Neuroscience Vol. 39, No. 3, Pp. 809-814, 1990 Printed in Great Britain
03064522/90 $3.00 + 0.00 Pergamon Press plc 0 1990 IBRO
EXPRESSION OF RECEPTOR FOR a-LATROTOXIN IN XENOPUS OOCYTES AFTER INJECTION OF mRNA FROM RAT BRAIN A. K. FILIPP~v,*~
E. M. KOBRINSKY,* G. P. TSURUPA,$
V. N. PA~HKOV
and E. V. GRI~HIN*§
*Institute of Biological Physics, U.S.S.R. Academy of Sciences, 142292 Pushchino, Moscow Region, U.S.S.R. SBranch of Shemyakin Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, U.S.S.R. &hemyakin Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, U.S.S.R. Abstract-a-Latrotoxin, the major toxin of black widow spider venom, was suggested to bind to the specific receptor on the membrane of presynaptic cells and to activate a nonselective cation channel. The aim of this investigation was to express the receptor to a-latrotoxin in the membrane of Xenopus laeuis oocytes. Responses to a-latrotoxin were studied using a double microelectrode voltage-clamp techniqe on X. laeuis oocytes previously injected with poly(A+f-RNA from rat brain. a-Latrotoxin (10 nM) was shown to induce a slow activating reversible inward membrane current at a clamp potential of -60 mV. A second application of a-latrotoxin immediately after washing out induced a smaller response. Reversal potential of this current was near to OmV, it hardly changed in low Cl- external solution. Response to a-latrotoxin did not depend si~ifi~ntly on the variation of Ca2+ concentration in external solution. Ethyleneglycolbis(amin~~yle~er)tetra-a~tate (EGTA) injection into oocytes did not decrease a-latrotoxin-induced current, but seemed to slow the kinetics of the response. Inorganic Ca-channel blocker Co2+ had no effect on a-latrotoxin response. These results indicate that a-latrotoxin-induced inward current flows mainly through cation nonspecific channel. A Win concanavalin A irreversibly inhibited a-latrotoxin-evoked inward current. Many of these observations are similar to those reported for nerve cells after a-latrotoxin application. The data obtained suggest that functional receptor to a-latrotoxin can be successively expressed in the membrane of Xenopus oocytes providing future search of DNA encoding receptor subunits and study of receptor structurefunction relationship.
a-Latrotoxin, a high molecular weight protein from the venom of the black widow spider, stimulates massive release of a variety of neurotransmitters both at peripheral and central synapses. This effect seems to be related to the activation of cation nonspecific channel in presynaptic membrane as a consequence of toxin binding to the specific membrane receptor.20 Receptor to cc-latrotoxin has been purified from bovine brain and reconstituted in active form in artificial membranes.3’.32 However, the primary structure of ar-latrotoxin receptor and gene encoding of this receptor are still unknown. Recently, a powerful approach has been developed which allows one both to indentify a set of DNA encoding the full structure of the membrane receptor or ion channel and to study the relation between the structure of a gene molecule and function of receptor or ion channel which this gene encodes. This approach is based on the functional expression of receptors and ion channels in the membrane of Xenopus laevis oocytes after microinjection of mRNA from different tissues into the oocyte cytoplasm.‘~5 For example, /I,and /I,-adrenergic receptors, M,-, M,- and a2-r ~To whom correspondence should be addressed. Abbreviations: EDTA, e~yl~~i~netetra-bite; e~ylene~y~lbis(a~n~~ylether~te~-agate; N-2-hydroxyethylpi~ra~ne-~-2~thanesulphonic
EGTA, HEPES, acid. 809
nicotinic acetylcholine receptors, receptors to serotonin, amino acids, dopamine, neuro~ptides, voltage-dependent sodium, calcium and potassium channels were expressed in the membrane of Xertopus oocytes. 1~5~13~15z30 Using this approach, cDNA encoding serotonin lC-receptor, neuropeptide substance K receptor, 16,17Na/glucose co-transporter’* and one of the potassium channels7 was identified. Moreover, the site-directed mutagenesis allowed one to determine localization of functional regions of nicotinic acetylcholine receptor subunits4,1’+‘4~23*24 and Nachannel.33 The goal of this investigation was to express functional receptor to a-latrotoxin in the membrane of Xenop~ oocytes. EXPERIMENTAL PROCEDURES Preparation
of mRNA
RNA was isolated from rat brain by the guanidine thiocyanate methods9 Poly(A+)-RNA was obtained by oligo(dT)-cellulose chromatography method with some modifications.‘s RNA was dissolved in water, treated at 65°C for 3 min, and mixed with an equal volume of 20 mM Tris-HCl, 2 mM EDTA, 800 mM NaCl (pH 7.5). It was loaded onto a pm-equilibrated column (volume, 1.5 ml). Eluate was heated to 65°C for 3min, cooled to room tem~ratu~ for 2min and re-applied to the column. Then the column was washed with 5 vol of IOmM Tri-HCl, 1 mM EDTA, 400 mM NaCl (pH 7.5), then with 5 vol of the
810
A. K.
FILIPPOVet al.
same buffer but containing 200 mM NaCl. Poly(A+)-RNA was eluted with 2 vol of buffer without NaCl. RNA content was determined spectroscopically assuming 40 pg RNA/A,, unit. The ethanol-precipitated RNA was stored at -20°C. For injection into oocytes mRNA was dissolved in water (1 mg/ml). Ability of poly(A+)-RNA to synthesize was assayed by in vitro translation using wheat-germ lysates in the presence of [t4C]valine and [14C]leucine.36Molecular weights of the products were analysed by dodecylsulphatepolyacrylamide gel electrophore@ followed by fluorography at -70°C.
