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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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KChIP1-labeled vesicles are not COPII coated. (A and B) HeLa cells were transfected to express KChIP1-EYFP or KChIP1-EYFP together with the constitutively active Sar1(H79G) mutant. The cells were immunostained with anti-Sec23, a COPII coat component, with visualization with Texas red–streptavidin. The color overlays show KChIP1-EYFP in green and Sec23 in red (colocalization appear in yellow). No colocalization between Sec23 and KChIP-EYFP can be observed in cells expressing Sar1(H79G). Bars, 10 μm. (C–F) Immunolabeling of frozen sections of transfected HeLa cells with anti-GFP to detect KChIP1-EYFP using 10 nm gold and anti-Sec23 using 15 nm colloidal gold. All micrographs are from cotransfected cells, but in some images, only labeling with one antibody is detectable. Small arrows indicate 10 nm gold and large arrows indicate 15 nm gold. Bars, 100 nm.
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fig4: KChIP1-labeled vesicles are not COPII coated. (A and B) HeLa cells were transfected to express KChIP1-EYFP or KChIP1-EYFP together with the constitutively active Sar1(H79G) mutant. The cells were immunostained with anti-Sec23, a COPII coat component, with visualization with Texas red–streptavidin. The color overlays show KChIP1-EYFP in green and Sec23 in red (colocalization appear in yellow). No colocalization between Sec23 and KChIP-EYFP can be observed in cells expressing Sar1(H79G). Bars, 10 μm. (C–F) Immunolabeling of frozen sections of transfected HeLa cells with anti-GFP to detect KChIP1-EYFP using 10 nm gold and anti-Sec23 using 15 nm colloidal gold. All micrographs are from cotransfected cells, but in some images, only labeling with one antibody is detectable. Small arrows indicate 10 nm gold and large arrows indicate 15 nm gold. Bars, 100 nm.

Mentions: We tested other intracellular markers for KChIP1-labeled vesicles, including P23 and P24, members of the P24 protein family. These proteins may be cargo receptors in ER-derived COPII transport vesicles, and therefore, were candidates to test for KChIP1-containing vesicles (Blum et al., 1999). EGFP-P23 and EGFP-P24 localized to punctate structures that were concentrated in the perinuclear region. However, they did not show overlap with KChIP1-hcRed in cotransfected HeLa cells (unpublished data). We also did not see a convincing overlap of KChIP1-EYFP and the COPII coat protein, Sec23 (Fig. 4 A). One problem with labeling COPII-coated transport vesicles is that the COPII coat can be lost rapidly after budding from the ER (Aridor et al., 1995; Stephens et al., 2000). Uncoating of the COPII-coated vesicles is initiated by GTP hydrolysis by Sar1, and can be inhibited by expression of the constitutively active Sar1(H79G) mutant (Aridor et al., 1995; Rowe et al., 1996). In the presence of the Sar1(H79G) mutant, Sec23-labeled structures accumulated in a perinuclear region (Fig. 4 B), as would be expected if COPII vesicles reached the ERGIC but could not uncoat and fuse with this compartment. In contrast, the KChIP1-EYFP–labeled vesicles did not have an altered distribution (Fig. 4 B). No colocalization was observed, at the light microscope level, between Sec23 and KChIP1 in cells that coexpressed Sar1(H79G) (Fig. 4 B). This suggested that the KChIP1-EYFP–labeled vesicles were not COPII-coated vesicles; however, to confirm this, immunoelectron microscopy was performed. Structures containing KChIP1-EYFP were detected using anti-GFP and were found to be vesicles that were similar in size (50–100 nm) to vesicles that were labeled for Sec23. Again, minimal overlap was observed, and in most cases, structures were labeled for YFP or Sec23 alone. Only 8 of 175 vesicles (4.8% colocalization) were labeled for YFP and Sec23 in control cells, and 7 of 151 vesicles (4.9%) in cells expression Sar1(H79G).


