[25]
REGULATION OF INTRACELLULARCa2+ IN OOCYTES
[25] Regulation
38 1
of Intracellular Calcium Activity in Xenopus O o c y t e s
By YORAM ORON and NATHAN DASCAL Introduction
Xenopus laevis oocytes serve as a useful model system for investigating the mechanism of signal transduction both of intrinsic responses in native oocytes and of acquired responses in oocytes injected with appropriate mRNAs from other tissues.t Native oocytes express several cell membrane receptors, stimulation of which leads to the mobilization of calcium both from cellular stores and from the medium (Table I). Following the injection of mRNAs, a number of foreign receptors are expressed in oocytes that also use calcium mobilization to produce the physiologic response, namely, activation of chloride channels (for partial list, see Table I). Several lines of evidence indicate that cellular calcium is indeed recruited to produce the physiological responses in Xenopus oocytes: (1) depletion of cellular calcium by injection of calcium chelators (e.g., EGTA), by repeated resonses in calcium-free medium, or by exposure to vanadate or divalent cation ionophores in calcium-free medium abolishes responses; (2) introduction of calcium into the oocyte either by microinjection or by pretreatment with divalent cation ionophores mimics responses; (3) injection of inositol 1,4,5-trisphosphate (IP3) mimics responses; (4) challenge with the physiological signals (hormones, neurotransmitters) or injection of IP 3 result in an increase in 45Ca2+ efllux from oocytes that, presumably, reflects an increase in cytosolic calcium concentration. This can be directly monitored by optical methods (fluorescence of Quin-2 or Fura-2 or luminescence of aequorin). In addition, evidence points to recruitment of extracellular calcium: (1) electrophysiologic responses are blunted in the absence of extracellular calcium; (2) metal cations that block calcium entry (i.e., Mn 2+) partially inhibit responses to signal or microinjection of IPa; (3) the second component of IP3-induced current is largely dependent on extracellular calcium; (4) following calcium depletion, receptor stimulation or IP 3 injection produce chloride current in response to challenge by extracellular calcium.
N. Dascal, Crit. Rev. Biochem. 4, 3 1 7 ( 1 9 8 7 ) . METHODS IN ENZYMOL(X3Y, VOL 207
Copyright © 1992 by Academic Press, Inc. All fights of reproduction in any form reserved.
TABLE I INTRINSIC AND ACQUIRED RESPONSES THAT UTILIZE CALCIUM
Stimulus Intrinsic Muscarinic (M3) Muscarinic (MI) Purinergic (P27) Angiotensin Acquired Muscarinic (M1, M3, M5) 5-Hydroxytryptamine (5-HT) Excitatory amino acids Tachykinins Vasopressin Thyrotropin-releasing hormone (TRH) Gonadotropin-releasing hormone (GnRH) Cholecystokinin a b c d e f s h J J k
Refs.
a, b c, d, e f, g h, i j-m k, n, o p-s t u v, w x y
K. Kusano, R. Miledi, and J. Stinnakre, Nature (London) 270, 739 (1977). N. Dascal and E. M. Landau, LifeSci. 27, 1423 (1980). E. Nadler, B. Gillo, Y. Lass, and Y. Oron, FEBSLett. 1999 208 (1986). y. Oron, B. Gillo, and M. C. Gershengorn, Proc. Natl. Acad. Sci. U.S.A. 85, 3820 (1988). A. Davidson, G. Mengod, N. Matus-Leibovitch, and Y. Oron, FEBSLett. 284, 252 (1991). I. Lotan, N. Dascal, S. Cohen, and Y. Lass, PfluegersArch. 406, 158 (1986). S. Gellerstein, H. Shapira, N. Dascal, R. Yekuel, and Y. Oron, Dee. Biol. 127, 25 (1988). E. M. Landau, personal communication. M. Lupu-Meiri and Y. Oron, unpublished. Y. Nomura, S. Kaneko, K. Kato, S. Yamagishi, and H. Sugiyama, Mol. Brain Res. 2, 113 (1987). N. Dascal, C. Ifune, R. Hopkins, T. P. Snutch, H. Lubbert, N. Davidson, M. Simon, and H. Lester, Mol. Brain Res. 1, 201 (1986). t K. Fukuda, T. Kubo, I. Akiba, A. Maeda, M. Mishina, and S. Numa, Nature (London) 327, 623 (1987). m H. Bujo, J. Nakai, T. Kubo, K. Fukuda, I. Akiba, A. Maeda, M. Mishina, and S. Numa, FEBS Lett. 240, 95 (1988). n C. B. Gundersen, R. Miledi, and I. Parker, Proc. R. Soc. London B 219, 103 (1983). o D. Julius, A. B. MacDermont, R. Axel, and T. Jessel, Science 241, 558 (1988). P C. B. Gundersen, R. Miledi, and I. Parker, Proc. R. Soc. London B 221, 127 (1984). q C. B. Gundersen, R. Miledi, and I. Parker, Proc. R. Soc. London B 221, 235 (1984). ' K. M. Houamed, G. Bribe, T. G. Smart, A. Constanti, D. A. Brown, E. A. Barnard, and B. M. Richard, Nature (London) 310, 318 (1984). s T.A. Verdoorn, N. W. Kleckner, and R. Dingledine, Mol. Pharmacol. 35, 360 (1989). t y. Harada, T. Takahashi, M. Kuno, K. Nakayama, Y. Masu, and S. Nakanishi, J. Neurosci. 7, 3265 (1987). u T. M. Moriarty, S. C. Sealfon, D. J. Carty, J. L. Roberts, R. Iyengar, and E. M. Landau, J. Biol. Chem. 264, 13524 (1989). v y. Oron, B. Gillo, R. E. Straub, and M. C. Gershcngorn, Mol. Endocrinol. 1, 918 (1987). w y. Oron, R. E. Straub, P. Traktman, and M. C. Crershengorn, Science 238, 1406 (1987). x S. Yoshida, S. Plant, P. L. Taylor, and K. A. Eidne, Mol. Endocrinol. 3, 1953 (1989). Y J. A. Williams, D. J. MeChesney, C. Calayag, V. R. Lingappa, and C. D. Logsdon, Proc. Natl. Acad. Sci. U.S.A. 85, 4939 (1988).