LTx
Injection info oocytes Oocytes were obtained from adult laboratory bred Xenopus laeuis. Pieces of the ovary were removed under general
anaesthesia through a small incision in the abdomen and placed into the physiological solution (ND 96) containing (in mM): NaCl 96, KC1 2, CaCl, 1.8, MgCl, 1, HEPESNaOH 5 (pH 7.5), and supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin. The abdominal musculature and then the skin of the frog was sutured. The frog was allowed to heal for at least four weeks before more oocytes were removed. Oocytes at maturation stage V or VI were separated manually from the ovary with forceps. Oocytes were injected with mRNA using the procedure of Gurdon.’ Injection pipettes with a tip-opening diameter of lo-15 pm were fabricated using a microforge. The water solution of poly(A+)-mRNA, 1 pg/nl was drawn into the injection pipette by slight suction. Each oocyte was injected with 50 nl of RNA solution, then placed in approximately 100~1 of sterile ND 96 solution with 100 U/ml penicillin and 100 pg/ ml streptomycin in a 96-well dish and cultured at 19-20°C five to seven days before electrophysiological experiments. The culture medium was changed daily. In some experiments the follicle cell layer of oocytes was removed two days after mRNA injection. Oocytes were treated with 1 mg/ml collagenase (form I, Koch-Light Laboratories, U.K.) in ND 96 solution for 1 h at room temperature and subsequently washed in ND 96 solution.*’ When necessary, the remaining follicle cell layer was removed manually with fine forceps.
Fig. 1. Whole-cell a-latrotoxin induced current record of voltage-clamped Xenopus laevis oocytes previously injected with poly(A+)-mRNA from rat brain. Horizontal bars above the traces show the time of application of a-latrotoxin, 10nM. Holding potential, -60mV. Downward deflection corresponds to inward current. LTx, cc-latrotoxin.
a-latrotoxin (10 nM) by slow membrane current, which was inward at clamp potential of -60 mV (Fig. 1). This current appeared with a delay of 1.5% 2 min after CI-1atrotoxin application, slowly increased and reached a plateau within 3-5 min. It slowly disappeared within the same time interval when oocytes were washed out with a-latrotoxin-free physiological solution. Delay and time-course of the response were similar in oocytes with and without follicle cell layer. This indicated that the effect of
2 1 Jr
LTx
-25
2 min
A+&
Electrophysiological measurements
Oocytes were voltage-clamped using a standard 24ntracellular microelectrode circuit with virtual ground current measurement. Borosilicate glass capillaries were pulled and filled with 3 M KC1 solution. Voltage recording and current injection electrodes had d.c. resistances between 0.5 and 2.0 MD. The oocytes were placed in a small groove in an experimental chamber and continuously perfused with ND 96 physiological solution. Solution changes were normally completed within 15 s. Current and voltage signals were observed on an oscilloscope and simultaneously recorded on a chart recorder. All experiments were done at room temperature (19-22°C). Low Cl- solution used in some experiments contained (in mM): NaCl 58, Na,SO, 19, CaCl, 1.8, KC1 2, MgCl,l, sucrose 19, HEPES 5 (titrated to pH 7.5). Concentrated stock solution of a-latrotoxin was purified from the venom of the Central Asia spider karakurt (black widow spider, Latrodectus mactans tredecimguttatus), by previously published methods2* It was divided into small aliquots and stored at -80°C. Concanavalin A (Pharmacia) was dissolved in the physiological solution immediately before the experiment. RESULTS
Induction
of a-latrotoxin
responses
Oocytes previously injected with rat brain poly(A+)-RNA responded to bath application of
+5 ._._1x- ._-
P+L-____--6_“__-_ 4
-
‘h-2
Fig. 2. Effect of holding potential changes on the response to a-latrotoxin. Holding potentials applied are indicated by numbers above the traces. Time of exposure to cc-latrotoxin is shown by horizontal bars. Dashed line shows resting current at a holding potential indicated on the left. The reversal potential of the a-latrotoxin-evoked current is near 0 mV. LTx, a-latrotoxin.
Expression of a-latrotoxin
did not depend on the presence of a follicular layer of oocytes. The responses to a-latrotoxin could be observed soon at 1 nM toxin concentration; however, they were noticeably smaller than at 10 nM. Therefore, 10 nM toxin concentration was used in most experiments. A second application of a-latrotoxin immediately after restoration of the current during washing out usually induced a 1.5-4 times smaller response (Fig. 1). The decrease varied from oocyte to oocyte. Washing out (2-3 h) was necessary to restore the same amplitude of the response. Hence, a partial desensitization is a property of a-latrotoxin-induced response in Xenopus oocytes. Control (non-mRNA-injected) oocytes from the same donors usually failed to show responses to a-latrotoxin. Only two of 10 frogs used in the experiments gave control oocytes which showed appreciable a -latrotoxin-induced current (15 +_1 nA; n = 21). However, this current was over three times smaller than that in the oocytes injected with mRNA (56 + 7 nA; n = 25). Oocytes from eight frogs responded to a-latrotoxin only if they were injected with mRNA. cl-latrotoxin
Reversal potential of a-latrotoxin induced response
The ionic nature of a-latrotoxin-evoked current was evaluated by measuring reversal potential of the response to a-latrotoxin. Because of partial desensitization of the response it was difficult to determine the reversal potential on single oocytes. Therefore, three oocytes were used for the determination of reversal potential. This allowed us to give no less than 1 h rest to the oocyte to restore the amplitude of response to a-latrotoxin. An example of the experiment is shown in Fig. 2. The oocytes were clamped at the holding potential indicated and then a-latrotoxin was applied. When holding potential was near to the reversal potential, an additional procedure was carried out in order to be sure that a-latrotoxin evoked a significant ionic conductance. When the response to a-latrotoxin reached a steady value, holding potential was changed to -60 mV and in 2-3 min wash-out was started. Significant inward current, which decreased during wash-out, indicated that a-latrotoxin was effective in inducing the ionic conductance. Reversal potential determined in this way was near to 0 mV (Fig. 2). It hardly changed in low Cl- solution (data not shown).
