Khvotchev M, Südhof T C
Center for Basic Neuroscience, Department of Molecular Genetics and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, TX 75235, USA.
EMBO J. 2000 Jul 3;19(13):3250-62. doi: 10.1093/emboj/19.13.3250.
alpha-latrotoxin, a component of black widow spider venom, binds to presynaptic nerve terminals and stimulates massive neurotransmitter release. Previous studies have demonstrated that alpha-latrotoxin first binds to two high-affinity receptors on nerve terminals, neurexins and CLs (CIRLs and latrophilins), and then executes a critical, second step of unknown nature that stimulates neurotransmitter release. We now demonstrate that incubation of alpha-latrotoxin with synaptosomes at 0 degrees C results in its peripheral membrane association. Incubation at 37 degrees C, however, converts the toxin into an operationally integral membrane protein, and induces generation of a protease-resistant fragment that consists of the entire N-terminal domain of alpha-latrotoxin and becomes protease sensitive after lysis of synaptosomes. Our data suggest that alpha-latrotoxin inserts into the presynaptic plasma membrane after receptor binding, resulting in an intracellular location of the N-terminal sequences. Membrane insertion of the N-terminal domain of alpha-latrotoxin occurs spontaneously, independently of membrane recycling or transmembrane ion gradients. We postulate that alpha-latrotoxin acts intracellularly in triggering release, and propose that non-selective cation channels induced by alpha-latrotoxin may be a by-product of membrane insertion.
α- latrotoxin是黑寡妇蜘蛛毒液的一种成分，它与突触前神经末梢结合并刺激大量神经递质释放。先前的研究表明，α- latrotoxin首先与神经末梢上的两种高亲和力受体——神经配素和CLs（CIRLs和促毒素亲和蛋白）结合，然后执行关键的第二步，其性质未知，但能刺激神经递质释放。我们现在证明，在0摄氏度下将α- latrotoxin与突触体一起孵育会导致其与外周膜结合。然而，在37摄氏度下孵育会将毒素转化为一种功能上的整合膜蛋白，并诱导产生一个蛋白酶抗性片段，该片段由α- latrotoxin的整个N端结构域组成，在突触体裂解后对蛋白酶敏感。我们的数据表明，α- latrotoxin在受体结合后插入突触前质膜，导致N端序列位于细胞内。α- latrotoxin的N端结构域的膜插入是自发发生的，与膜循环或跨膜离子梯度无关。我们推测α- latrotoxin在触发释放时在细胞内起作用，并提出由α- latrotoxin诱导的非选择性阳离子通道可能是膜插入的副产物。