[25]
383
REGULATION OF INTRACELLULAR C a 2+ IN OOCYTES
Methods
Injection of Active Substances All intracellular injections may be performed by two methods. When the injection is performed while the oocyte is voltage clamped, to observe the immediate effect of the injected substance, injections are done using a third pipette attached to regulated pressure source ( 1 - 3 psi) via a pneumatic valve controlled electronically by a time delay relay device. The micropipette is manually broken (tip diameter 2 - 5 gm) and backfilled with the desired solution. The volume of injection should not exceed 0.5% of the cell volume (i.e., - 5 nl). The values o f drop diameter, volume, and nominal concentration of injected substances are given in Table II. When long-term effects are to be tested, the oocyte can be injected using an adjustable automatic micropipette (e.g., D r u m m o n d type, total dispensed volume 5 - l 0 / d , can be adjusted to deliver 10- 50 nl). The glass capillary is pulled with an electrode puller and manually broken (diameter 1 0 - 2 5 TABLE II MICROINJECTION PARAMETERSa
Drop diameter (/tin)
Drop volume (nl)
% Oocyte volume
Amount injected (pmol)
Concentration (nM)
20 40 60 80 100 120 140 160 180 200
0.004 0.034 0.113 0.27 0.52 0.9 1.44 2.14 3.05 4.19
0.0005 0.0037 0.0125 0.030 0.058 0.1 0.16 0.24 0.34 0.46
0.004 0.034 0.113 0.27 0.52 0.9 1.44 2.14 3.05 4.19
9.3 74 250 593 1157 2000 3176 4740 6750 9260 (uM)
267 337 386 424 457 486
10 20 30 40 50 60
1.1 2.2 3.3 4.4 5.5 6.6
10 20 30 40 50 60
22 44 66 88 111 133
a Amounts refer to microinjected solution of any compound at 1 m M concentration in the pipette. Concentrations are nominal, assuming complete diffusion of the compound in the aqueous space of the cell (taken as 50% of eel volume for a 1.2-mm-diameter
oocyte).
384
EXPRESS,ON OF ION CHANNELS
[9.5]
TABLE III AMOUNTS AND EFFECTS OF INJECTED SUBSTANCES
Substance
Injected amount
Effect
EGTA
50 pmol 200- 300 pmol 100-300 pmol
CaCI2
0.1 pmol 5 - 50 pmol 100- 300 pmol
Inhibition of spontaneous fluctuations Inhibition of responses Inhibition of cellular calcium release to facilitate measurements of Ba2+ currents Threshold depolarizing fluctuations Medium monophasie responses Large responses, possibly biphasic, late fluctuations Threshold responses (5-10 hA, fluctuations) Small biphasie responses, fluctuations Large biphasic responses, fluctuations Close to saturating responses (0.5-1 #A), late fluctuations Desensitizing responses, 4sCa2+ efllux induction
IP3
1-5fmol 5-10 fmol 50-150 fmol 300-500 fmol 2-10 pmol
/.tm). The capillary is then pressure failed with light mineral or silicone oil and mounted on the wire plunger. It is back-filled with the desired solution. The volume of injected solution can be monitored by identical injection into oil under a microscope (10 × objective) equipped with a reticule. The concentrations of substances needed to achieve the desired effects are given in Table III. Remarks. The effect of intracellular injections depends to a large extent on the site and the depth of injection. To obtain a sharp and immediate effect, calcium or IPa should be injected as shallowly as possible (-50/zm deep). This can be performed only in manually or collagenase-defolliculated oocytes and using pipettes that have been broken to a small tip diameter. On deep injections, the resulting currents are delayed and much less sharp. The second component of responses to IP 3 is better observed using deep injections (150- 300/zm). 2 Responses obtained by injection near the animal (pigmented) pole of the oocyte differ from those observed near the vegetal pole. Chloride currents obtained by the injection of calcium are usually much sharper near the animal pole and decay faster than currents of similar amplitudes produced by injections near the vegetal pole. Injection of IPa at the two poles results in similar kinetics, except that the amplitude of responses 2 B. Gillo, Y. Lass, E. Nadler, and Y. Oron, J. Physiol. (London) 392, 349 (1987).
[25]
REGULATION OF INTRACELLULAR Ca 2+ IN OOCVTES
385
obtained near the animal pole is approximately 5 times greater than those obtained at the vegetal pole. 3 Injections of EGTA are more effective in suppressing spontaneous current fluctuations and the rapid component of the responses to various signals than the slow component of the response elicited by calcium influx? This may be because of the relatively slow kinetics of calcium chelation by EGTA. Once injected, EGTA is effective for a prolonged time (100-250 pmol will block spontaneous fluctuations for at least 30 hr). Injections of as little as 0.5 pmol of calcium produce measurable responses. These can be repeated several times without either desensitization or potentiation. A number of consecutive threshold injections results in delayed small depolarizing current with pronounced fluctuations. 2 This may be due to activation of a separate population of chloride channels or to calcium-induced calcium release. Deep injections of large quantities of calcium (100-300 pmol) result in large currents (-1 /tA, lasting 2 0 - 3 0 sec) which are potentiated by subsequent identical injection (possibly owing to the partial saturation of calcium storesS). Interestingly, unlike calcium introduced by ionophore, injected calcium does not inactivate chloride channels and is not subject to regulation by protein kinase C, indicating a possibly different site of action in the cell. 4-6
Measurement o f 45Ca2+ Fluxes Efflux Measurements. Ideally, to quantitate calcium movement, efflux studies should be performed at isotopic equilibrium. Practically, however, oocytes do not equilibrate their calcium even after 48 hr, and the studies are performed taking into account only the more rapidly exchangeable calcium pools. To measure calcium efflux in individual cells, oocytes are incubated overnight in a small volume ofNDE964 solution with 200 #Ci/ml of 45Ca2+ (specific radioactivity 100/tCi/pmol). The cells are washed several times in ND96 to remove adhering label. Efflux is monitored either by changing the solution every 0.5- 5 min or by holding the oocyte in a perfusion cell and perfusing the desired solution with a peristaltic p u m p at a rate of 1.0-5.0 ml/min. Fractions (0.5- 2.0 ml) are collected with a fraction collector and counted in 4 ml of scintillant. After a certain period needed to determine the basal rate of ettlux, oocytes are stimulated with a desired stimulus (hormone, IP3, etc.). At the end of the experiment, the oocyte is homoge3 M. Lupu-Meiri, H. Shapira, and Y. Oron, FEBSLetL 240, 83 (1988). 4 R. Boton, N. Dascal, B. Gillo, and Y. Lass, J. Physiol. (London) 408, 511 (1989). 5 N. Dascal and R. Boton, FEBSLett.267, 22 (1990). 6 M. Lupu-Meiri, H. Shapira, and Y. Oron, PfluegersArch. 413, 498 (1989).
