ca channel instead of the separate Na channel during excitation. Li+ substituted for Na+ in generating action potentials (Fig. 7 D). In contrast, Rb+ did not maintain action potentials (Fig. 7E), indicating that the permeability of Rb+ is negligible in the ovarian oocyte membrane of the mouse. Competition between monovalent and divalent cations The other evidence that the Na+ enters through the Ca channel is the presence of competition between Na+ and Ca2+ revealed in the present work. Fig. 5D-F and Fig. 6A-B show the peak level and the m.r.r. of the spike examined in the solutions containing various concentrations of Na+ and a fixed value of 5 mm-ca2+. Both parameters were smaller than those obtained when only Na+ was present in the bathing medium. The values ofthe peak level and the m.r.r. of the spikes were + 1 mv and 3'7 V/sec at 150 mm-na+, 5 mm-ca2+; + 2 mv and 3.9 V/sec at 300 mm-na+,

9 EXCITABILITY OF OVARIAN OOCYTES mm-ca2+; -2 mv and 30 V/sec at 450 mm-na+, 5 mm-ca2 and -3 mv and 2-8 V/sec at 600 mm-na+, 5 mm-ca2+. The values almost remain constant even though [Na+]. was changed in the wide range of concentration. This competition suggests that the Na+ goes through the Ca channel rather the Na channel and both ions compete at the same binding site of the Ca channel. It is supposed that the affinity of Na+ to the site is much smaller than that of Ca2. Competition was also observed between Na+ and Mn2+. Generation of Na spikes was suppressed by Mn2+ which passes through the Ca channel and produces Mn spikes (see below). 0 Na, 0 Ca A 300 mm-na, 0 Ca mm-na, 0 Ca, 10 mm-mn C 10 mm-mn, 155 mm-k 0 Na, 0 Ca D 0 Na, 0 Ca, 80 mm-mn E 80 mm-mn, 10 mm-co A C X S~~~~~~~~50mV T 2nA 0_2 na 0-5 sec Fig. 8. The role ofmn2+ as a Ca blocker and a charge carrier during excitation in the ovarian oocyte membrane of the mouse. The Na spike (A) is blocked by 10 mm-mn2+ (B), but small action potentials (Mn spikes) are revealed when the outward K current is suppressed by elevating the external K+ concentration in Na+-, Ca2+-free solution (C). The Mn spike also appears when Mn2+ concentration was raised up to 80 mm (D). The spike is blocked by Ca antagonist, 10 mm-co2+ (E). Voltage and time scales are the same in all records. Current calibration is 2 na for A and 0-2 na for other records. Mn spikes As is displayed in Fig. 8, Na spikes (A) were completely blocked by 10 mm-mn2+ and the ovarian oocyte showed a passive change of the membrane potential (B). Thus Mn2+ acted as a Ca blocker because it was already verified that the spikes were generated by the Na current through the Ca channels. When appropriate conditions were chosen, action potentials dependent on Mn2+ were revealed. Fig. 8C shows that the ovarian oocyte of the mouse produced small regenerative responses when the outward K current was suppressed by raising the [K+]o from normal 5 mm to 155 mm. The oocyte had a resting potential of -14 mv and the peak level of spike was -4 mv. An alternative technique to reveal Mn spikes