receptor in Xenopus oocytes
811 ca2+*5
LTx
w_lii_7 LTn
LTx
co2+ 2 min
N" +5---
'l,
.-*
'"
I
Fig. 3. Effect of 9 mM Ca2+ (upper trace), EGTA injection, 1 mM oocyte (middle trace) and 3 mM Co2+ (lower trace) on the response to a-latrotoxin. Holding potential, -60 mV. Horizontal bars indicate the time of application of a-latrotoxin and cations. Dashed line indicates resting current at holding potential. Upper and middle traces were obtained from one oocyte. LTx, a-latrotoxin.
external solution induced large progressive depolarization of the membrane and in some cases deterioration of the oocyte. This effect of EGTA was also observed when 20mM MgC12 was substituted for Ca*+ in the external solution in three experiments. In two experiments we studied the effect of EGTA injection into oocyte on a-latrotoxin response. The oocytes which showed large response to a-latrotoxin were left for 5 h to rest in ND 96 solution without a-latrotoxin, and then 15 mM EGTA, 50-75 nl/ oocyte (0.75-l mM/oocyte) buffered to pH 7.5 were injected into the oocytes 0.5 h before the application. EGTA. Injection did not decrease markedly the a-latrotoxin effect; however, the kinetics of a-latrotoxin response seemed to be slower (Fig. 3, middle). In contrast, EGTA injection greatly reduced (five to 10 times) the transient outward Ca-dependent chloride current Ic,(ca) measured on test rectangular pulses to + 5 mV from holding potential -60 mV. In the experiment shown in Fig. 3 Ic,(caJwas reduced from 160 to 20 nA. Control injection of buffered water (pH 7.5) did not show any influence on the a -1atrotoxin effect or on Ic,(ca). Inorganic Ca-channel blocker Co*+ (3 mM) had no effect on the a-latrotoxin response (three experiments). An example is shown in Fig. 3 (bottom). Effect of concanavalin A
Effect of Ca*+, EGTA and Co*+
The response to a-latrotoxin did not increase when Ca*+ concentration in the external solution was elevated by five times (Fig. 3, top). A significant effect of a-latrotoxin was observed when Ca*+ was not added to external physiological solution. Our attempts to study effect of a-latrotoxin in Ca-free solution using EGTA failed since EGTA (0.5-l mM) in the
Concanavalin A, 1 FM, slowly and irreversibly inhibited the effect of a-latrotoxin. Response to a-latrotoxin was not restored even after 15 h of washing out in concanavalin A-free ND 96 solution (Fig. 4). This was not due to the deterioration of the oocyte, because its resting potential remained unchanged (- 70 mV, for example, shown in Fig. 4) during this period of time.
812
A. K. LTx
FILIPPOV er a/.
Con A LTx
Fig. 4. Effect of I PM concanavalin A on response to cc-latrotoxin. Note that response to a-latrotoxin is slowly inhibited by concanavalin A (left trace) and does not restore even after 15h of wash-out in concanavalin A-free physiological solution (right trace). Holding potential, - 60 mV. Horizontal bars, time of exposure to cl-latrotoxin and concanavalin A. Dotted line indicates resting current at holding potential. Con A, concanavalin A; LTx, cc-latrotoxin.
Other RNA-directed responses Ionic current responses to GABA (100 FM), L-glutamate (100 FM), kainate (100 PM), neuropeptide substance P (1 PM) were also observed in mRNA-injected oocytes. These responses were never observed in control (non-injected) oocytes. Response to acetylcholine, serotonin, the appearance of voltageoperated sodium and Ca-channels were also directed by poly(A+)-mRNA injection in Xenopus oocytes in our experiments. All these responses were similar to those observed in other laboratories in RNA-injected Xenopus oocytes (see, e.g., Refs 5, 13, 27). DISCUSSION
The precise molecular mechanism underlying the a-latrotoxin effect is still a subject of some controversy. Some authors suggest that the action of the toxin is due to its spontaneous insertion across the membrane leading to a channel-forming activity.6 This suggestion leaves unexplained the strict tissue specificity of c(-1atrotoxin action.20 Other authors suggest the occurrence of a specific receptor to cc-latrotoxin on the presynaptic cell membrane.20*31*32 If cr-latrotoxin exerts its physiological effect through the activation of specific receptor on the membrane surface of presynaptic nerve cells then gene encoding of this receptor must exist. Indeed, we have observed that t(-1atrotoxin evokes significant inward transmembrane current at the resting membrane potential (- 60 mV) in Xenopus oocytes previously injected with poly(A+)RNA from rat brain. The properties of this current can be attributed in many aspects to cc-latrotoxin effect on synapses and neurosecretory cells of the PC 12 line. This current will depolarize the membranean effect typical for a-latrotoxin on synapses. Timecourse and reversal potential (near OmV) of the current activated by cc-latrotoxin in the oocytes are close to those obtained for the ionic current activated by cr-latrotoxin in PC 12 cells.39The reversal potential did not change when Cl- concentration was varied in the external solution. A similar result has been obtained on PC 12 cells indicating that cations, but not anions, are mainly responsible for the a-latrotoxininduced current.34 The reversal potential observed can be attributed to a nonspecific cation channel generally believed to be activated by cc-latrotoxin in