386
EXPRESSION O17 ION CHANNELS
[25]
TABLE IV STIMULATEDEFFLUXOF 45Ca2+
Stimulus
Concentration
Maximal fractional rate (%/min) a
Time of increased efllux (min)
Total stimulated efllux (%)
ACh-M3 ACh-MI Ade TRH 5-HT IP3 GTPrS
0.1 mM 0.1 mM 10#M 1/zM 1/zM > 2 pmol >20 pmol
1.0 6.4 0.5 11.7 14.0 2.9 3.3
4.6 12.2 1.9 15.0 10.0 9.9 13.7
2.2 35.1 2.0 45.6 41.0 32.2 40.7
a Maximal fractional rate denotes the net increase in the rate of efllux over the basal rate (0.2-1.0% of residual label/rain).
nized in an identical volume of perfusion solution and evaluated for residual counts. Both methods can be used on a voltage-clamped oocyte. In this way, parallel records of electrophysiologic responses and calcium efflux may be obtained. To normalize the data from different oocytes, data can be plotted as log(% residual cpm) versus time (first-order plot) or as percent of counts per minute (cpm) out of total counts per minute in the oocyte at that point (fractional rate of efflux). On short incubations, 45Ca2+ efflux appears to follow first-order kinetics. In prolonged incubations, however, the fractional rate of basal ettlux steadily decreases, indicating participation of another, slower pool. These methods are described in detail in Refs. 7 and 8. Reference values for various stimulations are given in Table IV. Oocytes take up 2000-10,000 cpm/cell, and basal efflux rates are 0.2-1.0%/min. Oocytes that exhibit much higher ettlux rates and/or take up much more 45Ca2+ should be discarded. Influx Measurements. Oocytes are incubated in ND96 solution with 200/zCi/ml 45Ca2+ for a desired period, then washed thoroughly of adhering label and counted. Previous stimulation with a hormone or a neurotransmitter in calcium-free medium results in an enhanced 45Ca2+ influx. To demonstrate this phenomenon, oocytes are incubated for 10 min with the agonist and washed free of it for an additional 20-30 min with calcium-free medium. They are then transferred to a solution that contains the label with or without the stimulus. Reference values are given in Table IV. 7 E. Nadler, B. Gillo, Y. Lass, and Y. Oron, FEBSLett. 199, 208 (1986). s H. Shapira, M. Lupu-Meiri, M. C. Gershengorn, and Y. Oron, Biophys. J. 57, 1281 (1990).
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REGULATION OF INTRACELLULAR Ca 2+ IN OOCYTES
387
Depletion of Cellular Calcium Stores Depletion of signal-sensitive calcium stores can be effected in calciumfree medium in three different ways: (1) by repeated exposures to the signala,9; (2) by exposure to divalent cation ionophores2; (3) by exposure to CaZ+-ATPase inhibitors (B. Gillo and Y. Oron, unpublished, 1985). It should be noted that only the first method is reversible. The effect can be measured indirectly by observing the physiological response (i.e., depolarizing chloride currents) or directly by following 4~Ca2+eftlux. These methods are briefly described below. In depletion experiments, any suitable medium (i.e., ND96 or OR21°) may be used, except that calcium addition is omitted and EGTA is added (usually 0.1 m M is enough). It is our experience that maintaining calcium below 50 p M is sufficient to cause calcium depletion. Depletion of calcium stores by repeated exposures is effected by challenging the oocyte with the signal for 1- 2 min at 30-rain intervals. This results in a complete disappearance of the response and of the associated 4~Ca2+ ettlux by the fourth exposure. Subsequent incubation of the oocyte for l0 min or more with a normal calcium concentration is sufficient to fully restore the response. 8,9 Depletion by ionophore can be effected by preincubation of oocytes in the presence of 0.2- 1 # M of A23187 [in 0.1% v/v ethanol or dimethyl sulfoxide (DMSO)] for 10-15 min. The cells are then washed free of the ionophore solution. It is notable that partial depletion (i.e., in the presence of low concentrations of A23187) affects the rapid component of the response to a larger extent than the second, slow component.TM This is in agreement with our findings that the second component of the responses requires a significant contribution of extracellular calcium. ~2 Depletion with ATPase inhibitors (e.g., vanadate) is effected by adding 0.5-5.0 m M o f t h e agent at pH 8.5-9.0. The initial response was a slowly developing depolarizing current with superimposed current fluctuations, accompanied by modesf 45Ca2+ ettlux. The cell is subsequently insensitive to stimulation (B. Gillo, E. Nadler, Y. Lass, and Y. Oron, unpublished results, 1985). Remarks. Oocytes maintained in calcium-free medium often deteriorate rapidly owing to the activation of unspecified channels.9,~3Addition of high magnesium (20 mM) or DMSO (0.1% v/v) sometimes stabilizes the 9 N. Dascal, B. Gillo, and Y. lass, J. Physiol. (London) 366, 299 (1985). 1oN. Dascal, T. P. Snutch, H. Lubbert, N. Davidson, and H. A. Lester, Science 231, 1147 0986). 11 D. Singer, R. Boton, O. Moran, and N. Dascal, PfluegersArch. 41, 7 (1990). 12 M. Lupu-Meiri, H. Shapira, and Y. Oron, FEBSLett. 262, 165 (1990). 13 N. Dascal, E. M. Landau, and Y. Lass, J. Physiol. (London) 352, 551 0984).
388
EXPRESSION OF ION CHANNELS
[25]
cells. It is our experience, however, that stability to low calcium is better in oocytes of some donors than in others. In oocytes of those donors the addition of high magnesium can be avoided. Also, oocytes kept in calcium-free medium develop pronounced oscillatory currents that can interfere with responses. These eventually disappear on prolonged incubation in the absence of calcium. A23187 is light-sensitive, and all operations should be conducted under diminished illumination and in light-protected vessels, Incubation of oocytes in the presence of calcium and A23187 results in slowly developing chloride current, reflecting most probably the kinetics of incorporation of the ionophore. Large responses can be obtained by incorporation of oocytes with A23187 in calcium-free medium, washing of the ionophore, and exposing the cells to short pulses of calcium.4,n The large currents can be elicited for at least 2 hr after washing out the ionophore, suggesting that it is incorporated into the membrane in a quasi-irreversible manner. The currents are biphasic, indicating two different conductances sensitive to different concentrations of extracellular calcium. The rapid conductance is subject to strong calcium-dependent inactivation.4,14
Calcium-Induced Chloride Currents Depletion of calcium stores greatly facilitates the detection of chloride currents evoked by entry of extracellular calcium. Oocytes challenged in the absence of calcium by an agonist or IP 3 respond to addition of calcium (20 see after the challenge) by a small depolarizing current. This current increases on continuous incubation in calcium-free medium (3 rain), indicating that calcium depletion potentiates the subsequent response to calcium. This phenomenon is often potentiated by inclusion of 0.1% v/v DMSO in the medium (M. Lupu-Meiri and Y. Oron, unpublished, 1991). This concentration of DMSO does not affect either the holding current or the magnitude of responses. The reason for these effects of DMSO is yet to be determined. Using DMSO-eontaining ND96, we have adopted a method which uses repeated challenges with the signal in calcium-free medium. On signficant depletion of the stores, large calcium-evoked chloride currents are observed (> 100 hA). The addition of a nonspeeific calcium entry blocker (e.g., Mn 2+, 1 raM) completely abolishes the calciuminduced chloride current. 12
Demonstration of Separate Calcium Stores Signal-evoked calcium depletion can be used to demonstrate the existence (or absence) of separate, dedicated calcium stores in the oocyte. 14 R. Boron, D. Singer, and N. Dascal, PfluegersArch. 41, 1 (1990).
