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Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway.

Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, Burgoyne RD - J. Cell Biol. (2005)

Bottom Line: Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP.The EF-hand mutant had no effect on general exocytic traffic.When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, England, UK.

ABSTRACT
The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

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Related in: MedlinePlus

Effect of disrupting COPII or COPI on the traffic of VSVG and Kv4.2 to the cell surface. (A) HeLa cells were transfected to express VSVG-GFP in the absence (A') or presence (A'') of Sar1(H79G). After transfection, cells were maintained at 37°C for 24 h. Sar1(H79G)-transfected cells were stained with anti-Sec23. In cells cotransfected with Sar1(H79G), its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of VSVG to the plasma membrane was inhibited by the Sar1 mutant. (B) HeLa cells were cotransfected with KChIP1-EYFP and ECFP-Kv4.2 in the absence (B') or presence (B'') of Sar1(H79G), and its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of Kv4.2 to the plasma membrane was not prevented by Sar1(H79G). (C) HeLa cells were transfected to express VSVG-GFP and ECFP-Kv4.2 in the presence of Sar1(H79G). The traffic of both proteins out of the ER was inhibited and they can be seen to be colocalized in the overlay. (D) HeLa cells were transfected to express VSVG-GFP in the absence (D') or presence (D'') of ARF1(Q71L). ARF1(Q71L)-transfected cells were stained with anti-HA to detect the ARF1(Q71L). Traffic of VSVG to the plasma membrane was inhibited by the ARF1 mutant. (E) HeLa cells were cotransfected with KChIP1-EYFP, ECFP-Kv4.2 and ARF1(Q71L). Traffic of Kv4.2 to the cell surface was blocked by ARF1(Q71L). Bars, 10 μm. The color overlays show VSVG-EGFP or ECFP-Kv4.2 in green and anti-Sec23 in red (A and B), VSVG-GFP in green and ECFP-Kv4.2 in red (C) or with anti-HA staining in red (D and E) with colocalization seen in yellow. (F) VSVG-EGFP or ECFP-Kv4.2 fluorescence was imaged in control cells or cells expressing Sar1(H79G) or ARF1(Q71L), and quantified by drawing regions of interest around the outside and the inside of the plasma membrane to allow determination of the percentage of total fluorescence at the plasma membrane. Data derived from the indicated number of cells are expressed as mean ± SEM.
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fig5: Effect of disrupting COPII or COPI on the traffic of VSVG and Kv4.2 to the cell surface. (A) HeLa cells were transfected to express VSVG-GFP in the absence (A') or presence (A'') of Sar1(H79G). After transfection, cells were maintained at 37°C for 24 h. Sar1(H79G)-transfected cells were stained with anti-Sec23. In cells cotransfected with Sar1(H79G), its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of VSVG to the plasma membrane was inhibited by the Sar1 mutant. (B) HeLa cells were cotransfected with KChIP1-EYFP and ECFP-Kv4.2 in the absence (B') or presence (B'') of Sar1(H79G), and its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of Kv4.2 to the plasma membrane was not prevented by Sar1(H79G). (C) HeLa cells were transfected to express VSVG-GFP and ECFP-Kv4.2 in the presence of Sar1(H79G). The traffic of both proteins out of the ER was inhibited and they can be seen to be colocalized in the overlay. (D) HeLa cells were transfected to express VSVG-GFP in the absence (D') or presence (D'') of ARF1(Q71L). ARF1(Q71L)-transfected cells were stained with anti-HA to detect the ARF1(Q71L). Traffic of VSVG to the plasma membrane was inhibited by the ARF1 mutant. (E) HeLa cells were cotransfected with KChIP1-EYFP, ECFP-Kv4.2 and ARF1(Q71L). Traffic of Kv4.2 to the cell surface was blocked by ARF1(Q71L). Bars, 10 μm. The color overlays show VSVG-EGFP or ECFP-Kv4.2 in green and anti-Sec23 in red (A and B), VSVG-GFP in green and ECFP-Kv4.2 in red (C) or with anti-HA staining in red (D and E) with colocalization seen in yellow. (F) VSVG-EGFP or ECFP-Kv4.2 fluorescence was imaged in control cells or cells expressing Sar1(H79G) or ARF1(Q71L), and quantified by drawing regions of interest around the outside and the inside of the plasma membrane to allow determination of the percentage of total fluorescence at the plasma membrane. Data derived from the indicated number of cells are expressed as mean ± SEM.

