A di-acidic sequence motif enhances the surface expression of the potassium channel TASK-3.
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.
Zuzarte
Marylou
M
Institute of Physiology, Marburg University, Deutschhausstr. 2, 35037 Marburg, Germany.
Rinné
Susanne
S
Schlichthörl
Günter
G
Schubert
Andrea
A
Daut
Jürgen
J
Preisig-Müller
Regina
R
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024342:Amino Acids, Acidic / Q000235:genetics* / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D024683:Potassium Channels, Tandem Pore Domain / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D014982:Xenopus laevis
D024342:Amino Acids, Acidic; C498655:KCNK9 protein, human; D024683:Potassium Channels, Tandem Pore Domain