Peter A B, Schittny J C, Niggli V, Reuter H, Sigel E
Department of Pharmacology, University of Bern, Switzerland.
J Cell Biol. 1991 Aug;114(3):455-64. doi: 10.1083/jcb.114.3.455.
Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.
外源信使核糖核酸(mRNA)在非洲爪蟾卵母细胞中表达。利用双电极电压钳技术结合一个特殊设计的小室,测量新表达的定位在特定质膜区域的离子电流,该小室仅使卵母细胞表面的一部分暴露于通道激动剂或抑制剂。发现新表达的电流在卵母细胞的表面膜中分布不均。这种不对称性在表达的早期最为明显,此时几乎只能在卵母细胞的动物半球检测到通道。注射mRNA后4天或更晚,动物极处通道的密度比植物极区域高三倍。观察到从粗组织mRNA和编码大鼠γ-氨基丁酸A型(GABAA)受体通道亚基的互补核糖核酸(cRNA)表达的各种离子通道的分布模式相似。电子显微镜分析显示,两个卵母细胞极区域的微绒毛模式非常相似。因此,电流分布的不对称不是由于表面结构的不对称。在表达期于秋水仙碱或细胞松弛素D中孵育后,发现两个极区域的电流密度相等。无活性的对照物质β-光秋水仙碱对分布的不对称性没有影响。秋水仙碱对表达的全细胞电流幅度没有影响。我们的测量揭示了非洲爪蟾卵母细胞内源性的质膜蛋白表达途径,这可能有助于这种高度组织化细胞极性的形成和维持。