Zuzarte Marylou, Rinné Susanne, Schlichthörl Günter, Schubert Andrea, Daut Jürgen, Preisig-Müller Regina
Institute of Physiology, Marburg University, Deutschhausstr. 2, 35037 Marburg, Germany.
Traffic. 2007 Aug;8(8):1093-100. doi: 10.1111/j.1600-0854.2007.00593.x. Epub 2007 Jun 5.
We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.
我们已对酸敏钾通道TASK-3近端C末端的一个序列基序EDE进行了特征分析。将人TASK-3通道在非洲爪蟾卵母细胞中表达，并用两种互补技术研究了通道在表面膜上的密度：对血凝素表位标记的TASK-3通道进行发光表面表达测定以及对酸敏钾电流进行电压钳测量。两种方法均表明，将EDE基序的两个谷氨酸残基突变为丙氨酸（ADA突变体）会显著降低TASK-3通道向细胞表面转运。EDE基序的中央天冬氨酸突变对表面表达无影响。在由截短的Kir2.1通道与TASK-3的C末端连接组成的嵌合构建体中，进一步对EDE基序的功能作用进行了特征分析。在这些构建体中，用ADA取代EDE基序也会强烈降低表面表达。对在COS-7细胞中表达的增强型绿色荧光蛋白标记通道进行活细胞成像显示，转染后24小时，野生型TASK-3主要定位于细胞表面，而ADA突变体大多保留在内质网（ER）中。TASK-3 C末端第二个双酸性基序（DAE）的突变对表面表达无影响。TASK-3与衣被募集GTP酶Sar1的GTP限制突变体（Sar1H79G）共表达导致通道在内质网中滞留。我们的数据表明，人TASK-3中的双酸性基序EDE是内质网输出速率的主要决定因素，也是通道有效表面表达所必需的。