[25]
REGULATION OF INTRACELLULAR Ca 2+ 1N OOCYTES
389
Briefly, oocytes possessing the intrinsic muscarinic response and an acquired response [e.g., to thyrotropin-releasing hormone (TRH) after injection with mRNA for TRH receptors] are repeatedly challenged with acetylcholine (ACh). In oocytes preloaded with 45Ca2+ in the presence of calcium, the label in ACh-specific store is lost, whereas that in the TRHspecific store remains intact. In the absence of extraceUular calcium, the response to ACh disappears, while that to TRH remains. The response to ACh can be restored by short incubation with calcium. 8 Measuring Free Cytoplasmic Calcium
Both calcium microelectrodes and optical methods with fluorescent or luminescent indicators have been used to qualitatively follow free cytoplasmic calcium in oocytes. The main drawbacks of these methods are that they are at best semiquantitative and require specialized equipment or specific training. We have, therefore, decided to include the appropriate references without elaborating these methods here. Although measurements of changes in oocyte free calcium with ion-selective electrodes have been reported, the only well-described work is by Busa et al. 15 It should be stressed that electrodes measure calcium in the immediate vicinity of the electrode tip, and this may not necessarily be the compartment of interest. We have used quin 2 to measure both resting and stimulated concentrations of free calcium in albino oocytes. The results were disappointing. The basal concentration of free calcium was indeed around 0.1 pM. However, the rises due to stimulation of native ACh receptors were negligible and implied that microinjected dye buffers the cytoplasmic calcium or that the rise occurs in a subcellular compartment which constitutes only a negligible fraction of the cytoplasmic space (Y. Oron and M. C. Gershengorn, unpublished, 1985). Moreau et aL have reported using aequorin to follow calcium elevation induced by progesterone) 6 These results were difficult to duplicate, t7,18 Parker and Miledi 19have demonstrated that injected aequorin can be used to monitor calcium elevation caused by injection of IP 3. Takahashi et aL 2° have described a method that used Fura-2 to monitor rises in free calcium following the stimulation of acquired 5-hydroxytryptamine (5-HT) recep-
15 W. B. Busa, J. E. Ferguson, S. K. Joseph, J. R. WiUiamson, and R. Nuccitelli, J. Cell Biol. 101, 677 (1985). 16 M. Moreau, J. P. Vilain, and P. Guerrier, Dev. Biol. 78, 201 (1980). 17 K. R. Robinson, Dev. BioL 109, 504 (1985). is R. J. Cork, M. F. Cicirelli, and K. R. Robinson, Dev. Biol. 121, 41 (1987). 19 I. Parker and R. Miledi, Proc. R. Soc. London B 228, 307 (1986). 20 T. Takahashi, E. Neher, and B. Sakmann, Proc. Natl. Acad. Sci. U.S.A. 84, 5063 (1987).
390
EXPRESSIONOF ION CaA~-mzI~S
[25]
tors. Recently, Brooker et al. 21 have described an elegant method to measure stimulated calcium changes in oocytes using Fura-2 fluorescence. Recording o f Currents Conducted by Plasma M e m b r a n e Calcium Channels
Native Xenopus oocytes exhibit low activities of intrinsic voltagedependent calcium channels? ° Much greater activities of voltage-dependent calcium channels are expressed in oocytes injected with mRNA from various excitable tissues? °,22-25 Direct recording of calcium current on depolarization is impossible due to the masking effect of large calcium-activated chloride currents. It may be possible to circumvent this problem by applying the internal perfusion method described elsewhere in this volume. 26 In intact oocytes, it is possible to record currents flowing through calcium channels using barium as the charge carrier, since Ba2+ does not activate chloride channels. 27 The standard medium for this type of experiments contains 30-40 m M Ba,2+ 60-70 m M Na + or N-methyl-o-glucamine, and 5 m M HEPES (pH 7.5) and substitutes methane sulfonate for chloride.~°a5 Methane sulfonate does not permeate through chloride channels and also partially blocks them? Chloride channels can be further inhibited by 9-anthracenecarboxylic acid (0.5-2 m M from a stock solution of 200 m M in 0.5-1.0 M NaOH) or niflumic of fluphenamic acids (0.01 0.10 m M from a 0.1- 1.0 M stock in DMSO). 4,2s Even in the presence of chloride channel blockers, large Ba2+ currents (> 100 nA) will produce late variable inward currents, probably owing to the release of cellular calcium and activation of chloride currents. These can be completely suppresed by injections of EGTA (see above). Note. In EGTA-injected oocytes, pure barium currents can be recorded even in solutions that contain C1- as the major anion. EGTA, however, fails to buffer cellular calcium fully (if Ca 2÷ is present in the medium), and the resulting chloride currents will interfere with the recording (N. Dascal, unpublished, 1985).
21G. Brooker, T. Seki, D. Croll, and C. Wahlstedt, Proc. Natl. Acad. Sci. U.S.A. 87, 2813 (1990). 22j. R. Moorman,Z. Zhou, G. E. Kitsch,A. E. Lacerda,J. M. Caffrey,D. M.-K.Lain,R. H. Joho, and A.M. Brown, Am. J. Physiol. 253, H-985 (1987). 23j. p. Leonard,J. Nargeot,T. P. Snutch,N. Davidson,and H. A. Lester,J. Neurosci. 7, 875 (1987). 24j. A. Umbachand C. B. Gundersen,Proc. Natl. Acad. Sci. U.S.A. 84, 5464 (1987). 25I. Lotan,P. Goelet,and N. Dascal,Science 243, 666 (1989). 26N. Dascal,G. Chilcott,and H. Lester,this volume[21]. 27M. E. Barish,J. PhysioL (London) 342, 309 (1983). 28M. M. Whiteand M. Aylwin,MoL Pharmacol. 37, 720 (1990).
