Filippov A K, Kobrinsky E M, Tsurupa G P, Pashkov V N, Grishin E V
Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region.
Neuroscience. 1990;39(3):809-14. doi: 10.1016/0306-4522(90)90263-4.
alpha-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 alpha-latrotoxin in the membrane of Xenopus laevis oocytes. Responses to alpha-latrotoxin were studied using a double microelectrode voltage-clamp technique on X. laevis oocytes previously injected with poly(A+)-RNA from rat brain. alpha-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 alpha-latrotoxin immediately after washing out induced a smaller response. Reversal potential of this current was near to 0 mV; it hardly changed in low Cl- external solution. Response to alpha-latrotoxin did not depend significantly on the variation of Ca2+ concentration in external solution. Ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) injection into oocytes did not decrease alpha-latrotoxin-induced current, but seemed to slow the kinetics of the response. Inorganic Ca-channel blocker Co2+ had no effect on alpha-latrotoxin response. These results indicate that alpha-latrotoxin-induced inward current flows mainly through cation nonspecific channel. A lectin concanavalin A irreversibly inhibited alpha-latrotoxin-evoked inward current. Many of these observations are similar to those reported for nerve cells after alpha-latrotoxin application. The data obtained suggest that functional receptor to alpha-latrotoxin can be successively expressed in the membrane of Xenopus oocytes providing future search of DNA encoding receptor subunits and study of receptor structure-function relationship.
α-拉毒素是黑寡妇蜘蛛毒液的主要毒素，据推测它可与突触前细胞膜上的特定受体结合，并激活一个非选择性阳离子通道。本研究的目的是在非洲爪蟾卵母细胞膜上表达α-拉毒素的受体。使用双微电极电压钳技术，对先前注射了来自大鼠脑的聚腺苷酸RNA（poly(A+) - RNA）的非洲爪蟾卵母细胞研究其对α-拉毒素的反应。结果表明，在钳制电位为-60 mV时，10 nM的α-拉毒素可诱导出一种缓慢激活的可逆内向膜电流。洗脱后立即再次施加α-拉毒素，诱导出的反应较小。该电流的反转电位接近0 mV；在低氯的细胞外溶液中几乎没有变化。对α-拉毒素的反应在很大程度上不依赖于细胞外溶液中钙离子浓度的变化。向卵母细胞注射乙二醇双（2-氨基乙醚）四乙酸（EGTA）并不会降低α-拉毒素诱导的电流，但似乎会减慢反应的动力学。无机钙通道阻滞剂Co2+对α-拉毒素的反应没有影响。这些结果表明，α-拉毒素诱导的内向电流主要通过阳离子非特异性通道流动。凝集素伴刀豆球蛋白A不可逆地抑制α-拉毒素诱发的内向电流。许多这些观察结果与α-拉毒素作用于神经细胞后所报道的结果相似。所获得的数据表明，α-拉毒素的功能性受体可以在非洲爪蟾卵母细胞膜上成功表达，这为未来寻找编码受体亚基的DNA以及研究受体结构-功能关系提供了可能。
