Tamkun Michael M, O'connell Kristen M S, Rolig Annah S
Department of Biomedical Sciences, Colorado State University, Ft. Collins, CO 80523, USA.
J Cell Sci. 2007 Jul 15;120(Pt 14):2413-23. doi: 10.1242/jcs.007351.
The Kv2.1 delayed-rectifier channel trafficks to 1-3 microm(2) clusters on the surface of neurons and transfected HEK cells. Single quantum dot (Qdot) tracking and FRAP approaches were used to quantify the diffusion of GFP-labeled Kv2.1 channels on the cell surface and address the mechanisms underlying the formation of these unique membrane structures. Mean square displacement analysis of single Kv2.1 channel tracks inside or outside the surface clusters yielded mean diffusion coefficients of 0.03+/-0.02 microm(2)/second and 0.06+/-0.05 microm(2)/second, respectively. Kv2.1 channels outside the clusters effectively ignore the cluster boundary, readily diffusing through these microdomains. However, in 5% of the tracks analyzed, single, non-clustered channels were observed to cross into a cluster and become corralled within the cluster perimeter. Alexa Fluor 594-labelled phalloidin staining and mCherry-Kv2.1 co-expression with GFP-actin indicated that the Kv2.1 surface clusters form where the cortical actin cytoskeleton is reduced. Kv2.1 channels lacking the C-terminus do not form clusters, freely diffusing over the cell surface with a mean diffusion coefficient of 0.07+/-0.04 microm(2)/second. These data support a model whereby the Kv2.1 clusters are formed by sub-membrane cytoskeletal structures that limit the lateral diffusion of only the sub-population of Kv2.1 channels carrying the appropriate modifications on the Kv2.1 C-terminus.
Kv2.1延迟整流通道会转运至神经元和转染的人胚肾（HEK）细胞表面上1-3微米²的聚集体。使用单量子点（Qdot）追踪和荧光漂白恢复（FRAP）方法来量化绿色荧光蛋白（GFP）标记的Kv2.1通道在细胞表面的扩散，并探究这些独特膜结构形成的潜在机制。对表面聚集体内部或外部单个Kv2.1通道轨迹的均方位移分析得出，其平均扩散系数分别为0.03±0.02微米²/秒和0.06±0.05微米²/秒。聚集体外部的Kv2.1通道实际上会忽略聚集体边界，很容易扩散通过这些微区。然而，在分析的5%的轨迹中，观察到单个非聚集体通道会进入一个聚集体并被限制在聚集体周边范围内。用Alexa Fluor 594标记的鬼笔环肽染色以及mCherry-Kv2.1与GFP-肌动蛋白共表达表明，Kv2.1表面聚集体形成于皮质肌动蛋白细胞骨架减少的位置。缺乏C末端的Kv2.1通道不会形成聚集体，而是以0.07±0.04微米²/秒的平均扩散系数在细胞表面自由扩散。这些数据支持了一种模型，即Kv2.1聚集体是由膜下细胞骨架结构形成的，这些结构仅限制了在Kv2.1 C末端带有适当修饰的Kv2.1通道亚群的横向扩散。