nerve cells.20It is well known that ct-latrotoxin effects are not modified by Ca-channel blockers and do not depend significantly on [Ca2+],Utconcentration if another divalent cation is present in the external solution.‘9~20~25~26~34 We observed this in our experiments. These data are also in favour of the hypothesis for the ion channel that has no specific selectivity for Ca2+ and is permeable to other cations such as Na+, K+, Mg* + as well. 2o,34Lectins such as concanavalin A are known to inhibit cc-latrotoxin effects2’ and this has also been observed in our experiments. Since Ca2+ ions seem to enter the cell through Elatrotoxin-activated channel in some preparations~6,~ one can suggest the oocyte’s endogenous [Cali,dependent chloride inward current &-,,,,5 to be part of the response to cr-latrotoxin observed in our experiments. Experiments with EGTA injection into the oocytes to prevent a rise of cytosolic Ca2+ concentration have shown that contribution of Iclcca, into the a-latrotoxin response does not seem to be significant. Indeed, injection of EGTA greatly reduced transient ZcI(caJ,but, however, did not change the amplitude of the a-latrotoxin-induced current. The data discussed are in favour of the suggestion that a-latrotoxin receptor has been successively expressed in the membrane of Xen~pu~ oocytes in our experiments as a consequence of mRNA injection into the oocytes. Several questions, however, remain to be answered. The effective concentration of oc-latrotoxin in our experiments was approximately one order higher than that observed at synapses, PC 12 cells or receptorThe delay in the reconstituted liposomes. 20*32~34 appearance of e-latrotoxin effect in our experiments seemed to be longer than in experiments on PC 12 cells34 and on planar lipid membranes infused with receptor-reconstituted liposomes3? taking into account even the “dead” time of our perfusion system. These points can probably bc explained if we assume that a-latrotoxin severely penetrates through the vitelline membrane of the oocyte which had not been removed in our experiments. Direct experiments on the oocytes without vitelline membrane can answer this question.
However, we have observed that the follicle cell layer which is usually considered as a barrier for large molecules2~3sdoes not affect the delay and the timecourse of the response to a-latrotoxin. Another point
Expression of x-latrotoxin receptor in Xenopus oocytes
is that a-latrotoxin effect is reversible in our experiments, whereas it does not reverse in synapses and PC 12 cell~.~*~ How can all these points be explained? The receptor to a-latrotoxin seems to have a complex subunit structure.3’ The functional role of the subunits is unknown. Besides, the complex chain of molecular events was suggested after a-latrotoxin binding to the receptor.3*20One can speculate that receptor subunits are not expressed properly or some other disturbances in receptor assembly take place in the oocyte membrane. Additional experiments are needed to answer this question. We suggest, however, that these disturbances, if they take place, are not too large since
813
many properties of the response to a-latrotoxin are preserved, Therefore, we conclude that functional cc-latrotoxin receptor can be expressed in the membrane of X. laevis oocytes after the mRNA injection from the brain into the oocyte cytoplasm. Hence, the goal of future investigations is to search for DNA encoding receptor subunits and to study structurefunction ~lationships of the a-latrotoxin receptor by a combined use of site-directed mutagenesis and electrophysiological recording. Acknowledgemenrs-We are grateful to Dr Viktor Uteshev for providing us with frogs, X. laeuis.
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18. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Moiec&r CIoning: A Laboru!ory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 19. Meldolesi J., Madeddu L., Torda M., Gatti G. and Niutta E. (1983) The effect of a-latrotoxin on the neurosecretory PC 12 cell line: studies on toxin binding and stimulation of transmitter release. Neuroscience 10, 997-1009. 20. Meldolesi J., Scheer H., Madeddu L. and Wanke E. (1986) Mechanism of action of a-latrotoxin: the presynaptic stimulatory toxin of the black widow spider venom. Trends’pharmac. Sci. 7, 151-155. 21. Methfessel C., Witzemann V., Takahashi T., Mishina M., Numa S. and Sakmann B. (1986) Patch clamp measurement on Xenopus Zaeuis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. PJugers Arch. ges. Physiol. 407, 577-588. 22. Meyerhof W., Morley S., Schwarz J. and Richter D. (1988) Receptors for neuropeptides are induced by exogenous poly(A+)-RNA in oocytes from Xenopus laevis. Proc. natn. Acad Sci. U.S.A. 85, 714-717. new approach to structure and mechanism. Neuron 2, 23. Miller C. (1989) Genetic manipulation of ion channels-a 1195-1205. 24. Mishina M., Tobimatsu T., Imoto K., Tanaka K., Fujita J., Fukuda K., Kurasaki M., Takahashi M., Morimoto J., Hirose T., Inavama S., Takahashi T., Kuno M. and Numa S. (1985) Location of functional regions of a~tylcholine receptor a-subunit by site-directed mutagenesis. Nature 313, 364369.