REGULATION OF INTRACELLULARCa2+ IN OOCYTES
[25] Regulation
38 1
of Intracellular Calcium Activity in Xenopus O o c y t e s
By YORAM ORON and NATHAN DASCAL Introduction
Xenopus laevis oocytes serve as a useful model system for investigating the mechanism of signal transduction both of intrinsic responses in native oocytes and of acquired responses in oocytes injected with appropriate mRNAs from other tissues.t Native oocytes express several cell membrane receptors, stimulation of which leads to the mobilization of calcium both from cellular stores and from the medium (Table I). Following the injection of mRNAs, a number of foreign receptors are expressed in oocytes that also use calcium mobilization to produce the physiologic response, namely, activation of chloride channels (for partial list, see Table I). Several lines of evidence indicate that cellular calcium is indeed recruited to produce the physiological responses in Xenopus oocytes: (1) depletion of cellular calcium by injection of calcium chelators (e.g., EGTA), by repeated resonses in calcium-free medium, or by exposure to vanadate or divalent cation ionophores in calcium-free medium abolishes responses; (2) introduction of calcium into the oocyte either by microinjection or by pretreatment with divalent cation ionophores mimics responses; (3) injection of inositol 1,4,5-trisphosphate (IP3) mimics responses; (4) challenge with the physiological signals (hormones, neurotransmitters) or injection of IP 3 result in an increase in 45Ca2+ efllux from oocytes that, presumably, reflects an increase in cytosolic calcium concentration. This can be directly monitored by optical methods (fluorescence of Quin-2 or Fura-2 or luminescence of aequorin). In addition, evidence points to recruitment of extracellular calcium: (1) electrophysiologic responses are blunted in the absence of extracellular calcium; (2) metal cations that block calcium entry (i.e., Mn 2+) partially inhibit responses to signal or microinjection of IPa; (3) the second component of IP3-induced current is largely dependent on extracellular calcium; (4) following calcium depletion, receptor stimulation or IP 3 injection produce chloride current in response to challenge by extracellular calcium.
N. Dascal, Crit. Rev. Biochem. 4, 3 1 7 ( 1 9 8 7 ) . METHODS IN ENZYMOL(X3Y, VOL 207
Copyright © 1992 by Academic Press, Inc. All fights of reproduction in any form reserved.
TABLE I INTRINSIC AND ACQUIRED RESPONSES THAT UTILIZE CALCIUM
Stimulus Intrinsic Muscarinic (M3) Muscarinic (MI) Purinergic (P27) Angiotensin Acquired Muscarinic (M1, M3, M5) 5-Hydroxytryptamine (5-HT) Excitatory amino acids Tachykinins Vasopressin Thyrotropin-releasing hormone (TRH) Gonadotropin-releasing hormone (GnRH) Cholecystokinin a b c d e f s h J J k
Refs.
a, b c, d, e f, g h, i j-m k, n, o p-s t u v, w x y
K. Kusano, R. Miledi, and J. Stinnakre, Nature (London) 270, 739 (1977). N. Dascal and E. M. Landau, LifeSci. 27, 1423 (1980). E. Nadler, B. Gillo, Y. Lass, and Y. Oron, FEBSLett. 1999 208 (1986). y. Oron, B. Gillo, and M. C. Gershengorn, Proc. Natl. Acad. Sci. U.S.A. 85, 3820 (1988). A. Davidson, G. Mengod, N. Matus-Leibovitch, and Y. Oron, FEBSLett. 284, 252 (1991). I. Lotan, N. Dascal, S. Cohen, and Y. Lass, PfluegersArch. 406, 158 (1986). S. Gellerstein, H. Shapira, N. Dascal, R. Yekuel, and Y. Oron, Dee. Biol. 127, 25 (1988). E. M. Landau, personal communication. M. Lupu-Meiri and Y. Oron, unpublished. Y. Nomura, S. Kaneko, K. Kato, S. Yamagishi, and H. Sugiyama, Mol. Brain Res. 2, 113 (1987). N. Dascal, C. Ifune, R. Hopkins, T. P. Snutch, H. Lubbert, N. Davidson, M. Simon, and H. Lester, Mol. Brain Res. 1, 201 (1986). t K. Fukuda, T. Kubo, I. Akiba, A. Maeda, M. Mishina, and S. Numa, Nature (London) 327, 623 (1987). m H. Bujo, J. Nakai, T. Kubo, K. Fukuda, I. Akiba, A. Maeda, M. Mishina, and S. Numa, FEBS Lett. 240, 95 (1988). n C. B. Gundersen, R. Miledi, and I. Parker, Proc. R. Soc. London B 219, 103 (1983). o D. Julius, A. B. MacDermont, R. Axel, and T. Jessel, Science 241, 558 (1988). P C. B. Gundersen, R. Miledi, and I. Parker, Proc. R. Soc. London B 221, 127 (1984). q C. B. Gundersen, R. Miledi, and I. Parker, Proc. R. Soc. London B 221, 235 (1984). ' K. M. Houamed, G. Bribe, T. G. Smart, A. Constanti, D. A. Brown, E. A. Barnard, and B. M. Richard, Nature (London) 310, 318 (1984). s T.A. Verdoorn, N. W. Kleckner, and R. Dingledine, Mol. Pharmacol. 35, 360 (1989). t y. Harada, T. Takahashi, M. Kuno, K. Nakayama, Y. Masu, and S. Nakanishi, J. Neurosci. 7, 3265 (1987). u T. M. Moriarty, S. C. Sealfon, D. J. Carty, J. L. Roberts, R. Iyengar, and E. M. Landau, J. Biol. Chem. 264, 13524 (1989). v y. Oron, B. Gillo, R. E. Straub, and M. C. Gershcngorn, Mol. Endocrinol. 1, 918 (1987). w y. Oron, R. E. Straub, P. Traktman, and M. C. Crershengorn, Science 238, 1406 (1987). x S. Yoshida, S. Plant, P. L. Taylor, and K. A. Eidne, Mol. Endocrinol. 3, 1953 (1989). Y J. A. Williams, D. J. MeChesney, C. Calayag, V. R. Lingappa, and C. D. Logsdon, Proc. Natl. Acad. Sci. U.S.A. 85, 4939 (1988).