Mentions: Certain cargos, such as procollagen, can traffic from the ER to the Golgi in non–COPII-coated structures. Nevertheless, these all require COPII for their budding from the ER (Mironov et al., 2003; Stephens and Pepperkok, 2004). To test whether Kv4.2/KChIP1 require COPII for traffic through the secretory pathway, the effect of Sar1(H79G) on traffic was examined. Its expression blocked traffic of all previously studied cargos to the cell surface (Kuge et al., 1994; Aridor et al., 1995, 2001; Stephens and Pepperkok, 2004), in some cases, by preventing export from the ER. We used the same experimental conditions to follow VSVG and Kv4.2/KChIP1, which resulted in a similar extent of traffic of each to the plasma membrane (Fig. 5 A', B', D', and F). To assess the effect of Sar1(H79G), the cells were stained with anti-Sec23 to verify the effectiveness of the mutant in redistributing Sec23 to the perinuclear region in the transfected cells. Only cells in which this could be established were examined for traffic of VSVG or Kv4.2/KChIP1. Expression of Sar1(H79G) effectively inhibited traffic of VSVG-EGFP to the plasma membrane (Fig. 5 A). In all cells that we examined, VSVG-EGFP remained in the ER and in perinuclear structures where it overlapped with Sec23. From the quantitative analysis of peripheral VSVG-EGFP, it was clear that inhibition of traffic to the plasma membrane essentially was complete, because the residual level of peripheral fluorescence (∼10% shown by the broken line in Fig. 5 E) is the same as that found using this assay with proteins that are unable to traffic out of the ER (Fig. S5; available at http://www.jcb.org/cgi/content/full/jcb.200506005/DC1) or beyond the Golgi (Fig. 1). In contrast, expression of Sar1(H79G) did not prevent Kv4.2 from reaching the plasma membrane when it was expressed in the presence of KChIP1, despite redistribution of Sec23 (Fig. 5 B). Plasma membrane expression was visible in 24/40 (60%) of the cells. Perinuclear ECFP-Kv4.2 fluorescence was observed often in Sar1(H79G)-expressing cells, as it was in control cells, but this did not overlap substantially with Sec23. Quantification supported the conclusion that significant traffic of Kv4.2/KChIP1 to the plasma membrane still occurred in the presence of Sar1(H79G) (Fig. 5 F). Interestingly, when ECFP-Kv4.2 was expressed without KChIP1, its exit from the ER was inhibited by Sar1(H79G). This also was seen in cells that expressed VSVG-GFP; in all cases examined in which traffic of VSVG-GFP was blocked, ECFP Kv4.2 was retained in reticular ER-like structures and overlapped completely with VSVG-GFP (Fig. 5 C), unlike the normal Golgi localization of the channel (Figs. S1 and S2).


Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway.

Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, Burgoyne RD - J. Cell Biol. (2005)

Effect of disrupting COPII or COPI on the traffic of VSVG and Kv4.2 to the cell surface. (A) HeLa cells were transfected to express VSVG-GFP in the absence (A') or presence (A'') of Sar1(H79G). After transfection, cells were maintained at 37°C for 24 h. Sar1(H79G)-transfected cells were stained with anti-Sec23. In cells cotransfected with Sar1(H79G), its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of VSVG to the plasma membrane was inhibited by the Sar1 mutant. (B) HeLa cells were cotransfected with KChIP1-EYFP and ECFP-Kv4.2 in the absence (B') or presence (B'') of Sar1(H79G), and its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of Kv4.2 to the plasma membrane was not prevented by Sar1(H79G). (C) HeLa cells were transfected to express VSVG-GFP and ECFP-Kv4.2 in the presence of Sar1(H79G). The traffic of both proteins out of the ER was inhibited and they can be seen to be colocalized in the overlay. (D) HeLa cells were transfected to express VSVG-GFP in the absence (D') or presence (D'') of ARF1(Q71L). ARF1(Q71L)-transfected cells were stained with anti-HA to detect the ARF1(Q71L). Traffic of VSVG to the plasma membrane was inhibited by the ARF1 mutant. (E) HeLa cells were cotransfected with KChIP1-EYFP, ECFP-Kv4.2 and ARF1(Q71L). Traffic of Kv4.2 to the cell surface was blocked by ARF1(Q71L). Bars, 10 μm. The color overlays show VSVG-EGFP or ECFP-Kv4.2 in green and anti-Sec23 in red (A and B), VSVG-GFP in green and ECFP-Kv4.2 in red (C) or with anti-HA staining in red (D and E) with colocalization seen in yellow. (F) VSVG-EGFP or ECFP-Kv4.2 fluorescence was imaged in control cells or cells expressing Sar1(H79G) or ARF1(Q71L), and quantified by drawing regions of interest around the outside and the inside of the plasma membrane to allow determination of the percentage of total fluorescence at the plasma membrane. Data derived from the indicated number of cells are expressed as mean ± SEM.
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Related In: Results  -  Collection

