Kaneko Shuji, Cooper Conan B, Nishioka Naoto, Yamasaki Hironobu, Suzuki Atsushi, Jarvis Scott E, Akaike Akinori, Satoh Masamichi, Zamponi Gerald W
Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Neurosci. 2002 Jan 1;22(1):82-92. doi: 10.1523/JNEUROSCI.22-01-00082.2002.
The physical interaction between the presynaptic vesicle release complex and the large cytoplasmic region linking domains II and III of N-type (Ca(v)2.2) calcium channel alpha(1)B subunits is considered to be of fundamental importance for efficient neurotransmission. By PCR analysis of human brain cDNA libraries and IMR32 cell mRNA, we have isolated novel N-type channel variants, termed Ca(v)2.2-Delta1 and Delta2, which lack large parts of the domain II-III linker region, including the synaptic protein interaction site. They appear to be widely expressed across the human CNS as indicated by RNase protection assays. When expressed in tsA-201 cells, both novel variants formed barium-permeable channels with voltage dependences and kinetics for activation that were similar to those observed with the full-length channel. All three channel types exhibited the hallmarks of prepulse facilitation, which interestingly occurred independently of G-protein betagamma subunits. By contrast, the voltage dependence of steady-state inactivation seen with both Delta1 and Delta2 channels was shifted toward more depolarized potentials, and recovery from inactivation of Delta1 and Delta2 channels occurred more rapidly than that of the full-length channel. Moreover, the Delta1 channel was dramatically less sensitive to both omega-conotoxin MVIIA and GVIA than either the Delta2 variant or the full-length construct. Finally, the domain II-III linker region of neither variant was able to effectively bind syntaxin in vitro. These results suggest that the structure of the II-III linker region is an important determinant of N-type channel function and pharmacology. The lack of syntaxin binding hints at a unique physiological function of these channels.
突触前囊泡释放复合物与连接N型（Ca(v)2.2）钙通道α(1)B亚基结构域II和III的大细胞质区域之间的物理相互作用被认为对高效神经传递至关重要。通过对人脑cDNA文库和IMR32细胞mRNA进行PCR分析，我们分离出了新的N型通道变体，称为Ca(v)2.2-Delta1和Delta2，它们缺少结构域II-III连接区域的大部分，包括突触蛋白相互作用位点。核糖核酸酶保护分析表明，它们似乎在人类中枢神经系统中广泛表达。当在tsA-201细胞中表达时，这两种新变体都形成了钡通透性通道，其电压依赖性和激活动力学与全长通道相似。所有三种通道类型都表现出预脉冲易化的特征，有趣的是，这种现象独立于G蛋白βγ亚基发生。相比之下，Delta1和Delta2通道的稳态失活电压依赖性向更去极化的电位偏移，并且Delta1和Delta2通道从失活状态恢复的速度比全长通道更快。此外，Delta1通道对ω-芋螺毒素MVIIA和GVIA的敏感性比Delta2变体或全长构建体都低得多。最后，两种变体的结构域II-III连接区域在体外都不能有效结合 syntaxin。这些结果表明，II-III连接区域的结构是N型通道功能和药理学的重要决定因素。缺乏与syntaxin的结合暗示了这些通道具有独特的生理功能。