[25]
383
REGULATION OF INTRACELLULAR C a 2+ IN OOCYTES
Methods
Injection of Active Substances All intracellular injections may be performed by two methods. When the injection is performed while the oocyte is voltage clamped, to observe the immediate effect of the injected substance, injections are done using a third pipette attached to regulated pressure source ( 1 - 3 psi) via a pneumatic valve controlled electronically by a time delay relay device. The micropipette is manually broken (tip diameter 2 - 5 gm) and backfilled with the desired solution. The volume of injection should not exceed 0.5% of the cell volume (i.e., - 5 nl). The values o f drop diameter, volume, and nominal concentration of injected substances are given in Table II. When long-term effects are to be tested, the oocyte can be injected using an adjustable automatic micropipette (e.g., D r u m m o n d type, total dispensed volume 5 - l 0 / d , can be adjusted to deliver 10- 50 nl). The glass capillary is pulled with an electrode puller and manually broken (diameter 1 0 - 2 5 TABLE II MICROINJECTION PARAMETERSa
Drop diameter (/tin)
Drop volume (nl)
% Oocyte volume
Amount injected (pmol)
Concentration (nM)
20 40 60 80 100 120 140 160 180 200
0.004 0.034 0.113 0.27 0.52 0.9 1.44 2.14 3.05 4.19
0.0005 0.0037 0.0125 0.030 0.058 0.1 0.16 0.24 0.34 0.46
0.004 0.034 0.113 0.27 0.52 0.9 1.44 2.14 3.05 4.19
9.3 74 250 593 1157 2000 3176 4740 6750 9260 (uM)
267 337 386 424 457 486
10 20 30 40 50 60
1.1 2.2 3.3 4.4 5.5 6.6
10 20 30 40 50 60
22 44 66 88 111 133
a Amounts refer to microinjected solution of any compound at 1 m M concentration in the pipette. Concentrations are nominal, assuming complete diffusion of the compound in the aqueous space of the cell (taken as 50% of eel volume for a 1.2-mm-diameter
oocyte).
384
EXPRESS,ON OF ION CHANNELS
[9.5]
TABLE III AMOUNTS AND EFFECTS OF INJECTED SUBSTANCES
Substance
Injected amount
Effect
EGTA
50 pmol 200- 300 pmol 100-300 pmol
CaCI2
0.1 pmol 5 - 50 pmol 100- 300 pmol
Inhibition of spontaneous fluctuations Inhibition of responses Inhibition of cellular calcium release to facilitate measurements of Ba2+ currents Threshold depolarizing fluctuations Medium monophasie responses Large responses, possibly biphasic, late fluctuations Threshold responses (5-10 hA, fluctuations) Small biphasie responses, fluctuations Large biphasic responses, fluctuations Close to saturating responses (0.5-1 #A), late fluctuations Desensitizing responses, 4sCa2+ efllux induction
IP3
1-5fmol 5-10 fmol 50-150 fmol 300-500 fmol 2-10 pmol
/.tm). The capillary is then pressure failed with light mineral or silicone oil and mounted on the wire plunger. It is back-filled with the desired solution. The volume of injected solution can be monitored by identical injection into oil under a microscope (10 × objective) equipped with a reticule. The concentrations of substances needed to achieve the desired effects are given in Table III. Remarks. The effect of intracellular injections depends to a large extent on the site and the depth of injection. To obtain a sharp and immediate effect, calcium or IPa should be injected as shallowly as possible (-50/zm deep). This can be performed only in manually or collagenase-defolliculated oocytes and using pipettes that have been broken to a small tip diameter. On deep injections, the resulting currents are delayed and much less sharp. The second component of responses to IP 3 is better observed using deep injections (150- 300/zm). 2 Responses obtained by injection near the animal (pigmented) pole of the oocyte differ from those observed near the vegetal pole. Chloride currents obtained by the injection of calcium are usually much sharper near the animal pole and decay faster than currents of similar amplitudes produced by injections near the vegetal pole. Injection of IPa at the two poles results in similar kinetics, except that the amplitude of responses 2 B. Gillo, Y. Lass, E. Nadler, and Y. Oron, J. Physiol. (London) 392, 349 (1987).
[25]
REGULATION OF INTRACELLULAR Ca 2+ IN OOCVTES
385
obtained near the animal pole is approximately 5 times greater than those obtained at the vegetal pole. 3 Injections of EGTA are more effective in suppressing spontaneous current fluctuations and the rapid component of the responses to various signals than the slow component of the response elicited by calcium influx? This may be because of the relatively slow kinetics of calcium chelation by EGTA. Once injected, EGTA is effective for a prolonged time (100-250 pmol will block spontaneous fluctuations for at least 30 hr). Injections of as little as 0.5 pmol of calcium produce measurable responses. These can be repeated several times without either desensitization or potentiation. A number of consecutive threshold injections results in delayed small depolarizing current with pronounced fluctuations. 2 This may be due to activation of a separate population of chloride channels or to calcium-induced calcium release. Deep injections of large quantities of calcium (100-300 pmol) result in large currents (-1 /tA, lasting 2 0 - 3 0 sec) which are potentiated by subsequent identical injection (possibly owing to the partial saturation of calcium storesS). Interestingly, unlike calcium introduced by ionophore, injected calcium does not inactivate chloride channels and is not subject to regulation by protein kinase C, indicating a possibly different site of action in the cell. 4-6
Measurement o f 45Ca2+ Fluxes Efflux Measurements. Ideally, to quantitate calcium movement, efflux studies should be performed at isotopic equilibrium. Practically, however, oocytes do not equilibrate their calcium even after 48 hr, and the studies are performed taking into account only the more rapidly exchangeable calcium pools. To measure calcium efflux in individual cells, oocytes are incubated overnight in a small volume ofNDE964 solution with 200 #Ci/ml of 45Ca2+ (specific radioactivity 100/tCi/pmol). The cells are washed several times in ND96 to remove adhering label. Efflux is monitored either by changing the solution every 0.5- 5 min or by holding the oocyte in a perfusion cell and perfusing the desired solution with a peristaltic p u m p at a rate of 1.0-5.0 ml/min. Fractions (0.5- 2.0 ml) are collected with a fraction collector and counted in 4 ml of scintillant. After a certain period needed to determine the basal rate of ettlux, oocytes are stimulated with a desired stimulus (hormone, IP3, etc.). At the end of the experiment, the oocyte is homoge3 M. Lupu-Meiri, H. Shapira, and Y. Oron, FEBSLetL 240, 83 (1988). 4 R. Boton, N. Dascal, B. Gillo, and Y. Lass, J. Physiol. (London) 408, 511 (1989). 5 N. Dascal and R. Boton, FEBSLett.267, 22 (1990). 6 M. Lupu-Meiri, H. Shapira, and Y. Oron, PfluegersArch. 413, 498 (1989).
