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First published online 12 April 2005
doi: 10.1242/jcs.02322

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Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers

Department of Physiology II, University of Freiburg, Hermann-Herder-Str. 7, 79104 Freiburg, Germany

View larger version (44K): [in a new window]   Fig. 1. Kir2.1 and Kir2.4 show different levels of surface expression. (A) Representative confocal images of OK cells expressing GFPKir2.1 and GFPKir2.4. Note that only GFPKir2.1 shows distinct plasma membrane fluorescence (arrow). (B) Surface expression of extracellularly HA-tagged GFPKir2.1 and GFPKir2.4 detected by -HA immunocytochemistry without membrane permeabilization (TX, Triton X-100; see Materials and Methods). (C) Cartoon depicting the membrane topology of Kir channels. The cytoplasmic N- and C-terminal domains contain trafficking signals for efficient ER and Golgi export, respectively (Ma et al., 2001; Stockklausner et al., 2001; Stockklausner and Klöcker, 2003). (D) Sequence alignment of the known trafficking signals in Kir2.1 and Kir2.4.

View larger version (55K): [in a new window]   Fig. 2. Kir2.4 accumulates in the Golgi complex. (A) Representative confocal images showing the subcellular distribution of GFPKir2.4 in OK cells. Golgi accumulation is shown by co-localization of GFPKir2.4 with DsRed2 fused to the targeting sequence of 1,4-galactosyltransferase (Golgi; upper panel) and redistribution of Kir2.4 into the ER by incubation with brefeldin A (BFA; lower panel). (B) Golgi accumulation of GFPKir2.4 is not due to retrograde transport to the Golgi complex. Extracellular epitope tagging does not reveal significant internalization of GFPKir2.4 after 16 hours (upper panel); inhibition of clathrin-dependent endocytosis by co-expression of a HA-tagged dominant-negative dynamin (dn-Dyn) mutant (dynamin-K44A) (Damke et al., 1994) does not affect the subcellular distribution of GFPKir2.4 (lower panel).

View larger version (33K): [in a new window]   Fig. 3. Sequence information in the Kir2.1 C-terminus is both necessary and sufficient to promote Golgi-to-plasma membrane transport. (A) Schematic drawing of Kir2.1/Kir2.4 chimeric constructs. Kir2.4-2.1(N): 1-77Kir2.4 was substituted by 1-69Kir2.1; Kir2.4-2.1(C): 212-439Kir2.4 was substituted by 204-428Kir2.1; Kir2.4-int(2.1): 241-260Kir2.4 was replaced by 233-252Kir2.1; Kir2.1-int(2.4): 233-252Kir2.1 was replaced by 241-260Kir2.4. (B) The C-terminal domain of Kir2.1 contains critical sequence information that is sufficient to increase Kir2.4 surface expression. Representative confocal images of OK cells expressing extracellularly HA-tagged GFPKir2.4 and GFPKir2.4-2.1(C), processed for -HA immunocytochemistry without membrane permeabilization (TX: Triton X-100). (C) A stretch of 20 amino acids of Kir2.1 (233-252Kir2.1) is sufficient to increase surface expression of Kir2.4 and necessary for efficient surface expression of Kir2.1. Representative confocal images of OK cells expressing the indicated extracellularly HA-tagged constructs, processed for detecting their surface expression. Total protein expression of the various constructs is similar as shown in -GFP immunoblot analysis below. (D) Quantification of the surface expression experiments in (C). (E) The Kir2.1 trafficking determinant actively promotes Golgi-to-plasma membrane transport. Substitution of 233-252Kir2.1 by a flexible glycine-serine linker peptide (Kir2.1-GS) reduces surface expression of Kir2.1, whereas Kir2.4 surface expression remains unaffected by homologous substitution of 241-260Kir2.4 by the linker peptide (Kir2.4-GS). *Statistically significant when compared with respective controls (P<0.01; unpaired Student's t-test; see Materials and Methods).

View larger version (21K): [in a new window]   Fig. 4. Sequence characterization of the Golgi-to-plasma membrane trafficking determinant in Kir2.1. (A) Primary sequence alignment of the identified trafficking determinant in Kir2.1 with the homologous sequence of Kir2.4. Non-conservative exchanges are in bold letters, the YXX consensus motif for adaptin binding is underlined. (B,C) Quantification of surface expression of Kir2.1 mutants and Kir2.4 mutants as indicated. Note that the Kir2.1 mutants in (B) show Golgi accumulation, indicating that the decrease in surface expression is not due to folding or ER export defects but to impaired post-Golgi transport. *Statistically significant when compared with respective controls (P<0.01; unpaired Student's t-test; see Materials and Methods).

View larger version (10K): [in a new window]   Fig. 5. The Kir2.1 Golgi-to-plasma membrane trafficking signal governs surface expression of Kir2.1/Kir2.4 heteromers. (A) Co-expression of GFPKir2.1 increases surface expression of HA-tagged Kir2.4. (B) Co-expression of Kir2.1 with the identified trafficking signal having been replaced by the homologous Kir2.4 sequence [Kir2.1-int(2.4)] reverses the effects of co-expressed wild-type GFPKir2.1 on promoting surface expression of HA-tagged Kir2.4. *Statistically significant when compared with respective control (P<0.01; unpaired Student's t-test; see Materials and Methods).

View larger version (29K): [in a new window]   Fig. 6. The identified Golgi-to-plasma membrane trafficking motif is recognized by the neuronal sorting machinery. (A) Representative confocal images of hippocampal neurons expressing extracellularly HA-tagged GFPKir2.4 and GFPKir2.4-int(2.1), respectively. Surface expression of channel constructs was determined by -HA immunocytochemistry without membrane permeabilization (TX, Triton X-100). The clustered distribution of GFPKir2.4-int(2.1) is artificially induced by the in vivo surface-labeling procedure. (B) Quantification of surface expression (n=10 neurons). Cy-3 fluorescence intensity values of neuronal surface stainings were corrected for background and related to total protein expression measured by GFP fluorescence. Data are given as relative intensity values per µm2. *Statistically significant when compared with respective control (P<0.01; unpaired Student's t-test; see Materials and Methods).

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