Singh Brij B, Lockwich Timothy P, Bandyopadhyay Bidhan C, Liu Xibao, Bollimuntha Sunitha, Brazer So-Ching, Combs Christian, Das Sunit, Leenders A G Miriam, Sheng Zu-Hang, Knepper Mark A, Ambudkar Suresh V, Ambudkar Indu S
Secretory Physiology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892, USA.
Mol Cell. 2004 Aug 27;15(4):635-46. doi: 10.1016/j.molcel.2004.07.010.
The mechanism(s) involved in agonist-stimulation of TRPC3 channels is not yet known. Here we demonstrate that TRPC3-N terminus interacts with VAMP2 and alphaSNAP. Further, endogenous and exogenously expressed TRPC3 colocalized and coimmunoprecipitated with SNARE proteins in neuronal and epithelial cells. Imaging of GFP-TRPC3 revealed its localization in the plasma membrane region and in mobile intracellular vesicles. Recovery of TRPC3-GFP fluorescence after photobleaching of the plasma membrane region was decreased by brefeldin-A or BAPTA-AM. Cleavage of VAMP2 with tetanus toxin (TeNT) did not prevent delivery of TRPC3 to the plasma membrane region but reduced its surface expression. TeNT also decreased carbachol and OAG, but not thapsigargin, stimulated Ca2+ influx. Importantly, carbachol, not thapsigargin, increased surface expression of TRPC3 that was attenuated by TeNT and not by BAPTA. In aggregate, these data suggest that VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to carbachol-stimulation of Ca2+ influx.
TRPC3通道激动剂刺激所涉及的机制尚不清楚。在此我们证明，TRPC3的N端与VAMP2和α-SNAP相互作用。此外，内源性和外源性表达的TRPC3在神经元和上皮细胞中与SNARE蛋白共定位并共免疫沉淀。GFP-TRPC3的成像显示其定位于质膜区域和可移动的细胞内囊泡中。布雷菲德菌素A或BAPTA-AM可降低质膜区域光漂白后TRPC3-GFP荧光的恢复。用破伤风毒素（TeNT）切割VAMP2并不妨碍TRPC3向质膜区域的转运，但会降低其表面表达。TeNT还可减少卡巴胆碱和油酸甘油酯（OAG）刺激的Ca2+内流，但对毒胡萝卜素刺激的Ca2+内流无影响。重要的是，卡巴胆碱而非毒胡萝卜素可增加TRPC3的表面表达，而TeNT可减弱这种增加，BAPTA则无此作用。总的来说，这些数据表明，VAMP2依赖的胞吐作用调节TRPC3通道向质膜的插入，并促进卡巴胆碱刺激的Ca2+内流。
