Bravo-Zehnder M, Orio P, Norambuena A, Wallner M, Meera P, Toro L, Latorre R, González A
Departamento de Inmunologia Clinica y Reumatologia, Facultad de Medicina, Departamento de Biologia Celular y Molecular, Pontificia Universidad Católica de Chile, Santiago.
Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13114-9. doi: 10.1073/pnas.240455697.
The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.
电压和钙激活的钾离子通道（K(V,Ca)）在多种极化上皮细胞中表达，电生理学研究表明，其似乎呈现出组织依赖性的从顶端到基底外侧的区域化分布。利用结构域特异性生物素化和免疫荧光技术，我们发现人类通道K(V,Ca)α亚基（人类慢poke通道，hSlo）主要存在于永久转染的Madin-Darby犬肾细胞的顶端质膜区域。野生型和缺乏唯一潜在N-糖基化位点的突变型hSlo蛋白均能有效地转运至细胞表面，并集中在顶端区域，即使它们的过表达水平比内源性Slo通道密度高200至300倍。此外，衣霉素处理并不能阻止hSlo的顶端分选，这表明不需要内源性糖基化蛋白（如K(V,Ca)β亚基）。从用去污剂或碳酸钠提取后在蔗糖梯度中的漂浮分布判断，hSlo似乎具有靶向脂筏的特性。证据表明，hSlo蛋白拥有通过一种可能涉及与脂筏结合的机制转运至顶端细胞表面的内在信息，且该机制独立于通道本身或相关蛋白的糖基化。因此，这种特定的多跨膜模型蛋白表明，Madin-Darby犬肾细胞中内源性膜蛋白存在不依赖糖基化的顶端转运途径。