386
EXPRESSION O17 ION CHANNELS
[25]
TABLE IV STIMULATEDEFFLUXOF 45Ca2+
Stimulus
Concentration
Maximal fractional rate (%/min) a
Time of increased efllux (min)
Total stimulated efllux (%)
ACh-M3 ACh-MI Ade TRH 5-HT IP3 GTPrS
0.1 mM 0.1 mM 10#M 1/zM 1/zM > 2 pmol >20 pmol
1.0 6.4 0.5 11.7 14.0 2.9 3.3
4.6 12.2 1.9 15.0 10.0 9.9 13.7
2.2 35.1 2.0 45.6 41.0 32.2 40.7
a Maximal fractional rate denotes the net increase in the rate of efllux over the basal rate (0.2-1.0% of residual label/rain).
nized in an identical volume of perfusion solution and evaluated for residual counts. Both methods can be used on a voltage-clamped oocyte. In this way, parallel records of electrophysiologic responses and calcium efflux may be obtained. To normalize the data from different oocytes, data can be plotted as log(% residual cpm) versus time (first-order plot) or as percent of counts per minute (cpm) out of total counts per minute in the oocyte at that point (fractional rate of efflux). On short incubations, 45Ca2+ efflux appears to follow first-order kinetics. In prolonged incubations, however, the fractional rate of basal ettlux steadily decreases, indicating participation of another, slower pool. These methods are described in detail in Refs. 7 and 8. Reference values for various stimulations are given in Table IV. Oocytes take up 2000-10,000 cpm/cell, and basal efflux rates are 0.2-1.0%/min. Oocytes that exhibit much higher ettlux rates and/or take up much more 45Ca2+ should be discarded. Influx Measurements. Oocytes are incubated in ND96 solution with 200/zCi/ml 45Ca2+ for a desired period, then washed thoroughly of adhering label and counted. Previous stimulation with a hormone or a neurotransmitter in calcium-free medium results in an enhanced 45Ca2+ influx. To demonstrate this phenomenon, oocytes are incubated for 10 min with the agonist and washed free of it for an additional 20-30 min with calcium-free medium. They are then transferred to a solution that contains the label with or without the stimulus. Reference values are given in Table IV. 7 E. Nadler, B. Gillo, Y. Lass, and Y. Oron, FEBSLett. 199, 208 (1986). s H. Shapira, M. Lupu-Meiri, M. C. Gershengorn, and Y. Oron, Biophys. J. 57, 1281 (1990).
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REGULATION OF INTRACELLULAR Ca 2+ IN OOCYTES
387
Depletion of Cellular Calcium Stores Depletion of signal-sensitive calcium stores can be effected in calciumfree medium in three different ways: (1) by repeated exposures to the signala,9; (2) by exposure to divalent cation ionophores2; (3) by exposure to CaZ+-ATPase inhibitors (B. Gillo and Y. Oron, unpublished, 1985). It should be noted that only the first method is reversible. The effect can be measured indirectly by observing the physiological response (i.e., depolarizing chloride currents) or directly by following 4~Ca2+eftlux. These methods are briefly described below. In depletion experiments, any suitable medium (i.e., ND96 or OR21°) may be used, except that calcium addition is omitted and EGTA is added (usually 0.1 m M is enough). It is our experience that maintaining calcium below 50 p M is sufficient to cause calcium depletion. Depletion of calcium stores by repeated exposures is effected by challenging the oocyte with the signal for 1- 2 min at 30-rain intervals. This results in a complete disappearance of the response and of the associated 4~Ca2+ ettlux by the fourth exposure. Subsequent incubation of the oocyte for l0 min or more with a normal calcium concentration is sufficient to fully restore the response. 8,9 Depletion by ionophore can be effected by preincubation of oocytes in the presence of 0.2- 1 # M of A23187 [in 0.1% v/v ethanol or dimethyl sulfoxide (DMSO)] for 10-15 min. The cells are then washed free of the ionophore solution. It is notable that partial depletion (i.e., in the presence of low concentrations of A23187) affects the rapid component of the response to a larger extent than the second, slow component.TM This is in agreement with our findings that the second component of the responses requires a significant contribution of extracellular calcium. ~2 Depletion with ATPase inhibitors (e.g., vanadate) is effected by adding 0.5-5.0 m M o f t h e agent at pH 8.5-9.0. The initial response was a slowly developing depolarizing current with superimposed current fluctuations, accompanied by modesf 45Ca2+ ettlux. The cell is subsequently insensitive to stimulation (B. Gillo, E. Nadler, Y. Lass, and Y. Oron, unpublished results, 1985). Remarks. Oocytes maintained in calcium-free medium often deteriorate rapidly owing to the activation of unspecified channels.9,~3Addition of high magnesium (20 mM) or DMSO (0.1% v/v) sometimes stabilizes the 9 N. Dascal, B. Gillo, and Y. lass, J. Physiol. (London) 366, 299 (1985). 1oN. Dascal, T. P. Snutch, H. Lubbert, N. Davidson, and H. A. Lester, Science 231, 1147 0986). 11 D. Singer, R. Boton, O. Moran, and N. Dascal, PfluegersArch. 41, 7 (1990). 12 M. Lupu-Meiri, H. Shapira, and Y. Oron, FEBSLett. 262, 165 (1990). 13 N. Dascal, E. M. Landau, and Y. Lass, J. Physiol. (London) 352, 551 0984).
388
EXPRESSION OF ION CHANNELS
[25]
cells. It is our experience, however, that stability to low calcium is better in oocytes of some donors than in others. In oocytes of those donors the addition of high magnesium can be avoided. Also, oocytes kept in calcium-free medium develop pronounced oscillatory currents that can interfere with responses. These eventually disappear on prolonged incubation in the absence of calcium. A23187 is light-sensitive, and all operations should be conducted under diminished illumination and in light-protected vessels, Incubation of oocytes in the presence of calcium and A23187 results in slowly developing chloride current, reflecting most probably the kinetics of incorporation of the ionophore. Large responses can be obtained by incorporation of oocytes with A23187 in calcium-free medium, washing of the ionophore, and exposing the cells to short pulses of calcium.4,n The large currents can be elicited for at least 2 hr after washing out the ionophore, suggesting that it is incorporated into the membrane in a quasi-irreversible manner. The currents are biphasic, indicating two different conductances sensitive to different concentrations of extracellular calcium. The rapid conductance is subject to strong calcium-dependent inactivation.4,14
Calcium-Induced Chloride Currents Depletion of calcium stores greatly facilitates the detection of chloride currents evoked by entry of extracellular calcium. Oocytes challenged in the absence of calcium by an agonist or IP 3 respond to addition of calcium (20 see after the challenge) by a small depolarizing current. This current increases on continuous incubation in calcium-free medium (3 rain), indicating that calcium depletion potentiates the subsequent response to calcium. This phenomenon is often potentiated by inclusion of 0.1% v/v DMSO in the medium (M. Lupu-Meiri and Y. Oron, unpublished, 1991). This concentration of DMSO does not affect either the holding current or the magnitude of responses. The reason for these effects of DMSO is yet to be determined. Using DMSO-eontaining ND96, we have adopted a method which uses repeated challenges with the signal in calcium-free medium. On signficant depletion of the stores, large calcium-evoked chloride currents are observed (> 100 hA). The addition of a nonspeeific calcium entry blocker (e.g., Mn 2+, 1 raM) completely abolishes the calciuminduced chloride current. 12
Demonstration of Separate Calcium Stores Signal-evoked calcium depletion can be used to demonstrate the existence (or absence) of separate, dedicated calcium stores in the oocyte. 14 R. Boron, D. Singer, and N. Dascal, PfluegersArch. 41, 1 (1990).
