O'Connell Kristen M S, Tamkun Michael M
Department of Biomedical Sciences, Colorado State University, Ft Collins, CO 80523, USA.
J Cell Sci. 2005 May 15;118(Pt 10):2155-66. doi: 10.1242/jcs.02348. Epub 2005 Apr 26.
Voltage-gated potassium (Kv) channels regulate action potential duration in nerve and muscle; therefore changes in the number and location of surface channels can profoundly influence electrical excitability. To investigate trafficking of Kv2.1, 1.4 and 1.3 within the plasma membrane, we combined the expression of fluorescent protein-tagged Kv channels with live cell confocal imaging. Kv2.1 exhibited a clustered distribution in HEK cells similar to that seen in hippocampal neurons, whereas Kv1.4 and Kv1.3 were evenly distributed over the plasma membrane. Using FRAP, surface Kv2.1 displayed limited mobility; approximately 40% of the fluorescence recovered within 20 minutes of photobleach (M(f)=0.41+/-0.04). Recovery occurred not by diffusion from adjacent membrane but probably by transport of nascent channel from within the cell. By contrast, the Kv1 family members Kv1.4 and Kv1.3 were highly mobile, both showing approximately 80% recovery (Kv 1.4 M(f)=0.78+/-0.07; Kv1.3 M(f)=0.78+/-0.04; without correction for photobleach); unlike Kv2.1, recovery was consistent with diffusion of channel from membrane adjacent to the bleach region. Studies using PA-GFP-tagged channels were consistent with the FRAP results. Following photoactivation of a small region of plasma membrane PA-GFP-Kv2.1 remained restricted to the photoactivation ROI, while PA-GFP-Kv1.4 rapidly diffused throughout the cell surface. Additionally, PA-GFP-Kv2.1 moved into regions of the cell membrane not adjacent to the original photoactivation ROI. Sucrose density gradient analysis indicated that half of Kv2.1 is part of a large, macromolecular complex while Kv1.4 sediments as predicted for the tetrameric channel complex. Disruption of membrane cholesterol by cyclodextrin minimally altered Kv2.1 mobility (M(f)=0.32+/-0.03), but significantly increased surface cluster size by at least fourfold. By comparison, the mobility of Kv1.4 decreased following cholesterol depletion with no change in surface distribution. The mobility of Kv1.3 was slightly increased following cyclodextrin treatment. These results indicate that (1) Kv2.1, Kv1.4 and Kv1.3 exist in distinct compartments that exhibit different trafficking properties, (2) membrane cholesterol levels differentially modulate the trafficking and localization of Kv channels and (3) Kv2.1 expressed in HEK cells exhibits a surface distribution similar to that seen in native cells.
电压门控钾（Kv）通道调节神经和肌肉中的动作电位持续时间；因此，表面通道数量和位置的变化会深刻影响电兴奋性。为了研究Kv2.1、1.4和1.3在质膜内的运输，我们将荧光蛋白标记的Kv通道的表达与活细胞共聚焦成像相结合。Kv2.1在HEK细胞中呈现出簇状分布，类似于在海马神经元中观察到的分布，而Kv1.4和Kv1.3则均匀分布在质膜上。使用荧光漂白恢复技术（FRAP），表面Kv2.1的流动性有限；在光漂白后20分钟内约40%的荧光恢复（M(f)=0.41±0.04）。恢复不是通过从相邻膜扩散发生的，而是可能通过细胞内新生通道的运输发生的。相比之下，Kv1家族成员Kv1.4和Kv1.3具有高度的流动性，两者都显示出约80%的恢复率（Kv 1.4 M(f)=0.78±0.07；Kv1.3 M(f)=0.78±0.04；未校正光漂白）；与Kv2.1不同，恢复与通道从漂白区域相邻的膜扩散一致。使用PA-GFP标记通道的研究结果与FRAP结果一致。在质膜的一个小区域进行光激活后，PA-GFP-Kv2.1仍局限于光激活感兴趣区域（ROI），而PA-GFP-Kv1.4则迅速扩散到整个细胞表面。此外，PA-GFP-Kv2.1移动到不与原始光激活ROI相邻的细胞膜区域。蔗糖密度梯度分析表明，一半的Kv2.1是一个大型大分子复合物的一部分，而Kv1.4的沉降情况符合四聚体通道复合物的预测。用环糊精破坏膜胆固醇对Kv2.1的流动性影响最小（M(f)=0.32±0.03），但显著增加了表面簇的大小至少四倍。相比之下，胆固醇耗竭后Kv1.4的流动性降低，表面分布没有变化。环糊精处理后Kv1.3的流动性略有增加。这些结果表明：（1）Kv2.1、Kv1.4和Kv1.3存在于具有不同运输特性的不同区室中；（2）膜胆固醇水平对Kv通道的运输和定位有不同的调节作用；（3）在HEK细胞中表达的Kv2.1表现出与天然细胞中相似的表面分布。