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fig5: Effect of disrupting COPII or COPI on the traffic of VSVG and Kv4.2 to the cell surface. (A) HeLa cells were transfected to express VSVG-GFP in the absence (A') or presence (A'') of Sar1(H79G). After transfection, cells were maintained at 37°C for 24 h. Sar1(H79G)-transfected cells were stained with anti-Sec23. In cells cotransfected with Sar1(H79G), its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of VSVG to the plasma membrane was inhibited by the Sar1 mutant. (B) HeLa cells were cotransfected with KChIP1-EYFP and ECFP-Kv4.2 in the absence (B') or presence (B'') of Sar1(H79G), and its effectiveness was checked by examining distribution of Sec23, which became concentrated in a perinuclear region. Traffic of Kv4.2 to the plasma membrane was not prevented by Sar1(H79G). (C) HeLa cells were transfected to express VSVG-GFP and ECFP-Kv4.2 in the presence of Sar1(H79G). The traffic of both proteins out of the ER was inhibited and they can be seen to be colocalized in the overlay. (D) HeLa cells were transfected to express VSVG-GFP in the absence (D') or presence (D'') of ARF1(Q71L). ARF1(Q71L)-transfected cells were stained with anti-HA to detect the ARF1(Q71L). Traffic of VSVG to the plasma membrane was inhibited by the ARF1 mutant. (E) HeLa cells were cotransfected with KChIP1-EYFP, ECFP-Kv4.2 and ARF1(Q71L). Traffic of Kv4.2 to the cell surface was blocked by ARF1(Q71L). Bars, 10 μm. The color overlays show VSVG-EGFP or ECFP-Kv4.2 in green and anti-Sec23 in red (A and B), VSVG-GFP in green and ECFP-Kv4.2 in red (C) or with anti-HA staining in red (D and E) with colocalization seen in yellow. (F) VSVG-EGFP or ECFP-Kv4.2 fluorescence was imaged in control cells or cells expressing Sar1(H79G) or ARF1(Q71L), and quantified by drawing regions of interest around the outside and the inside of the plasma membrane to allow determination of the percentage of total fluorescence at the plasma membrane. Data derived from the indicated number of cells are expressed as mean ± SEM.
Mentions: Certain cargos, such as procollagen, can traffic from the ER to the Golgi in non–COPII-coated structures. Nevertheless, these all require COPII for their budding from the ER (Mironov et al., 2003; Stephens and Pepperkok, 2004). To test whether Kv4.2/KChIP1 require COPII for traffic through the secretory pathway, the effect of Sar1(H79G) on traffic was examined. Its expression blocked traffic of all previously studied cargos to the cell surface (Kuge et al., 1994; Aridor et al., 1995, 2001; Stephens and Pepperkok, 2004), in some cases, by preventing export from the ER. We used the same experimental conditions to follow VSVG and Kv4.2/KChIP1, which resulted in a similar extent of traffic of each to the plasma membrane (Fig. 5 A', B', D', and F). To assess the effect of Sar1(H79G), the cells were stained with anti-Sec23 to verify the effectiveness of the mutant in redistributing Sec23 to the perinuclear region in the transfected cells. Only cells in which this could be established were examined for traffic of VSVG or Kv4.2/KChIP1. Expression of Sar1(H79G) effectively inhibited traffic of VSVG-EGFP to the plasma membrane (Fig. 5 A). In all cells that we examined, VSVG-EGFP remained in the ER and in perinuclear structures where it overlapped with Sec23. From the quantitative analysis of peripheral VSVG-EGFP, it was clear that inhibition of traffic to the plasma membrane essentially was complete, because the residual level of peripheral fluorescence (∼10% shown by the broken line in Fig. 5 E) is the same as that found using this assay with proteins that are unable to traffic out of the ER (Fig. S5; available at http://www.jcb.org/cgi/content/full/jcb.200506005/DC1) or beyond the Golgi (Fig. 1). In contrast, expression of Sar1(H79G) did not prevent Kv4.2 from reaching the plasma membrane when it was expressed in the presence of KChIP1, despite redistribution of Sec23 (Fig. 5 B). Plasma membrane expression was visible in 24/40 (60%) of the cells. Perinuclear ECFP-Kv4.2 fluorescence was observed often in Sar1(H79G)-expressing cells, as it was in control cells, but this did not overlap substantially with Sec23. Quantification supported the conclusion that significant traffic of Kv4.2/KChIP1 to the plasma membrane still occurred in the presence of Sar1(H79G) (Fig. 5 F). Interestingly, when ECFP-Kv4.2 was expressed without KChIP1, its exit from the ER was inhibited by Sar1(H79G). This also was seen in cells that expressed VSVG-GFP; in all cases examined in which traffic of VSVG-GFP was blocked, ECFP Kv4.2 was retained in reticular ER-like structures and overlapped completely with VSVG-GFP (Fig. 5 C), unlike the normal Golgi localization of the channel (Figs. S1 and S2).

Bottom Line: Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP.The EF-hand mutant had no effect on general exocytic traffic.When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, England, UK.

ABSTRACT
The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

Show MeSH
Related in: MedlinePlus