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25 Misler S. and Hurlbut W. P. (1979) Action of black widow spider venom on quantized release of acetylcholine at the
frog neuromuscular junction: dependence upon external Mg’+. Proc. natn. Acad. Sci. U.S.A. 76, 99-995. of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx and stimulates transmitter release in guinea pig brain synaptosomes. Proc. natn. Acad. Sci. U.S.A. 79, 7924-7928. 27. Parker f., Sumikawa K. and Miledi R. (1986) Neurotensin and substance P receptors expressed in Xenonus oocytes by messenger RNA from rat brain. Proc. R. SQC. Land., B 229, 151-159. 28. Pashkov V. N., Kovalevskava G. I.. Krasnoeerov V. G. and Bulnakov 0. V. (19891 Isolation of a-latrotoxin from the venom of Latr~dectus m&ans tredecimguttkus by means of rr&oclonal antibodies. Bioorg. Chem. 15, 1281-1283 (in Russian). 29. Rubin L. L., Gorio A. and Mauro A. (1978) Effect of convcanavalin A on black widow spider venom action at the neuromuscular junction: implications for mechanisms of venom action. Bruin Rex 143, 107-124. 30. Sakai Y., Sekiguchi M., Okamoto K., Kuwano R. and Takahashi Y. (1990) Dopamine receptors expressed in the Xenopus oocytes injected with bovine striatal mRNA. Molec. Brain Res. 7, 183-187. of the putative a-latrotoxin receptor from bovine synaptosomal 31. Scheer H. and Meldolesi J. (1985) Pu~fi~tion membranes in an active binding form. Eur. molec. Biol. Org. J. 4, 323-327. 32. Scheer H., Prestipino G. and Meldolesi J. (1986) Reconstitution of the purified a-latrotoxin receptor in liposomes and planar lipid membranes. Clues to the mechanism of toxin action. Eur. molec. Biol. Org. J. 5, 2643-2648. 33. Stuhmer W., Conti F., Suzuki H., Wang X., Noda M., Jahagi N., Kubo H. and Numa S. (1989) Structural parts involved in activation and inactivation of the sodium channel. Nature 339, 597-603. 34. Wanke E., Ferroni A., Gattanini P. and Meldolesi J. (1986) a-Latrotoxin of the black widow spider venom opens a small non-closing cation channel. Riochem. Biuphys. Res. Commun. 134, 320-325. 35. Werner R., Miller T., Azamia R. and Dahl G. (1985) Translation and functional expression of cell-to-ceil channel mRNA in Xenopus oocytes. .I. Membrane Bioi. 87, 2.53-268. 36. Zozulya S. A., Gurevich V. V., Shmukler B. E., Natochin M. Yu., Zvyaga T. A., Gryaznov S. M. and Shirokova E. P. (1988) Synthesis of visual rhodopsin in a cell-free translation system. Bioorg. Chem. 14, 1663-1670 (in Russian). 26. Nicholls D. G., Rugolo M., Scott J. G. and Meldolesi J. (1982) a-Latrotoxin
(Accepted 19 June 1990)
03064522/90 $3.00 + 0.00 Pergamon Press plc 0 1990 IBRO
EXPRESSION OF RECEPTOR FOR a-LATROTOXIN IN XENOPUS OOCYTES AFTER INJECTION OF mRNA FROM RAT BRAIN A. K. FILIPP~v,*~
E. M. KOBRINSKY,* G. P. TSURUPA,$
V. N. PA~HKOV
and E. V. GRI~HIN*§
*Institute of Biological Physics, U.S.S.R. Academy of Sciences, 142292 Pushchino, Moscow Region, U.S.S.R. SBranch of Shemyakin Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, U.S.S.R. &hemyakin Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, U.S.S.R. Abstract-a-Latrotoxin, the major toxin of black widow spider venom, was suggested to bind to the specific receptor on the membrane of presynaptic cells and to activate a nonselective cation channel. The aim of this investigation was to express the receptor to a-latrotoxin in the membrane of Xenopus laeuis oocytes. Responses to a-latrotoxin were studied using a double microelectrode voltage-clamp techniqe on X. laeuis oocytes previously injected with poly(A+f-RNA from rat brain. a-Latrotoxin (10 nM) was shown to induce a slow activating reversible inward membrane current at a clamp potential of -60 mV. A second application of a-latrotoxin immediately after washing out induced a smaller response. Reversal potential of this current was near to OmV, it hardly changed in low Cl- external solution. Response to a-latrotoxin did not depend si~ifi~ntly on the variation of Ca2+ concentration in external solution. Ethyleneglycolbis(amin~~yle~er)tetra-a~tate (EGTA) injection into oocytes did not decrease a-latrotoxin-induced current, but seemed to slow the kinetics of the response. Inorganic Ca-channel blocker Co2+ had no effect on a-latrotoxin response. These results indicate that a-latrotoxin-induced inward current flows mainly through cation nonspecific channel. A Win concanavalin A irreversibly inhibited a-latrotoxin-evoked inward current. Many of these observations are similar to those reported for nerve cells after a-latrotoxin application. The data obtained suggest that functional receptor to a-latrotoxin can be successively expressed in the membrane of Xenopus oocytes providing future search of DNA encoding receptor subunits and study of receptor structurefunction relationship.