[25]
REGULATION OF INTRACELLULAR Ca 2+ 1N OOCYTES
389
Briefly, oocytes possessing the intrinsic muscarinic response and an acquired response [e.g., to thyrotropin-releasing hormone (TRH) after injection with mRNA for TRH receptors] are repeatedly challenged with acetylcholine (ACh). In oocytes preloaded with 45Ca2+ in the presence of calcium, the label in ACh-specific store is lost, whereas that in the TRHspecific store remains intact. In the absence of extraceUular calcium, the response to ACh disappears, while that to TRH remains. The response to ACh can be restored by short incubation with calcium. 8 Measuring Free Cytoplasmic Calcium
Both calcium microelectrodes and optical methods with fluorescent or luminescent indicators have been used to qualitatively follow free cytoplasmic calcium in oocytes. The main drawbacks of these methods are that they are at best semiquantitative and require specialized equipment or specific training. We have, therefore, decided to include the appropriate references without elaborating these methods here. Although measurements of changes in oocyte free calcium with ion-selective electrodes have been reported, the only well-described work is by Busa et al. 15 It should be stressed that electrodes measure calcium in the immediate vicinity of the electrode tip, and this may not necessarily be the compartment of interest. We have used quin 2 to measure both resting and stimulated concentrations of free calcium in albino oocytes. The results were disappointing. The basal concentration of free calcium was indeed around 0.1 pM. However, the rises due to stimulation of native ACh receptors were negligible and implied that microinjected dye buffers the cytoplasmic calcium or that the rise occurs in a subcellular compartment which constitutes only a negligible fraction of the cytoplasmic space (Y. Oron and M. C. Gershengorn, unpublished, 1985). Moreau et aL have reported using aequorin to follow calcium elevation induced by progesterone) 6 These results were difficult to duplicate, t7,18 Parker and Miledi 19have demonstrated that injected aequorin can be used to monitor calcium elevation caused by injection of IP 3. Takahashi et aL 2° have described a method that used Fura-2 to monitor rises in free calcium following the stimulation of acquired 5-hydroxytryptamine (5-HT) recep-
15 W. B. Busa, J. E. Ferguson, S. K. Joseph, J. R. WiUiamson, and R. Nuccitelli, J. Cell Biol. 101, 677 (1985). 16 M. Moreau, J. P. Vilain, and P. Guerrier, Dev. Biol. 78, 201 (1980). 17 K. R. Robinson, Dev. BioL 109, 504 (1985). is R. J. Cork, M. F. Cicirelli, and K. R. Robinson, Dev. Biol. 121, 41 (1987). 19 I. Parker and R. Miledi, Proc. R. Soc. London B 228, 307 (1986). 20 T. Takahashi, E. Neher, and B. Sakmann, Proc. Natl. Acad. Sci. U.S.A. 84, 5063 (1987).
390
EXPRESSIONOF ION CaA~-mzI~S
[25]
tors. Recently, Brooker et al. 21 have described an elegant method to measure stimulated calcium changes in oocytes using Fura-2 fluorescence. Recording o f Currents Conducted by Plasma M e m b r a n e Calcium Channels
Native Xenopus oocytes exhibit low activities of intrinsic voltagedependent calcium channels? ° Much greater activities of voltage-dependent calcium channels are expressed in oocytes injected with mRNA from various excitable tissues? °,22-25 Direct recording of calcium current on depolarization is impossible due to the masking effect of large calcium-activated chloride currents. It may be possible to circumvent this problem by applying the internal perfusion method described elsewhere in this volume. 26 In intact oocytes, it is possible to record currents flowing through calcium channels using barium as the charge carrier, since Ba2+ does not activate chloride channels. 27 The standard medium for this type of experiments contains 30-40 m M Ba,2+ 60-70 m M Na + or N-methyl-o-glucamine, and 5 m M HEPES (pH 7.5) and substitutes methane sulfonate for chloride.~°a5 Methane sulfonate does not permeate through chloride channels and also partially blocks them? Chloride channels can be further inhibited by 9-anthracenecarboxylic acid (0.5-2 m M from a stock solution of 200 m M in 0.5-1.0 M NaOH) or niflumic of fluphenamic acids (0.01 0.10 m M from a 0.1- 1.0 M stock in DMSO). 4,2s Even in the presence of chloride channel blockers, large Ba2+ currents (> 100 nA) will produce late variable inward currents, probably owing to the release of cellular calcium and activation of chloride currents. These can be completely suppresed by injections of EGTA (see above). Note. In EGTA-injected oocytes, pure barium currents can be recorded even in solutions that contain C1- as the major anion. EGTA, however, fails to buffer cellular calcium fully (if Ca 2÷ is present in the medium), and the resulting chloride currents will interfere with the recording (N. Dascal, unpublished, 1985).
21G. Brooker, T. Seki, D. Croll, and C. Wahlstedt, Proc. Natl. Acad. Sci. U.S.A. 87, 2813 (1990). 22j. R. Moorman,Z. Zhou, G. E. Kitsch,A. E. Lacerda,J. M. Caffrey,D. M.-K.Lain,R. H. Joho, and A.M. Brown, Am. J. Physiol. 253, H-985 (1987). 23j. p. Leonard,J. Nargeot,T. P. Snutch,N. Davidson,and H. A. Lester,J. Neurosci. 7, 875 (1987). 24j. A. Umbachand C. B. Gundersen,Proc. Natl. Acad. Sci. U.S.A. 84, 5464 (1987). 25I. Lotan,P. Goelet,and N. Dascal,Science 243, 666 (1989). 26N. Dascal,G. Chilcott,and H. Lester,this volume[21]. 27M. E. Barish,J. PhysioL (London) 342, 309 (1983). 28M. M. Whiteand M. Aylwin,MoL Pharmacol. 37, 720 (1990).