10 640 S. YOSHIDA was to increase the external Mn2+ concentration under Na+-, Ca2+-free conditions (Fig. 8D). In such a solution, the ovarian oocyte showed a resting potential of -10 mv and an overshoot of 2 mv. The spikes disappeared either in the presence of Ca blockers, e.g. 10 mm-co2+ (Fig. 8E) or in the absence of Mn2+ (not illustrated). Thus the action potentials are considered to be dependent on Mn2+ which goes through the Ca channel. This observation corresponds to the report that Mn2+ carried small inward current under voltage-clamp technique in the ovulated mouse oocytes (Okamoto et al. 1977). It is concluded that the Mn2+ has two actions in the ovarian oocyte membrane of the mouse: (1) action of Mn2+ as a Ca blocker, and (2) action of Mn2+ as a charge carrier during excitation (Ochi, 1976). Resting potential of the ovarian oocyte membrane It is shown above that the resting membrane potential varied with [Ca2+]0 with a negative slope (Fig. 3A) and with [Na+]. with a positive slope (Fig. 6A). Further study of the resting membrane was carried out by varying the [K+]. in the presence of 10 mm-mn2+ under Na+-and Ca2+-free conditions (not displayed). The resting potential changed with a very small positive slope when [K+]. was increased mv (n = 3) at 5 mm-k+, mv (n = 3) at 55 mm-k+ and mv at 155 mm-k+ (n = 3). It is suggested that the resting membrane of the ovarian oocyte is permeable to not only Na+ but also to some extent to K+, and Ca2+ reduces the leakage due to stabilizing effect (Okamoto et al. 1977). DISCUSSION In the present study, it was shown for the first time that mammalian oocytes, even before ovulation, are electrically excitable. The Ca channels are present in the ovarian oocyte membrane of the mouse. Such voltage-dependent Ca channels have been identified in all eggs of vertebrate and invertebrate animals examined so far (Hagiwara & Miyazaki, 1977; Hagiwara & Jaffe, 1979; Hagiwara & Byerly, 1981). It is very characteristic that the ovarian oocytes revealed action potentials dependent on either Na+ or Li+ under Ca2+-free conditions. In eggs of the starfish Mediaster, a Na-dependent current was found but the study concluded that Na+ just played a facilitating role and an actual current was carried only by Ca2+ (Hagiwara, Ozawa & Sand, 1975). Eggs of the tunicate Halocynthia are known to have the Na channels in addition to the Ca channels (Okamoto, Takahashi & Yoshii, 1976). Xenopus oocytes have the Na channels which are induced by depolarization. These Na channels do not have an inactivation mechanism (Baud et al. 1982). Okamoto and his colleagues investigated ovulated and unfertilized oocytes of the mouse and concluded that the contribution of Na+ to the inward current was negligible (Okamoto et al. 1977). They compared the I-V relations taken in media containing Na+ and in media without Na+, and they were negative as to the passage of the Na+ through the membrane during excitation because no substantial change could be detected in the I- V curves. However, those workers replaced Ca2+ with Mn2+ in making Ca2+-free, Na+ solutions. This replacement is not appropriate for estimating

11 EXCITABILITY OF OVARIAN OOCYTES the permeation of Na+ through the oocyte membrane because it was found in the present study that Mn2+ suppresses the action potentials dependent on monovalent cations. In fact, further study revealed that the ovulated oocytes of the mouse produced Na or Li spikes under Ca2+-, MnS+-free conditions (S. Yoshida, in preparation). The inward current carried by monovalent cations was insensitive to TTX and was blocked by Ca antagonists such as Co2+, Cd2+, Mn2+ and La3+ in the ovarian oocytes, suggesting that monovalent cations enter through the Ca channels. Such rather unselective Ca channels permeable both to divalent and monovalent cations have been reported in insect muscles (Yamamoto & Washio, 1979), and cardiac preparations (Vitek & Trautwein, 1971; Goldman & Morak, 1977; Reuter & Scholz, 1977). The idea that monovalent cations pass through the Ca channels was further verified by the competition phenomenon observed between monovalent and divalent cations, both between Na+ and Ca2+ (Figs. 5 and 6), and Na+ and Mn2+ (Fig. 8). It is suggested that Ca2+ is more permeable than Na+ through the Ca channels in the mouse ovarian oocytes. Since the competition presumably takes place at the binding site connected with the Ca channel, the affinity of this site for divalent cations is probably larger than for monovalent cations. However, it should be taken into account that the concentration of monovalent cations cannot be raised so much as can be done in the case of divalent cations. Further study is necessary to conclude that monovalent cations surely pass through the Ca channels because the block of Na spikes by Co2+ etc. might be explained by an influence of a Ca-antagonist on a separate Na+-selective channel. Development of ion channels have been studied in many excitable cells including cleavage-arrested embryos of an ascidian (Takahashi & Yoshii, 1981). In general, inward current is carried in large part by Ca2+ at first, a Na+ current appears later, and in some instances the Ca2+ current disappears at late stages of development (Spitzer, 1979). Similar results were obtained in mouse dorsal root ganglion cells, and it was proposed that the number of Ca channels per unit area of the membrane decreases with time while that of Na channels increases (Matsuda, Yoshida & Yonezawa, 1978; Yoshida, Matsuda & Samejima, 1978). Present work shows that an unselective or immature Ca channel is present in the early stage of development, i.e. ovarian oocytes. This kind of Ca channel may be a prototype of Na channel which is observed in the later stages of development. I thank Drs W. J. Moody, Jr and Y. Matsuda for their comments on the manuscript and Mr M. Yogata and Mrs N. Momosaki for making figures. This work was supported by a research grant from the Japan Ministry of Education. REFERENCES BAUD, C., KADO, R. T. & MARCHER, K. (1982). Sodium channels induced by depolarization of the XenopuB laevis oocyte. Proc. natn. Acad. Sci. U.S.A. 79, GOLDMAN, Y. & MORAD, M. (1977). Ionic membrane conductance during the time course of the cardiac action potential. J. Physiol. 268, HAGIWARA, S. (1973). Ca spike. Adv. Biophya. 4, HAGIWARA, S. & BYERLY, L. (1981). Calcium channel. Ann. Rev. Neurowci. 4, HAGIWARA, S. & JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Ann. Rev. Biophys. Bioeng. 8, PHY

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