a-Latrotoxin, a high molecular weight protein from the venom of the black widow spider, stimulates massive release of a variety of neurotransmitters both at peripheral and central synapses. This effect seems to be related to the activation of cation nonspecific channel in presynaptic membrane as a consequence of toxin binding to the specific membrane receptor.20 Receptor to cc-latrotoxin has been purified from bovine brain and reconstituted in active form in artificial membranes.3’.32 However, the primary structure of ar-latrotoxin receptor and gene encoding of this receptor are still unknown. Recently, a powerful approach has been developed which allows one both to indentify a set of DNA encoding the full structure of the membrane receptor or ion channel and to study the relation between the structure of a gene molecule and function of receptor or ion channel which this gene encodes. This approach is based on the functional expression of receptors and ion channels in the membrane of Xenopus laevis oocytes after microinjection of mRNA from different tissues into the oocyte cytoplasm.‘~5 For example, /I,and /I,-adrenergic receptors, M,-, M,- and a2-r ~To whom correspondence should be addressed. Abbreviations: EDTA, e~yl~~i~netetra-bite; e~ylene~y~lbis(a~n~~ylether~te~-agate; N-2-hydroxyethylpi~ra~ne-~-2~thanesulphonic
EGTA, HEPES, acid. 809
nicotinic acetylcholine receptors, receptors to serotonin, amino acids, dopamine, neuro~ptides, voltage-dependent sodium, calcium and potassium channels were expressed in the membrane of Xertopus oocytes. 1~5~13~15z30 Using this approach, cDNA encoding serotonin lC-receptor, neuropeptide substance K receptor, 16,17Na/glucose co-transporter’* and one of the potassium channels7 was identified. Moreover, the site-directed mutagenesis allowed one to determine localization of functional regions of nicotinic acetylcholine receptor subunits4,1’+‘4~23*24 and Nachannel.33 The goal of this investigation was to express functional receptor to a-latrotoxin in the membrane of Xenop~ oocytes. EXPERIMENTAL PROCEDURES Preparation
of mRNA
RNA was isolated from rat brain by the guanidine thiocyanate methods9 Poly(A+)-RNA was obtained by oligo(dT)-cellulose chromatography method with some modifications.‘s RNA was dissolved in water, treated at 65°C for 3 min, and mixed with an equal volume of 20 mM Tris-HCl, 2 mM EDTA, 800 mM NaCl (pH 7.5). It was loaded onto a pm-equilibrated column (volume, 1.5 ml). Eluate was heated to 65°C for 3min, cooled to room tem~ratu~ for 2min and re-applied to the column. Then the column was washed with 5 vol of IOmM Tri-HCl, 1 mM EDTA, 400 mM NaCl (pH 7.5), then with 5 vol of the
810
A. K.
FILIPPOVet al.
same buffer but containing 200 mM NaCl. Poly(A+)-RNA was eluted with 2 vol of buffer without NaCl. RNA content was determined spectroscopically assuming 40 pg RNA/A,, unit. The ethanol-precipitated RNA was stored at -20°C. For injection into oocytes mRNA was dissolved in water (1 mg/ml). Ability of poly(A+)-RNA to synthesize was assayed by in vitro translation using wheat-germ lysates in the presence of [t4C]valine and [14C]leucine.36Molecular weights of the products were analysed by dodecylsulphatepolyacrylamide gel electrophore@ followed by fluorography at -70°C.
LTx
Injection info oocytes Oocytes were obtained from adult laboratory bred Xenopus laeuis. Pieces of the ovary were removed under general
anaesthesia through a small incision in the abdomen and placed into the physiological solution (ND 96) containing (in mM): NaCl 96, KC1 2, CaCl, 1.8, MgCl, 1, HEPESNaOH 5 (pH 7.5), and supplemented with 100 U/ml penicillin and 100 pg/ml streptomycin. The abdominal musculature and then the skin of the frog was sutured. The frog was allowed to heal for at least four weeks before more oocytes were removed. Oocytes at maturation stage V or VI were separated manually from the ovary with forceps. Oocytes were injected with mRNA using the procedure of Gurdon.’ Injection pipettes with a tip-opening diameter of lo-15 pm were fabricated using a microforge. The water solution of poly(A+)-mRNA, 1 pg/nl was drawn into the injection pipette by slight suction. Each oocyte was injected with 50 nl of RNA solution, then placed in approximately 100~1 of sterile ND 96 solution with 100 U/ml penicillin and 100 pg/ ml streptomycin in a 96-well dish and cultured at 19-20°C five to seven days before electrophysiological experiments. The culture medium was changed daily. In some experiments the follicle cell layer of oocytes was removed two days after mRNA injection. Oocytes were treated with 1 mg/ml collagenase (form I, Koch-Light Laboratories, U.K.) in ND 96 solution for 1 h at room temperature and subsequently washed in ND 96 solution.*’ When necessary, the remaining follicle cell layer was removed manually with fine forceps.
Fig. 1. Whole-cell a-latrotoxin induced current record of voltage-clamped Xenopus laevis oocytes previously injected with poly(A+)-mRNA from rat brain. Horizontal bars above the traces show the time of application of a-latrotoxin, 10nM. Holding potential, -60mV. Downward deflection corresponds to inward current. LTx, cc-latrotoxin.
a-latrotoxin (10 nM) by slow membrane current, which was inward at clamp potential of -60 mV (Fig. 1). This current appeared with a delay of 1.5% 2 min after CI-1atrotoxin application, slowly increased and reached a plateau within 3-5 min. It slowly disappeared within the same time interval when oocytes were washed out with a-latrotoxin-free physiological solution. Delay and time-course of the response were similar in oocytes with and without follicle cell layer. This indicated that the effect of
2 1 Jr
LTx
-25
2 min
A+&
Electrophysiological measurements
Oocytes were voltage-clamped using a standard 24ntracellular microelectrode circuit with virtual ground current measurement. Borosilicate glass capillaries were pulled and filled with 3 M KC1 solution. Voltage recording and current injection electrodes had d.c. resistances between 0.5 and 2.0 MD. The oocytes were placed in a small groove in an experimental chamber and continuously perfused with ND 96 physiological solution. Solution changes were normally completed within 15 s. Current and voltage signals were observed on an oscilloscope and simultaneously recorded on a chart recorder. All experiments were done at room temperature (19-22°C). Low Cl- solution used in some experiments contained (in mM): NaCl 58, Na,SO, 19, CaCl, 1.8, KC1 2, MgCl,l, sucrose 19, HEPES 5 (titrated to pH 7.5). Concentrated stock solution of a-latrotoxin was purified from the venom of the Central Asia spider karakurt (black widow spider, Latrodectus mactans tredecimguttatus), by previously published methods2* It was divided into small aliquots and stored at -80°C. Concanavalin A (Pharmacia) was dissolved in the physiological solution immediately before the experiment. RESULTS
Induction
of a-latrotoxin
responses
Oocytes previously injected with rat brain poly(A+)-RNA responded to bath application of
+5 ._._1x- ._-
P+L-____--6_“__-_ 4
-
‘h-2
Fig. 2. Effect of holding potential changes on the response to a-latrotoxin. Holding potentials applied are indicated by numbers above the traces. Time of exposure to cc-latrotoxin is shown by horizontal bars. Dashed line shows resting current at a holding potential indicated on the left. The reversal potential of the a-latrotoxin-evoked current is near 0 mV. LTx, a-latrotoxin.
