Khanna R, Sun L, Li Q, Guo L, Stanley E F
Cellular and Molecular Biology Division, Toronto Western Research Institute, University Health Network, 399 Bathurst Street, MP14-320, Toronto, Ontario, Canada M5T 2S8.
Neuroscience. 2006;138(4):1115-25. doi: 10.1016/j.neuroscience.2005.12.050. Epub 2006 Feb 13.
The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.
突触前N型钙通道（CaV2.2）与递质释放位点装置相关，在递质释放的门控过程中起关键作用。有人提出，一种独特的CaV2.2长C末端剪接变体靶向神经末梢，并通过钙/钙调蛋白依赖性丝氨酸蛋白激酶（CASK）和Munc-18相互作用蛋白（MINT）这两种模块化衔接蛋白锚定在释放面。我们使用分离的鸡睫状神经节花萼末梢以及两种对CaV2.2长C末端剪接变体具有选择性的新抗体（L4569、L4570）来检验这些假设。通过Rab3a相互作用分子（RIM）共染色和定量免疫细胞化学鉴定，发现CaV2.2长C末端剪接变体存在于突触前递质释放位点。CASK也存在于末梢，与它的结合伴侣MINT结合或独立存在。从脑裂解物中免疫沉淀CaV2.2长C末端剪接变体可共沉淀出CASK，证实这两种蛋白可形成复合物。然而，在花萼末梢释放面，CASK既不与CaV2.2长C末端剪接变体也不与递质释放位点标记物RIM共定位。MINT也不与CaV2.2长C末端剪接变体共定位。我们的结果表明，天然的CaV2.2长C末端剪接变体靶向完整突触前末梢的递质释放位点。然而，CASK在释放位点缺乏富集，加上该蛋白或其伴侣MINT未能与CaV2.2共定位，这与这些模块化衔接蛋白将CaV2.2锚定在突触前神经末梢的观点相悖。