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)

KChIP1-labeled vesicles are not COPII coated. (A and B) HeLa cells were transfected to express KChIP1-EYFP or KChIP1-EYFP together with the constitutively active Sar1(H79G) mutant. The cells were immunostained with anti-Sec23, a COPII coat component, with visualization with Texas red–streptavidin. The color overlays show KChIP1-EYFP in green and Sec23 in red (colocalization appear in yellow). No colocalization between Sec23 and KChIP-EYFP can be observed in cells expressing Sar1(H79G). Bars, 10 μm. (C–F) Immunolabeling of frozen sections of transfected HeLa cells with anti-GFP to detect KChIP1-EYFP using 10 nm gold and anti-Sec23 using 15 nm colloidal gold. All micrographs are from cotransfected cells, but in some images, only labeling with one antibody is detectable. Small arrows indicate 10 nm gold and large arrows indicate 15 nm gold. Bars, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171252&req=5

fig4: KChIP1-labeled vesicles are not COPII coated. (A and B) HeLa cells were transfected to express KChIP1-EYFP or KChIP1-EYFP together with the constitutively active Sar1(H79G) mutant. The cells were immunostained with anti-Sec23, a COPII coat component, with visualization with Texas red–streptavidin. The color overlays show KChIP1-EYFP in green and Sec23 in red (colocalization appear in yellow). No colocalization between Sec23 and KChIP-EYFP can be observed in cells expressing Sar1(H79G). Bars, 10 μm. (C–F) Immunolabeling of frozen sections of transfected HeLa cells with anti-GFP to detect KChIP1-EYFP using 10 nm gold and anti-Sec23 using 15 nm colloidal gold. All micrographs are from cotransfected cells, but in some images, only labeling with one antibody is detectable. Small arrows indicate 10 nm gold and large arrows indicate 15 nm gold. Bars, 100 nm.
Mentions: We tested other intracellular markers for KChIP1-labeled vesicles, including P23 and P24, members of the P24 protein family. These proteins may be cargo receptors in ER-derived COPII transport vesicles, and therefore, were candidates to test for KChIP1-containing vesicles (Blum et al., 1999). EGFP-P23 and EGFP-P24 localized to punctate structures that were concentrated in the perinuclear region. However, they did not show overlap with KChIP1-hcRed in cotransfected HeLa cells (unpublished data). We also did not see a convincing overlap of KChIP1-EYFP and the COPII coat protein, Sec23 (Fig. 4 A). One problem with labeling COPII-coated transport vesicles is that the COPII coat can be lost rapidly after budding from the ER (Aridor et al., 1995; Stephens et al., 2000). Uncoating of the COPII-coated vesicles is initiated by GTP hydrolysis by Sar1, and can be inhibited by expression of the constitutively active Sar1(H79G) mutant (Aridor et al., 1995; Rowe et al., 1996). In the presence of the Sar1(H79G) mutant, Sec23-labeled structures accumulated in a perinuclear region (Fig. 4 B), as would be expected if COPII vesicles reached the ERGIC but could not uncoat and fuse with this compartment. In contrast, the KChIP1-EYFP–labeled vesicles did not have an altered distribution (Fig. 4 B). No colocalization was observed, at the light microscope level, between Sec23 and KChIP1 in cells that coexpressed Sar1(H79G) (Fig. 4 B). This suggested that the KChIP1-EYFP–labeled vesicles were not COPII-coated vesicles; however, to confirm this, immunoelectron microscopy was performed. Structures containing KChIP1-EYFP were detected using anti-GFP and were found to be vesicles that were similar in size (50–100 nm) to vesicles that were labeled for Sec23. Again, minimal overlap was observed, and in most cases, structures were labeled for YFP or Sec23 alone. Only 8 of 175 vesicles (4.8% colocalization) were labeled for YFP and Sec23 in control cells, and 7 of 151 vesicles (4.9%) in cells expression Sar1(H79G).

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