Expression of a-latrotoxin
did not depend on the presence of a follicular layer of oocytes. The responses to a-latrotoxin could be observed soon at 1 nM toxin concentration; however, they were noticeably smaller than at 10 nM. Therefore, 10 nM toxin concentration was used in most experiments. A second application of a-latrotoxin immediately after restoration of the current during washing out usually induced a 1.5-4 times smaller response (Fig. 1). The decrease varied from oocyte to oocyte. Washing out (2-3 h) was necessary to restore the same amplitude of the response. Hence, a partial desensitization is a property of a-latrotoxin-induced response in Xenopus oocytes. Control (non-mRNA-injected) oocytes from the same donors usually failed to show responses to a-latrotoxin. Only two of 10 frogs used in the experiments gave control oocytes which showed appreciable a -latrotoxin-induced current (15 +_1 nA; n = 21). However, this current was over three times smaller than that in the oocytes injected with mRNA (56 + 7 nA; n = 25). Oocytes from eight frogs responded to a-latrotoxin only if they were injected with mRNA. cl-latrotoxin
Reversal potential of a-latrotoxin induced response
The ionic nature of a-latrotoxin-evoked current was evaluated by measuring reversal potential of the response to a-latrotoxin. Because of partial desensitization of the response it was difficult to determine the reversal potential on single oocytes. Therefore, three oocytes were used for the determination of reversal potential. This allowed us to give no less than 1 h rest to the oocyte to restore the amplitude of response to a-latrotoxin. An example of the experiment is shown in Fig. 2. The oocytes were clamped at the holding potential indicated and then a-latrotoxin was applied. When holding potential was near to the reversal potential, an additional procedure was carried out in order to be sure that a-latrotoxin evoked a significant ionic conductance. When the response to a-latrotoxin reached a steady value, holding potential was changed to -60 mV and in 2-3 min wash-out was started. Significant inward current, which decreased during wash-out, indicated that a-latrotoxin was effective in inducing the ionic conductance. Reversal potential determined in this way was near to 0 mV (Fig. 2). It hardly changed in low Cl- solution (data not shown).
receptor in Xenopus oocytes
811 ca2+*5
LTx
w_lii_7 LTn
LTx
co2+ 2 min
N" +5---
'l,
.-*
'"
I
Fig. 3. Effect of 9 mM Ca2+ (upper trace), EGTA injection, 1 mM oocyte (middle trace) and 3 mM Co2+ (lower trace) on the response to a-latrotoxin. Holding potential, -60 mV. Horizontal bars indicate the time of application of a-latrotoxin and cations. Dashed line indicates resting current at holding potential. Upper and middle traces were obtained from one oocyte. LTx, a-latrotoxin.
external solution induced large progressive depolarization of the membrane and in some cases deterioration of the oocyte. This effect of EGTA was also observed when 20mM MgC12 was substituted for Ca*+ in the external solution in three experiments. In two experiments we studied the effect of EGTA injection into oocyte on a-latrotoxin response. The oocytes which showed large response to a-latrotoxin were left for 5 h to rest in ND 96 solution without a-latrotoxin, and then 15 mM EGTA, 50-75 nl/ oocyte (0.75-l mM/oocyte) buffered to pH 7.5 were injected into the oocytes 0.5 h before the application. EGTA. Injection did not decrease markedly the a-latrotoxin effect; however, the kinetics of a-latrotoxin response seemed to be slower (Fig. 3, middle). In contrast, EGTA injection greatly reduced (five to 10 times) the transient outward Ca-dependent chloride current Ic,(ca) measured on test rectangular pulses to + 5 mV from holding potential -60 mV. In the experiment shown in Fig. 3 Ic,(caJwas reduced from 160 to 20 nA. Control injection of buffered water (pH 7.5) did not show any influence on the a -1atrotoxin effect or on Ic,(ca). Inorganic Ca-channel blocker Co*+ (3 mM) had no effect on the a-latrotoxin response (three experiments). An example is shown in Fig. 3 (bottom). Effect of concanavalin A
Effect of Ca*+, EGTA and Co*+
The response to a-latrotoxin did not increase when Ca*+ concentration in the external solution was elevated by five times (Fig. 3, top). A significant effect of a-latrotoxin was observed when Ca*+ was not added to external physiological solution. Our attempts to study effect of a-latrotoxin in Ca-free solution using EGTA failed since EGTA (0.5-l mM) in the
Concanavalin A, 1 FM, slowly and irreversibly inhibited the effect of a-latrotoxin. Response to a-latrotoxin was not restored even after 15 h of washing out in concanavalin A-free ND 96 solution (Fig. 4). This was not due to the deterioration of the oocyte, because its resting potential remained unchanged (- 70 mV, for example, shown in Fig. 4) during this period of time.
812
A. K. LTx
FILIPPOV er a/.
Con A LTx
Fig. 4. Effect of I PM concanavalin A on response to cc-latrotoxin. Note that response to a-latrotoxin is slowly inhibited by concanavalin A (left trace) and does not restore even after 15h of wash-out in concanavalin A-free physiological solution (right trace). Holding potential, - 60 mV. Horizontal bars, time of exposure to cl-latrotoxin and concanavalin A. Dotted line indicates resting current at holding potential. Con A, concanavalin A; LTx, cc-latrotoxin.
Other RNA-directed responses Ionic current responses to GABA (100 FM), L-glutamate (100 FM), kainate (100 PM), neuropeptide substance P (1 PM) were also observed in mRNA-injected oocytes. These responses were never observed in control (non-injected) oocytes. Response to acetylcholine, serotonin, the appearance of voltageoperated sodium and Ca-channels were also directed by poly(A+)-mRNA injection in Xenopus oocytes in our experiments. All these responses were similar to those observed in other laboratories in RNA-injected Xenopus oocytes (see, e.g., Refs 5, 13, 27). DISCUSSION
The precise molecular mechanism underlying the a-latrotoxin effect is still a subject of some controversy. Some authors suggest that the action of the toxin is due to its spontaneous insertion across the membrane leading to a channel-forming activity.6 This suggestion leaves unexplained the strict tissue specificity of c(-1atrotoxin action.20 Other authors suggest the occurrence of a specific receptor to cc-latrotoxin on the presynaptic cell membrane.20*31*32 If cr-latrotoxin exerts its physiological effect through the activation of specific receptor on the membrane surface of presynaptic nerve cells then gene encoding of this receptor must exist. Indeed, we have observed that t(-1atrotoxin evokes significant inward transmembrane current at the resting membrane potential (- 60 mV) in Xenopus oocytes previously injected with poly(A+)RNA from rat brain. The properties of this current can be attributed in many aspects to cc-latrotoxin effect on synapses and neurosecretory cells of the PC 12 line. This current will depolarize the membranean effect typical for a-latrotoxin on synapses. Timecourse and reversal potential (near OmV) of the current activated by cc-latrotoxin in the oocytes are close to those obtained for the ionic current activated by cr-latrotoxin in PC 12 cells.39The reversal potential did not change when Cl- concentration was varied in the external solution. A similar result has been obtained on PC 12 cells indicating that cations, but not anions, are mainly responsible for the a-latrotoxininduced current.34 The reversal potential observed can be attributed to a nonspecific cation channel generally believed to be activated by cc-latrotoxin in
nerve cells.20It is well known that ct-latrotoxin effects are not modified by Ca-channel blockers and do not depend significantly on [Ca2+],Utconcentration if another divalent cation is present in the external solution.‘9~20~25~26~34 We observed this in our experiments. These data are also in favour of the hypothesis for the ion channel that has no specific selectivity for Ca2+ and is permeable to other cations such as Na+, K+, Mg* + as well. 2o,34Lectins such as concanavalin A are known to inhibit cc-latrotoxin effects2’ and this has also been observed in our experiments. Since Ca2+ ions seem to enter the cell through Elatrotoxin-activated channel in some preparations~6,~ one can suggest the oocyte’s endogenous [Cali,dependent chloride inward current &-,,,,5 to be part of the response to cr-latrotoxin observed in our experiments. Experiments with EGTA injection into the oocytes to prevent a rise of cytosolic Ca2+ concentration have shown that contribution of Iclcca, into the a-latrotoxin response does not seem to be significant. Indeed, injection of EGTA greatly reduced transient ZcI(caJ,but, however, did not change the amplitude of the a-latrotoxin-induced current. The data discussed are in favour of the suggestion that a-latrotoxin receptor has been successively expressed in the membrane of Xen~pu~ oocytes in our experiments as a consequence of mRNA injection into the oocytes. Several questions, however, remain to be answered. The effective concentration of oc-latrotoxin in our experiments was approximately one order higher than that observed at synapses, PC 12 cells or receptorThe delay in the reconstituted liposomes. 20*32~34 appearance of e-latrotoxin effect in our experiments seemed to be longer than in experiments on PC 12 cells34 and on planar lipid membranes infused with receptor-reconstituted liposomes3? taking into account even the “dead” time of our perfusion system. These points can probably bc explained if we assume that a-latrotoxin severely penetrates through the vitelline membrane of the oocyte which had not been removed in our experiments. Direct experiments on the oocytes without vitelline membrane can answer this question.
However, we have observed that the follicle cell layer which is usually considered as a barrier for large molecules2~3sdoes not affect the delay and the timecourse of the response to a-latrotoxin. Another point
Expression of x-latrotoxin receptor in Xenopus oocytes
is that a-latrotoxin effect is reversible in our experiments, whereas it does not reverse in synapses and PC 12 cell~.~*~ How can all these points be explained? The receptor to a-latrotoxin seems to have a complex subunit structure.3’ The functional role of the subunits is unknown. Besides, the complex chain of molecular events was suggested after a-latrotoxin binding to the receptor.3*20One can speculate that receptor subunits are not expressed properly or some other disturbances in receptor assembly take place in the oocyte membrane. Additional experiments are needed to answer this question. We suggest, however, that these disturbances, if they take place, are not too large since
813
many properties of the response to a-latrotoxin are preserved, Therefore, we conclude that functional cc-latrotoxin receptor can be expressed in the membrane of X. laevis oocytes after the mRNA injection from the brain into the oocyte cytoplasm. Hence, the goal of future investigations is to search for DNA encoding receptor subunits and to study structurefunction ~lationships of the a-latrotoxin receptor by a combined use of site-directed mutagenesis and electrophysiological recording. Acknowledgemenrs-We are grateful to Dr Viktor Uteshev for providing us with frogs, X. laeuis.
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(Accepted 19 June 1990)
